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Antimicrobial activity of n-butyl lactate obtained via enzymatic esterification of lactic acid with n-butanol in supercritical trifluoromethane
J. of Supercritical Fluids 85 (2014) 143–150
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Antimicrobial activity of n-butyl lactate obtained via enzymatic esterification of lactic acid with n-butanol in supercritical trifluoromethane Sabina Kavˇciˇc, Zˇ eljko Knez, Maja Leitgeb ∗ University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Separation Processes and Product Design, Smetanova 17, 2000 Maribor, Slovenia
a r t i c l e
i n f o
Article history: Received 21 August 2013 Received in revised form 7 November 2013 Accepted 8 November 2013 Keywords: n-Butyl lactate Candida antarctica lipase B Supercritical trifluoromethane Antimicrobial tests
a b s t r a c t The lipase-catalyzed synthesis of n-butyl lactate by esterification was performed in supercritical trifluoromethane. Immobilized lipase B from Candida antarctica (Novozym 435) was used as a biocatalyst. Process conditions (pressure and temperature) were optimized performing experiments in a highpressure batch stirred-tank reactor. Experiments were carried out in the operative pressure ranges from 7.5 to 30 MPa and at temperatures 35 ◦ C and 55 ◦ C. For this purpose phase behavior for d,l-lactic acid/n-butanol/Novozym 435/supercritical fluid system at temperature 55 ◦ C and different pressures was studied. The highest conversion of lactic acid after 26 h of reaction performance was obtained in supercritical trifluoromethane at 30 MPa and 55 ◦ C. The n-butyl lactate (standard and enzymatically synthesized) and d,l-lactic acid were tested against four food-borne fungi: Saccharomyces cerevisiae, Aspergillus niger, Trichoderma viride and Penicillium cyclopium and three health-damaging bacteria: Escherichia coli, Pseudomonas fluorescens and Bacillus cereus by the agar well diffusion. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lactic acid (LA) and lactate esters are used in wide applications in food industry for preservation, emulsifiers and flavoring purposes, as well as in pharmaceutical and cosmetic industries [1–4]. LA is also used as an important raw material for the manufacture of biodegradable polymers. Its esters including n-butyl lactate are used as flavors and solvents with excellent properties [3]. The Food and Drug Administration (FDA) included LA on its list of substances considered Generally Recognized As Safe (GRAS) as direct food additives. Ethyl lactate and butyl lactate are also approved for use as direct food additives [5]. Furthermore, broad spectrum disinfecting and microbicidal compositions of biodegradable and environmentally friendly compositions containing esters include butyl lactate. These compositions display activities against
Abbreviations: CLEAs, cross-linking of enzyme aggregates; CLECs, cross-linking of enzyme crystals; FDA, Food and Drug Administration; GC, gas chromatography; GRAS, Generally Recognized As Safe; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; LA, lactic acid; LA1, monomer of lactic acid; LA2, lactoyllactic acid; LA3, lactide; PDA, potato dextrose agar; SCF, supercritical fluid; SC CHF3 , supercritical trifluoromethane; SC C2 H6 , supercritical ethane; SC CO2 , supercritical carbon dioxide. ∗ Corresponding author. Tel.: +386 2 2294 462; fax: +386 2 2527 774. E-mail address: [email protected] (M. Leitgeb). 0896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2013.11.003
the most resistant microbial forms which use safe and non-toxic chemical agents selected from alcohol esters of butyl lactate and its homologs for preparing products with cleaning, solubilizing, antimicrobial and microbicidal properties. Most common antimicrobial products such as chlorine, chlorine dioxide peracetic acid, ozone, hydrogen peroxide, UV- and other radiations, used to reduce microbial population on food and other contact surfaces possess highly oxidizing and sometimes destructive properties. These oxidizing chemicals and physical agents inactivate microorganisms by reacting with their organic material. These antimicrobial products do not have detergent action or cleaning properties. Some other cleaning preparations that are allowed on food either do not have antimicrobial and microbicidal properties or are not safe enough as the ingredients are not considered by the FDA as GRAS or food additive safe. Some preparations have disinfecting properties without the solubilizing properties to remove harmful pesticide residues. Still some other cleaning products need to incorporate antibacterial compounds in these preparations to inhibit or kill microorganisms [6–9]. Biocatalysts are extensively used in the industrial production for the production of novel compounds and natural products [10]. Enzymatic synthesis can play a major role, since it avoids the use of acid catalysts and high temperatures and, mainly, because, as enzymes are nature’s catalyst, they may be a path to ‘natural’ products. In fact, US and EU legislations consider as ‘natural’ the products
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resulting from enzymatic synthesis [11]. Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3), due to their versatility and selectivity, are among the most versatile of the enzyme classes and are used in a number of applications in various industries, including the pharmaceutical, food, detergent, cosmetic, oleochemical, fat-processing, leather, textile and paper industries [11–14]. Applications of lipases include chemo-, regio- and stereoselective processes, as well as the desymmetrization of prochiral or meso compounds and as aldol reactions or Michael additions [14,15]. The use of lipases as an alternative to chemical catalysts, to catalyze esterification reactions aimed at the production of flavoring esters for food purposes, has been developed. Their ability to catalyze synthesis in non-aqueous media has made them extensively used to produce useful esters [16]. A key technology for successful realization of enzyme-based industrial processes is immobilization. Basically, three methods of enzyme immobilization can be distinguished: (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross-linking (carrier free) [17,18]. For binding to a support example, the widely used is commercially available lipase B from Candida antarctica in immobilized form as Novozym 435 which consists of the enzyme adsorbed on a macroporous acrylic resin [18]. In industrial processes it is used as a biocatalyst in the synthesis of simple esters and amides, but primary it is used as a highly enantioselective catalyst in the synthesis of optically active alcohols, amines and carboxylic acids [14,19]. Enzymes can be immobilized by entrapment in sol–gel matrices formed by hydrolytic polymerization of metal alkoxides. The third immobilization method is cross-linking of enzyme aggregates (CLEAs) or crystals (CLECs), using a bifunctional reagent, ˇ et al. [20] preto prepare carrierless macroparticles [18]. Sulek sented the successful preparation of catalytically active CLEAs of peroxidase (HRP) from horseradish roots using aggregation agents, followed by cross-linking with glutaraldehyde. A percent activity recovery of 83% was obtained. The immobilized enzyme can be reused, provided it is stable enough [21–23]. Therefore, using an immobilized enzyme allows the enzyme to be used repeatedly, resulting in a simple process and lower production costs [24]. The immobilization of enzymes has a very powerful tool to improve almost all enzyme properties; stability (including the possibility of enzyme reactivation) is the most common, but also activity, specificity, selectivity, enzyme purity and inhibitions [21,23,25,26]. Since many organic solvents are toxic and are being progressively restricted for use in food processes, the use of non-conventional solvents has gained attention. Supercritical fluids (SCF) have been one of the choices to replace the conventional organic solvents. Besides the reduction of organic waste, SCF have the additional benefit that separation after reaction is relatively simple, which offers several process advantages. Another useful characteristic of SCF is their adjustable solvation ability and properties that facilitate mass transfer. There is an interest in the use of SCF making it suitable for biocatalysis. Moreover, solubilities of solutes can be tuned by changing temperature and pressure and it can be used in food and pharmaceutical processes without major regulatory issues [11,27–30]. So, the combination of a sustainable and clean technology, as biocatalysis, with a green/natural solvent, is very attractive for the development of ‘green’ processes and seems to be a good choice for the synthesis of ‘natural’ substances, as shown by the several works that have been published on enzymatic reactions in SCF [11]. Although many lipase-catalyzed esterification reactions have been reported in supercritical carbon dioxide (SC CO2 ) [11,19,28–33], we are unaware of any study on the synthesis of n-butyl lactate in supercritical trifluoromethane (SC CHF3 ) and supercritical ethane (SC C2 H6 ). SC CHF3 is chemically inert, nonflammable, has low toxicity and a low critical temperature (26.14 ◦ C) and pressure (4.83 MPa). SC CHF3 can solubilize pharmaceutical compounds, because CHF3 has a strong permanent dipole moment (1.56 D)
Table 1 Physico-chemical properties of CO2 , CHF3 and C2 H6 [12,18]. Gas
Mw (g/mol)
Tc (◦ C)
pc (MPa)
CO2 CHF3 C2 H6
44.01 70.01 30.07
30.98 26.14 32.18
7.38 4.83 4.87
[34,35]. n-Butyl lactate can generally be produced by esterification. Reaction can be catalyzed enzymatically, by lipases. The esterification route consists in: d, l-LA + n-butanol ↔ n-butyllactate + water The reaction is enantioselective esterification of racemic LA, but in our case, the individual enantiomers (butyl l- and d-lactate) were not monitored. Ohara et al. [24] reported the optical resolution of butyl l-lactate and butyl d-lactate using an immobilized lipase. This report showed that a mixture containing 90.4% of butyl l-lactate and 9.6% of butyl d-lactate. In present research, lipase-catalyzed synthesis of n-butyl lactate was performed in a high-pressure batch stirred-tank reactor, which has been described in previous work [31]. Synthesis was catalyzed by C. antarctica lipase B (Novozym 435). In this research esterification of n-butanol and d,l-LA in SC CHF3 was studied, since the use of d,l-LA is more important for its applicative reasons. Optimization of the reaction parameters, such as pressure and temperature, was the aim of the preliminary study of the esterification of d,l-LA. Furthermore, the impact of different SCFs on the phase behavior of the whole reaction system was examined. Antimicrobial activity of enzymatically synthesized n-butyl lactate against test microorganisms was determined. In addition, antifungal and antibacterial activities of enzymatically synthesized n-butyl lactate were compared to antifungal and antibacterial activities of commercial d,l-LA and n-butyl lactate.
2. Materials and methods 2.1. Enzymes and chemicals Novozym 435 [C. antarctica lipase B immobilized on a macroporous acrylic resin], was kindly donated from Novo Nordisk AS (Copenhagen, Denmark) and was used directly without any pre-treatment. d,l-LA (90%, w/w), (n)-butyl l-lactate (≥97% (GC)), sodium chloride (99.8%), d-(+)-glucose (99% (GC)) and agar were purchased from Sigma–Aldrich (Deisenhofen, Germany). Phosphoric acid (min 85%) was purchased from Kemika (Zagreb, Croatia). n-Butanol (99.5%), acetonitrile (≥99.8%), peptone from meat, meat extract and potato dextrose agar were purchased from Merck (Darmstadt, Germany). n-Hexane (95%) was purchased from Carlo Erba (Rodano, Italy). Trifluoromethane 2.8 (99.8%) and ethane (>99%) were obtained from Linde plin (Celje, Slovenia) and helium 6.0 for gas chromatography (GC) was supplied from Messer MG (Ruˇse, Slovenia).
2.2. Phase behavior for d,l-LA/n-butanol/Novozym 43 system in different SCF Apparatus, consisting of a view cell, has been used to observe phase behavior for d,l-LA/n-butanol/Novozym 435 system in different SCFs. A sketch of a high-pressure view cell has been already described elsewhere [36]. Physico-chemical properties of SCFs which were used in this work are presented in the Table 1.
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2.3. Enzymatic reaction in SCF Lipase-catalyzed esterification of d,l-LA with n-butanol in different SCFs was performed. Reaction was performed in a high-pressure batch stirred-tank reactor (V = 60 mL) with constant stirring rate of 700 rpm at desired temperature and pressure. The reaction mixture contained 1 mmol of LA and 3.6 mmol of n-butanol and procedure was described in previous work [31]. After the synthesis performed in SCF, immobilized lipase was separated from the reaction mixture by filtration. 2.4. Analyses 2.4.1. Composition of the lactic acid solution The commercially available d,l-LA (90%, w/w) solution contains beside the monomeric form a significant amount of lactoyllactic acid [37]. The composition was characterized using a high performance liquid chromatography (HPLC) from Agilent Technologies 1200 Series. As a stationary phase the Lichrospherer 100, Chromsep, RP8 column, with dimension 250 mm × 4.6 mm and 5 m particle size was used. The mobile phase consisted of two solvents; water and acetonitrile which were both acidifed using 2 mL of 85% (w/v) phosphoric acid in 1 L of solvent. The column oven temperature was maintained at 40 ◦ C. The flow rate was 1.0 ml/min and the detection was performed at 210 nm. The quantification was made with an external standard. Water content was measured using a Mettler Toledo DL31 KarlFisher titrator. 2.4.2. Assay of n-butyl lactate The reaction was monitored using a HP 6890 gas chromatograph equipped with a HP-Ultra 2 column and a flame ionization detector. The GC column with internal diameter 0.2 mm, 25 m long and film thickness 0.33 m was used. Helium was used as the carrier gas. The temperature of injector was 260 ◦ C and 300 ◦ C of FID detector. For the analysis 50 l sample of reaction mixture was taken. 10 L of water and 1 mL of n-hexane were added in the Eppendorf tubes. After centrifugation supernatant was diluted with n-hexane. Conversion of LA (X) was calculated on the basic of the monomer acid content. From the following equation: X (%) =
nbutyl lactate nLAt=0
× 100
(1)
where X (%) is conversion of LA, nbutyl lactate are mmol of n-butyl lactate and nLAt = 0 are mmol of LA monomer at the beginning of the reaction. 2.4.3. Antimicrobial tests 2.4.3.1. Microbial strains. Microorganisms were obtained as lyophilized cultures from the National Collection of Agricultural and Industrial Microorganisms (Hungary). Organisms were as follows: four species of molds, Saccharomyces cerevisiae, Aspergillus niger, Trichoderma viride and Penicillium cyclopium, and two species of Gram negative bacteria, Escherichia coli and Pseudomonas fluorescens and one species of Gram positive bacteria, Bacillus cereus. 2.4.3.2. Preparation of test microorganisms. The test fungi were maintained on PDA slopes and stored at 4 ◦ C. Conidia were harvested in sterile physiological salt solution containing approximately 105 –107 conidia/mL. These conidial suspensions were used immediately after preparation for determining the antifungal activities of the samples. In the antibacterial experiments, test bacteria were grown on meat nutrient agar slopes at 28 ◦ C, except for E. coli, that was
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grown at 37 ◦ C and then stored at 4 ◦ C. Before the bacterial experiments were carried out, liquid medium was inoculated with freshly harvested bacteria. These bacterial suspensions (approximately 107 –108 cell/mL) were used to inoculate the test medium containing the samples. 2.4.3.3. Antifungal activity test and antibacterial activity test. Two different methods were employed for the determination of antimicrobial activities; agar well-diffusion method for the antifungal activity and disk diffusion method for the antibacterial activity [38]. Enzymatically synthesized n-butyl lactate, commercial d,l-LA and n-butyl lactate were used directly without any dilution. For antifungal activity test each PDA sterile plate contained 19 mL of molten medium and 1 mL of a sample (A: physiological salt solution, B: d,lLA, C: standard of n-butyl lactate or D: synthesized n-butyl lactate). For all test fungi, PDA plates were made and were used as control plates. The solid plates were inoculated with 0.1 mL of conidial suspension, measuring it into the holes (diameter 12 mm) in the center of the medium. The plate was left undisturbed to allow diffusion of the sample into agar, and then the plate was incubated in the dark at 28 ◦ C and the diameter of the mycelial growth was measured. The antibacterial activity of all samples was carried out by disk diffusion method. The Petri dishes containing 19 mL of meat nutrient agar were cultured with diluted bacterial strain. Then the sterile disk papers (diameter 6 mm) were placed on the culture medium. 10 L of each sample was injected to the prepared disk. Negative controls were prepared using the physiological salt solution (9 g/L NaCl). Then the inoculated plates were incubated at 28 ◦ C, except for E. coli, that was grown at 37 ◦ C, for 48 h for the tested microorganisms. The diameter of the zone around the disc was measured. Four disk per plate and each test was run in triplicate. The antifungal activity and antibacterial activity were determined in terms of percentage mycelial inhibition calculated by the following formula: I = (C − T ) × 100
(2)
where I is inhibition (%), C is the colony diameter of the mycelium on the physiological salt solution control plate (mm), and T is the colony diameter of the mycelium on the test Petri plate (mm). Each test was run in triplicate and averages were calculated. 3. Results and discussions Esterification of d,l-LA with n-butanol over Novozym 435 was performed in different SCFs. Experiments were planned to elucidate the effect of pressure and temperature on the conversion of LA. Process conditions were optimized performing experiments in a high-pressure batch stirred-tank reactor, which has been described elsewhere [31]. Visual observations in a high-pressure view cell were performed in order to determine phase behavior of the reaction mixture. 3.1. Phase behavior for d,l-LA/n-butanol/Novozym 435 system in different SCF The results of phase-behavior visual observation of d,lLA/n-butanol/Novozym 435/SC CO2 system at different reaction conditions are depicted in previous work [31]. The next step of esterification of d,l-LA with n-butanol catalyzed by immobilized lipase B from C. antarctica was to choose another SCF where higher conversion of LA at low process pressure and temperature could be achieved. The phase behavior for reaction mixture SC CHF3 and SC C2 H6 was observed visually in a high-pressure view cell. Before studying the temperature and pressure effect on the lipase-catalyzed esterification of n-butyl lactate in SC CHF3 , the
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Fig. 1. Phase behavior of d,l-LA/n-butanol/Novozym 435/SC CHF3 system at different reaction conditions. Temperature: 55 ◦ C; pressures: 7.5, 10, 20 and 30 MPa.
phase behavior for d,l-LA/n-butanol/Novozym 435/SC CHF3 system at temperature 55 ◦ C and at pressure between 7.5 MPa and 30 MPa was observed. The results at 55 ◦ C and different pressures are depicted in Fig. 1. The reaction mixture (substrates and CHF3 ) can be seen as a two-phase system (supercritical phase and solid enzyme) at 20 MPa and 30 MPa, while at 7.5 MPa and 10 MPa, the reaction mixture contained solid, liquid and gas phase with a distinct meniscus between the liquid and gas phase. In the pressure region from 16 MPa to 17 MPa the reaction mixture turned to supercritical. Solubility of the substrates in SC CHF3 depends on CHF3 density, which can be changed by small changes in temperature and/or pressure particularly near the critical point [34,39]. Usually solubility of substrates increases with increase in pressure because of higher fluid density. The results indicate that phase behavior of the reaction mixture in SC CHF3 was similar to the one in SC CO2 as described in our previous work [31]. Furthermore, the phase behavior for d,l-LA/nbutanol/Novozym 435/SC CHF3 system at temperature 35 ◦ C and at pressure between 7.5 MPa and 30 MPa were similar to those at 55 ◦ C. In SC C2 H6 for the pressure region from 2 MPa to 30 MPa the reaction mixture contained solid, liquid and gas phase. On the basis of the results obtained by investigating the phase behavior of d,l-LA/n-butanol/Novozym 435/SC CHF3 system at different pressures, the temperature and pressure effect on lipasecatalyzed esterification was studied in the pressure range between 7.5 MPa and 30 MPa at 35 ◦ C and 55 ◦ C. 3.2. Esterification of LA In recent years, SCFs are used as media for biosynthesis due to their advantages compared to conventional organic solvents. Therefore, esterification of n-butanol with d,l-LA, catalyzed by immobilized lipase B from C. antarctica, was performed in different SCFs. The effect of the two most important reaction parameters, which affect the reaction performance, temperature and pressure, on the conversion of LA was studied. The most significant findings are summarized below. Lipase-catalyzed esterification of n-butanol with d,l-LA was previously performed in SC C2 H6 and SC CO2 . The comparison of the results was done with esterification, performed in SC CHF3 , at 35 ◦ C and 20 MPa. Fig. 2 shows the comparison between conversion for esterification of n-butanol with d,l-LA obtained when the three above mentioned SCFs were used. Knez et al. [31] describes our previous research work, which was upgraded with new reaction medium (SC CHF3 ). Lipase-catalyzed esterification of n-butanol with d,l-LA performed in SC CHF3 gave much higher conversion after 26 h at temperatures 35 ◦ C and 55 ◦ C and pressures between 7.5 MPa and 30 MPa in the comparison to the same reaction where SC CO2 was used as the reaction medium. When SC CHF3 was used as the reaction medium the highest conversion of LA (63.28%) was obtained, while the lowest conversion of LA was performed in SC C2 H6 (38.86%).
Fig. 2. The conversion of LA as a function of time of reaction performance at 35 ◦ C and 20 MPa in SC C2 H6 , SC CO2 and SC CHF3 . Reaction conditions: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm.
Furthermore, the study of the pressure influence on the total conversion of LA, yield (gBL /(gE ·h)) and initial rate (((gBL /gsample )/gE )/h) in SC CHF3 was performed at two different temperatures, modulating the pressure in the range from 7.5 MPa to 30 MPa at 55 ◦ C and 35 ◦ C. Influence of temperature and pressure on the conversion of LA is shown in Fig. 3, which clearly shows that the conversion is pressure and temperature dependant. The effect of the pressure and temperature on the yield after 26 h of reaction performance and initial rate in d,l-LA/n-butanol/Novozym 435/SC CHF3 system is shown in Fig. 4. At 55 ◦ C, by increasing the pressure from 10 MPa to 20 MPa significant increase in the
Fig. 3. Effect of pressure on the conversion of LA after 26 h of the reaction performance using SC CHF3 and SC CO2 as reaction medium. Reaction conditions: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm.
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Fig. 4. Effect of pressure and temperature on the yield and initial rate after 26 h of the reaction performance using SC CHF3 as reaction medium. Reaction conditions: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm.
conversion of LA (from 65.3% to 87.5%) and in yield (from 7.7 gBL /(gE ·h) to 10.3 gBL /(gE ·h)) were obtained. The reaction mixture in this pressure region turned from three phase system to two phase system. Figs. 3 and 4 also show that the conversion and yield slightly increased when increasing the pressure from 7.5 MPa to 10 MPa and from 20 MPa to 30 MPa, which was due to the fact that an increase in pressure leads to enhanced solvent density, improving its solvating power in the reaction bulk [40]. Compared to the reaction, performed in SC CO2 [31], much higher conversion after 26 h of reaction was achieved when SC CHF3 was used at 55 ◦ C and pressure between 7.5 MPa and 30 MPa, because of different properties in density, polarity and dipole moment of SC CHF3 and SC CO2 . A maximum yield of 10.4 gBL /(gE ·h) after 26 h of reaction performance was attained at 55 ◦ C and 30 MPa (Fig. 4). At 7.5 MPa and 10 MPa at 35 ◦ C for both SCFs and at 55 ◦ C in SC CO2 a slight decrease in the conversion of LA was observed. Furthermore, the increase in temperature from 35 ◦ C to 55 ◦ C enhances the enzyme activity for reaction performance in SC CHF3 and SC CO2 . The increase in initial rate with higer temperature at certain pressure was detected. Before studying the lipase-catalyzed esterification of n-butyl lactate, the exact composition of the d,l-LA solution (53.3% (w/w) monomer (LA1), 28.7% (w/w) lactoyllactic acid (LA2), and 11.7% (w/w) water) was determined by HPLC and Karl Fisher titrator. Remaining 6.3% (w/w) represents lactide (LA3), the cyclic diester of LA. For the synthesis of n-butyl lactate monomeric form (LA1) was used. The composition of the d,l-LA in the reaction system was monitored for each reaction, because the composition of the d,lLA during reaction performance was changing. The content of LA1 and LA2 decreased during esterification, because they were used for the formation of the product n-butyl lactate. According to the results of HPLC analysis (Fig. 5) for enzymatic esterification of d,lLA in SC CHF3 at 55 ◦ C and 30 MPa the LA1 and LA2 content of the reaction mixture decreased, due to the formation of the product n-butyl lactate. The amount of LA3 remained practically constant during the reaction. Water content in reaction mixture at the beginning of the reaction was 3.54% (w/w). As can be seen in Fig. 5, water content increased during reaction, because water is a by-product of synthesis of n-butyl lactate. Pressure influence on the concentration of d,l-LA and n-butyl lactate (BL) was studied at two different temperatures (55 ◦ C and 35 ◦ C) and in the pressure range from 7.5 MPa to 30 MPa. Fig. 6 represents the concentration of d,l-LA and BL in the reaction mixture after 26 h in SC CHF3 at 55 ◦ C and pressure ranges from 7.5 MPa to
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Fig. 5. Composition of d,l-LA solution, water content and content of BL in the reaction mixture versus reaction time. Reaction conditions in SC CHF3 for BL: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm, 55 ◦ C, 30 MPa.
Fig. 6. Concentration of d,l-LA and BL in the reaction mixture after 26 h. Reaction conditions in SC CHF3 for BL: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm, 55 ◦ C, 30 MPa.
30 MPa. While the concentration of BL increased from 13.95% (w/w) to 19.00% (w/w), the concentration of LA1 decreased for about 10% (w/w). At 7.5 MPa the concentration of LA1 was 19.55% (w/w) and with higher pressure decreased due to the enhanced product formation. At 20 MPa and 30 MPa the concentration of LA1 was found to be lower compared to the concentration of LA2. The reason for increased concentration of BL is that the LA2 (open chain dimmer of d,l-LA) is able to decompose to monomers and form n-butyl lactate. Concentration of LA2 was the same for 7.5 MPa and 10 MPa. By increasing the pressure from 10 MPa to 30 MPa a decrease in concentration of LA2 from 16.94% (w/w) to 11.90% (w/w) was oserved, which could be due to the fact that an increase in pressure leads to decomposition of LA2 to monomers. The concentration of LA3 remained approx. the same for all tested pressures (5.6%, w/w), and we assume that higher temperatures [41] are necessary for the decomposition of the LA3 cyclic di-ester to lactic acid monomers. 3.3. Antifungal and antibacterial activities In spite of the modern improvements in food hygiene and food production techniques, food safety is an increasing important public healt issue. Therefore, the reducing or inhibiting foodborne pathogens, possibly in combination with the existing methods is still needed [42]. Antifungal and antibacterial activities of d,l-LA and n-butyl lactate (standard and synthesized) were determined. Till now, the studies of antifungal and antibacterial activities on microorganisms (A. niger, T. viride, P. cyclopium, P. flourescens and B. cereus) were not reported. Therefore, presented results are unique.
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Table 2 Antimicrobial activities of selected fungi strains. Fungal strains
72 h Blankb
S. cerevisiae A. niger T. viride P. cyclopium
14 46 70 18
Percentage mycelial zone inhibitiona LA
n-Butyl lactate
Sample of BL
100 100 100 100
100 100 100 100
100 100 100 100
a Diameter of zones including diameter of disk 12 mm (average of three replicates). b Colony diameter of the mycelium on the physiological salt solution plate (mm).
Lopes et al. [6] reported the microbicidal activity and the minimum lethal activity of butyl lactate on Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhimurium). E. coli and S. cerevisiae were investigated by both groups with a different protocol, where the method prepared by our group for antifungal activity was agar well-diffusion method and the method for antibacterial activity was disk diffusion method. S. cerevisiae is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like E. coli as the model prokaryote [43,44]. The agricultural pathogenic fungus A. niger causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food [45]. P. cyclopium is the main contaminant fungi detected in seeds harvested. Such contaminants are fearsome, since they affect the seeds before harvest time and may find optimal developing conditions when the seeds are stored, leading to alteration of the germination quality of these seeds [46]. E. coli is a Gram-negative bacterium, which is one of the most extensively studied micro-organisms because of its wide use as a model organism in research and microbial genetics and physiology as well as its use in industrial applications [44]. Although E. coli are normal inhabitants of the colons of all warm-blooded mammals, some pathogenic strains can cause urinary tract infections, diarrhea and also new born meningitis [44,47]. Another used a Gram-negative bacterium is P. fluorescens. This rod-shaped bacterium presents biocontrol properties and protecting the roots of some plant species against parasitic fungi [48,49]. B. cereus is a Gam-positive, rodshaped bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals [50]. Antifungal activities of d,l-LA and n-butyl lactate (standard and synthesized) against S. cerevisiae, A. niger, T. viride and P. cyclopium are presented in Table 2, where the results of the microbial growth, applying three parallel measurements, are given. The results of antifungal activity assay showed that all samples exhibited 100% inhibitory effects on the growth of three tested mycelial fungi. The results of antifungal activity suggest the use of d,l-LA and n-butyl lactate (standard and enzymatically synthesized) as an antimicrobial aditive in food processing to provide improve food safety.
Fig. 7. Growth inhibition of T. viride after 72 h growth: (A) physiological salt solution, (B) d,l-LA, (C) standard of n-butyl lactate and (D) synthesized n-butyl lactate.
Antifungal activities of d,l-LA and n-butyl lactate (standard and synthesized) against T. viride after 72 h growth are presented in Fig. 7. They showed 100% inhibitory effects on the growth of fungus T. viride in comparison with control of physiological salt solution. Antibacterial activity of substances was determined on the paper discs and expressed in terms of an inhibition zone. The diameter of the clear zone of inhibition around a paper disk agar is shown in Table 3, which summarizes antibacterial activities of d,l-LA and n-butyl lactate (standard and synthesized) in comparison with a physiological salt solution control after a 24 h and 48 h growth. Studies have suggested that d,l-LA [51] and n-butyl lactate (standard and synthesized) [6] have antibacterial activity against the tested bacteria. Comparisons of d,l-LA and n-butyl lactate showed that the clear zone around the paper disk on the plate agar was wider for the d,l-LA than for n-butyl lactate, demonstrating that the d,l-LA has higher antimicrobial activity than n-butyl lactate. Diameter of inhibition zones of enzymatically synthesized n-butyl lactate was determined to be in all cases slightly higher than those of commercial n-butyl lactate. The reason could be the fact that the samples contain some unreacted d,l-LA. Fig. 8 shows inhibition zones including discs of 6 mm in diameter for d,l-LA, standard of n-butyl lactate and synthesized n-butyl lactate against B. cereus after 24 h growth. The comparison of antimicrobial activity between standard (which is 97% pure and used any dilutions) and synthesized n-butyl lactate (used immediately after synthesis without any treatement and any dilutions) was done owing to confirmation of the similar antimicrobial properties of both n-butyl lactates. The aim was to examine how the n-butyl lactate itself affects the inhibition of the growth. d,l-LA shows the highest antifungal and antibacterial activities but the point of the research was to examine the
Table 3 Antimicrobial activities of selected bacterial strains. Bacterial strains
Inhibition zones diametera 24 h
E. coli P. flourescens B. cereus
48 h
Blankb
LA
n-Butyl lactate
Sample of BL
Blankb
LA
n-Butyl lactate
Sample of BL
– – –
43.0 ± 0.6 40.3 ± 2.5 45.3 ± 0.6
20.0 ± 1.0 19.0 ± 3.6 18.3 ± 1.2
23.3 ± 2.3 23.0 ± 3.6 23.0 ± 1.0
– – –
43.7 ± 0.6 40.0 ± 2.6 41.3 ± 3.2
22.0 ± 1.0 17.5 ± 2.2 10.7 ± 0.6
23.3 ± 2.3 21.3 ± 3.5 18.8 ± 1.4
Each value represents the mean S.D: (n = 3). –, no inhibition. a Diameter of inhibition zones including diameter of disk 6 mm. b Physiological salt solution (9 g/L NaCl).
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149
Fig. 8. Growth inhibition of B. cereus after 24 h growth: (1/1 and 1/2) physiological salt solution, (2/1 and 2/2) d,l-LA, (3/1 and 3/2) standard of n-butyl lactate and (4/1 and 4/2) synthesized n-butyl lactate.
antifungal and antibacterial activities of n-butyl lactate, since it could be used as a food additive, therefore its antimicrobial activity is of great importance.
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The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Antimicrobial activity of n-butyl lactate obtained via enzymatic esterification of lactic acid with n-butanol in supercritical trifluoromethane Sabina Kavˇciˇc, Zˇ eljko Knez, Maja Leitgeb ∗ University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Separation Processes and Product Design, Smetanova 17, 2000 Maribor, Slovenia
a r t i c l e
i n f o
Article history: Received 21 August 2013 Received in revised form 7 November 2013 Accepted 8 November 2013 Keywords: n-Butyl lactate Candida antarctica lipase B Supercritical trifluoromethane Antimicrobial tests
a b s t r a c t The lipase-catalyzed synthesis of n-butyl lactate by esterification was performed in supercritical trifluoromethane. Immobilized lipase B from Candida antarctica (Novozym 435) was used as a biocatalyst. Process conditions (pressure and temperature) were optimized performing experiments in a highpressure batch stirred-tank reactor. Experiments were carried out in the operative pressure ranges from 7.5 to 30 MPa and at temperatures 35 ◦ C and 55 ◦ C. For this purpose phase behavior for d,l-lactic acid/n-butanol/Novozym 435/supercritical fluid system at temperature 55 ◦ C and different pressures was studied. The highest conversion of lactic acid after 26 h of reaction performance was obtained in supercritical trifluoromethane at 30 MPa and 55 ◦ C. The n-butyl lactate (standard and enzymatically synthesized) and d,l-lactic acid were tested against four food-borne fungi: Saccharomyces cerevisiae, Aspergillus niger, Trichoderma viride and Penicillium cyclopium and three health-damaging bacteria: Escherichia coli, Pseudomonas fluorescens and Bacillus cereus by the agar well diffusion. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lactic acid (LA) and lactate esters are used in wide applications in food industry for preservation, emulsifiers and flavoring purposes, as well as in pharmaceutical and cosmetic industries [1–4]. LA is also used as an important raw material for the manufacture of biodegradable polymers. Its esters including n-butyl lactate are used as flavors and solvents with excellent properties [3]. The Food and Drug Administration (FDA) included LA on its list of substances considered Generally Recognized As Safe (GRAS) as direct food additives. Ethyl lactate and butyl lactate are also approved for use as direct food additives [5]. Furthermore, broad spectrum disinfecting and microbicidal compositions of biodegradable and environmentally friendly compositions containing esters include butyl lactate. These compositions display activities against
Abbreviations: CLEAs, cross-linking of enzyme aggregates; CLECs, cross-linking of enzyme crystals; FDA, Food and Drug Administration; GC, gas chromatography; GRAS, Generally Recognized As Safe; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; LA, lactic acid; LA1, monomer of lactic acid; LA2, lactoyllactic acid; LA3, lactide; PDA, potato dextrose agar; SCF, supercritical fluid; SC CHF3 , supercritical trifluoromethane; SC C2 H6 , supercritical ethane; SC CO2 , supercritical carbon dioxide. ∗ Corresponding author. Tel.: +386 2 2294 462; fax: +386 2 2527 774. E-mail address: [email protected] (M. Leitgeb). 0896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2013.11.003
the most resistant microbial forms which use safe and non-toxic chemical agents selected from alcohol esters of butyl lactate and its homologs for preparing products with cleaning, solubilizing, antimicrobial and microbicidal properties. Most common antimicrobial products such as chlorine, chlorine dioxide peracetic acid, ozone, hydrogen peroxide, UV- and other radiations, used to reduce microbial population on food and other contact surfaces possess highly oxidizing and sometimes destructive properties. These oxidizing chemicals and physical agents inactivate microorganisms by reacting with their organic material. These antimicrobial products do not have detergent action or cleaning properties. Some other cleaning preparations that are allowed on food either do not have antimicrobial and microbicidal properties or are not safe enough as the ingredients are not considered by the FDA as GRAS or food additive safe. Some preparations have disinfecting properties without the solubilizing properties to remove harmful pesticide residues. Still some other cleaning products need to incorporate antibacterial compounds in these preparations to inhibit or kill microorganisms [6–9]. Biocatalysts are extensively used in the industrial production for the production of novel compounds and natural products [10]. Enzymatic synthesis can play a major role, since it avoids the use of acid catalysts and high temperatures and, mainly, because, as enzymes are nature’s catalyst, they may be a path to ‘natural’ products. In fact, US and EU legislations consider as ‘natural’ the products
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resulting from enzymatic synthesis [11]. Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3), due to their versatility and selectivity, are among the most versatile of the enzyme classes and are used in a number of applications in various industries, including the pharmaceutical, food, detergent, cosmetic, oleochemical, fat-processing, leather, textile and paper industries [11–14]. Applications of lipases include chemo-, regio- and stereoselective processes, as well as the desymmetrization of prochiral or meso compounds and as aldol reactions or Michael additions [14,15]. The use of lipases as an alternative to chemical catalysts, to catalyze esterification reactions aimed at the production of flavoring esters for food purposes, has been developed. Their ability to catalyze synthesis in non-aqueous media has made them extensively used to produce useful esters [16]. A key technology for successful realization of enzyme-based industrial processes is immobilization. Basically, three methods of enzyme immobilization can be distinguished: (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross-linking (carrier free) [17,18]. For binding to a support example, the widely used is commercially available lipase B from Candida antarctica in immobilized form as Novozym 435 which consists of the enzyme adsorbed on a macroporous acrylic resin [18]. In industrial processes it is used as a biocatalyst in the synthesis of simple esters and amides, but primary it is used as a highly enantioselective catalyst in the synthesis of optically active alcohols, amines and carboxylic acids [14,19]. Enzymes can be immobilized by entrapment in sol–gel matrices formed by hydrolytic polymerization of metal alkoxides. The third immobilization method is cross-linking of enzyme aggregates (CLEAs) or crystals (CLECs), using a bifunctional reagent, ˇ et al. [20] preto prepare carrierless macroparticles [18]. Sulek sented the successful preparation of catalytically active CLEAs of peroxidase (HRP) from horseradish roots using aggregation agents, followed by cross-linking with glutaraldehyde. A percent activity recovery of 83% was obtained. The immobilized enzyme can be reused, provided it is stable enough [21–23]. Therefore, using an immobilized enzyme allows the enzyme to be used repeatedly, resulting in a simple process and lower production costs [24]. The immobilization of enzymes has a very powerful tool to improve almost all enzyme properties; stability (including the possibility of enzyme reactivation) is the most common, but also activity, specificity, selectivity, enzyme purity and inhibitions [21,23,25,26]. Since many organic solvents are toxic and are being progressively restricted for use in food processes, the use of non-conventional solvents has gained attention. Supercritical fluids (SCF) have been one of the choices to replace the conventional organic solvents. Besides the reduction of organic waste, SCF have the additional benefit that separation after reaction is relatively simple, which offers several process advantages. Another useful characteristic of SCF is their adjustable solvation ability and properties that facilitate mass transfer. There is an interest in the use of SCF making it suitable for biocatalysis. Moreover, solubilities of solutes can be tuned by changing temperature and pressure and it can be used in food and pharmaceutical processes without major regulatory issues [11,27–30]. So, the combination of a sustainable and clean technology, as biocatalysis, with a green/natural solvent, is very attractive for the development of ‘green’ processes and seems to be a good choice for the synthesis of ‘natural’ substances, as shown by the several works that have been published on enzymatic reactions in SCF [11]. Although many lipase-catalyzed esterification reactions have been reported in supercritical carbon dioxide (SC CO2 ) [11,19,28–33], we are unaware of any study on the synthesis of n-butyl lactate in supercritical trifluoromethane (SC CHF3 ) and supercritical ethane (SC C2 H6 ). SC CHF3 is chemically inert, nonflammable, has low toxicity and a low critical temperature (26.14 ◦ C) and pressure (4.83 MPa). SC CHF3 can solubilize pharmaceutical compounds, because CHF3 has a strong permanent dipole moment (1.56 D)
Table 1 Physico-chemical properties of CO2 , CHF3 and C2 H6 [12,18]. Gas
Mw (g/mol)
Tc (◦ C)
pc (MPa)
CO2 CHF3 C2 H6
44.01 70.01 30.07
30.98 26.14 32.18
7.38 4.83 4.87
[34,35]. n-Butyl lactate can generally be produced by esterification. Reaction can be catalyzed enzymatically, by lipases. The esterification route consists in: d, l-LA + n-butanol ↔ n-butyllactate + water The reaction is enantioselective esterification of racemic LA, but in our case, the individual enantiomers (butyl l- and d-lactate) were not monitored. Ohara et al. [24] reported the optical resolution of butyl l-lactate and butyl d-lactate using an immobilized lipase. This report showed that a mixture containing 90.4% of butyl l-lactate and 9.6% of butyl d-lactate. In present research, lipase-catalyzed synthesis of n-butyl lactate was performed in a high-pressure batch stirred-tank reactor, which has been described in previous work [31]. Synthesis was catalyzed by C. antarctica lipase B (Novozym 435). In this research esterification of n-butanol and d,l-LA in SC CHF3 was studied, since the use of d,l-LA is more important for its applicative reasons. Optimization of the reaction parameters, such as pressure and temperature, was the aim of the preliminary study of the esterification of d,l-LA. Furthermore, the impact of different SCFs on the phase behavior of the whole reaction system was examined. Antimicrobial activity of enzymatically synthesized n-butyl lactate against test microorganisms was determined. In addition, antifungal and antibacterial activities of enzymatically synthesized n-butyl lactate were compared to antifungal and antibacterial activities of commercial d,l-LA and n-butyl lactate.
2. Materials and methods 2.1. Enzymes and chemicals Novozym 435 [C. antarctica lipase B immobilized on a macroporous acrylic resin], was kindly donated from Novo Nordisk AS (Copenhagen, Denmark) and was used directly without any pre-treatment. d,l-LA (90%, w/w), (n)-butyl l-lactate (≥97% (GC)), sodium chloride (99.8%), d-(+)-glucose (99% (GC)) and agar were purchased from Sigma–Aldrich (Deisenhofen, Germany). Phosphoric acid (min 85%) was purchased from Kemika (Zagreb, Croatia). n-Butanol (99.5%), acetonitrile (≥99.8%), peptone from meat, meat extract and potato dextrose agar were purchased from Merck (Darmstadt, Germany). n-Hexane (95%) was purchased from Carlo Erba (Rodano, Italy). Trifluoromethane 2.8 (99.8%) and ethane (>99%) were obtained from Linde plin (Celje, Slovenia) and helium 6.0 for gas chromatography (GC) was supplied from Messer MG (Ruˇse, Slovenia).
2.2. Phase behavior for d,l-LA/n-butanol/Novozym 43 system in different SCF Apparatus, consisting of a view cell, has been used to observe phase behavior for d,l-LA/n-butanol/Novozym 435 system in different SCFs. A sketch of a high-pressure view cell has been already described elsewhere [36]. Physico-chemical properties of SCFs which were used in this work are presented in the Table 1.
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2.3. Enzymatic reaction in SCF Lipase-catalyzed esterification of d,l-LA with n-butanol in different SCFs was performed. Reaction was performed in a high-pressure batch stirred-tank reactor (V = 60 mL) with constant stirring rate of 700 rpm at desired temperature and pressure. The reaction mixture contained 1 mmol of LA and 3.6 mmol of n-butanol and procedure was described in previous work [31]. After the synthesis performed in SCF, immobilized lipase was separated from the reaction mixture by filtration. 2.4. Analyses 2.4.1. Composition of the lactic acid solution The commercially available d,l-LA (90%, w/w) solution contains beside the monomeric form a significant amount of lactoyllactic acid [37]. The composition was characterized using a high performance liquid chromatography (HPLC) from Agilent Technologies 1200 Series. As a stationary phase the Lichrospherer 100, Chromsep, RP8 column, with dimension 250 mm × 4.6 mm and 5 m particle size was used. The mobile phase consisted of two solvents; water and acetonitrile which were both acidifed using 2 mL of 85% (w/v) phosphoric acid in 1 L of solvent. The column oven temperature was maintained at 40 ◦ C. The flow rate was 1.0 ml/min and the detection was performed at 210 nm. The quantification was made with an external standard. Water content was measured using a Mettler Toledo DL31 KarlFisher titrator. 2.4.2. Assay of n-butyl lactate The reaction was monitored using a HP 6890 gas chromatograph equipped with a HP-Ultra 2 column and a flame ionization detector. The GC column with internal diameter 0.2 mm, 25 m long and film thickness 0.33 m was used. Helium was used as the carrier gas. The temperature of injector was 260 ◦ C and 300 ◦ C of FID detector. For the analysis 50 l sample of reaction mixture was taken. 10 L of water and 1 mL of n-hexane were added in the Eppendorf tubes. After centrifugation supernatant was diluted with n-hexane. Conversion of LA (X) was calculated on the basic of the monomer acid content. From the following equation: X (%) =
nbutyl lactate nLAt=0
× 100
(1)
where X (%) is conversion of LA, nbutyl lactate are mmol of n-butyl lactate and nLAt = 0 are mmol of LA monomer at the beginning of the reaction. 2.4.3. Antimicrobial tests 2.4.3.1. Microbial strains. Microorganisms were obtained as lyophilized cultures from the National Collection of Agricultural and Industrial Microorganisms (Hungary). Organisms were as follows: four species of molds, Saccharomyces cerevisiae, Aspergillus niger, Trichoderma viride and Penicillium cyclopium, and two species of Gram negative bacteria, Escherichia coli and Pseudomonas fluorescens and one species of Gram positive bacteria, Bacillus cereus. 2.4.3.2. Preparation of test microorganisms. The test fungi were maintained on PDA slopes and stored at 4 ◦ C. Conidia were harvested in sterile physiological salt solution containing approximately 105 –107 conidia/mL. These conidial suspensions were used immediately after preparation for determining the antifungal activities of the samples. In the antibacterial experiments, test bacteria were grown on meat nutrient agar slopes at 28 ◦ C, except for E. coli, that was
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grown at 37 ◦ C and then stored at 4 ◦ C. Before the bacterial experiments were carried out, liquid medium was inoculated with freshly harvested bacteria. These bacterial suspensions (approximately 107 –108 cell/mL) were used to inoculate the test medium containing the samples. 2.4.3.3. Antifungal activity test and antibacterial activity test. Two different methods were employed for the determination of antimicrobial activities; agar well-diffusion method for the antifungal activity and disk diffusion method for the antibacterial activity [38]. Enzymatically synthesized n-butyl lactate, commercial d,l-LA and n-butyl lactate were used directly without any dilution. For antifungal activity test each PDA sterile plate contained 19 mL of molten medium and 1 mL of a sample (A: physiological salt solution, B: d,lLA, C: standard of n-butyl lactate or D: synthesized n-butyl lactate). For all test fungi, PDA plates were made and were used as control plates. The solid plates were inoculated with 0.1 mL of conidial suspension, measuring it into the holes (diameter 12 mm) in the center of the medium. The plate was left undisturbed to allow diffusion of the sample into agar, and then the plate was incubated in the dark at 28 ◦ C and the diameter of the mycelial growth was measured. The antibacterial activity of all samples was carried out by disk diffusion method. The Petri dishes containing 19 mL of meat nutrient agar were cultured with diluted bacterial strain. Then the sterile disk papers (diameter 6 mm) were placed on the culture medium. 10 L of each sample was injected to the prepared disk. Negative controls were prepared using the physiological salt solution (9 g/L NaCl). Then the inoculated plates were incubated at 28 ◦ C, except for E. coli, that was grown at 37 ◦ C, for 48 h for the tested microorganisms. The diameter of the zone around the disc was measured. Four disk per plate and each test was run in triplicate. The antifungal activity and antibacterial activity were determined in terms of percentage mycelial inhibition calculated by the following formula: I = (C − T ) × 100
(2)
where I is inhibition (%), C is the colony diameter of the mycelium on the physiological salt solution control plate (mm), and T is the colony diameter of the mycelium on the test Petri plate (mm). Each test was run in triplicate and averages were calculated. 3. Results and discussions Esterification of d,l-LA with n-butanol over Novozym 435 was performed in different SCFs. Experiments were planned to elucidate the effect of pressure and temperature on the conversion of LA. Process conditions were optimized performing experiments in a high-pressure batch stirred-tank reactor, which has been described elsewhere [31]. Visual observations in a high-pressure view cell were performed in order to determine phase behavior of the reaction mixture. 3.1. Phase behavior for d,l-LA/n-butanol/Novozym 435 system in different SCF The results of phase-behavior visual observation of d,lLA/n-butanol/Novozym 435/SC CO2 system at different reaction conditions are depicted in previous work [31]. The next step of esterification of d,l-LA with n-butanol catalyzed by immobilized lipase B from C. antarctica was to choose another SCF where higher conversion of LA at low process pressure and temperature could be achieved. The phase behavior for reaction mixture SC CHF3 and SC C2 H6 was observed visually in a high-pressure view cell. Before studying the temperature and pressure effect on the lipase-catalyzed esterification of n-butyl lactate in SC CHF3 , the
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Fig. 1. Phase behavior of d,l-LA/n-butanol/Novozym 435/SC CHF3 system at different reaction conditions. Temperature: 55 ◦ C; pressures: 7.5, 10, 20 and 30 MPa.
phase behavior for d,l-LA/n-butanol/Novozym 435/SC CHF3 system at temperature 55 ◦ C and at pressure between 7.5 MPa and 30 MPa was observed. The results at 55 ◦ C and different pressures are depicted in Fig. 1. The reaction mixture (substrates and CHF3 ) can be seen as a two-phase system (supercritical phase and solid enzyme) at 20 MPa and 30 MPa, while at 7.5 MPa and 10 MPa, the reaction mixture contained solid, liquid and gas phase with a distinct meniscus between the liquid and gas phase. In the pressure region from 16 MPa to 17 MPa the reaction mixture turned to supercritical. Solubility of the substrates in SC CHF3 depends on CHF3 density, which can be changed by small changes in temperature and/or pressure particularly near the critical point [34,39]. Usually solubility of substrates increases with increase in pressure because of higher fluid density. The results indicate that phase behavior of the reaction mixture in SC CHF3 was similar to the one in SC CO2 as described in our previous work [31]. Furthermore, the phase behavior for d,l-LA/nbutanol/Novozym 435/SC CHF3 system at temperature 35 ◦ C and at pressure between 7.5 MPa and 30 MPa were similar to those at 55 ◦ C. In SC C2 H6 for the pressure region from 2 MPa to 30 MPa the reaction mixture contained solid, liquid and gas phase. On the basis of the results obtained by investigating the phase behavior of d,l-LA/n-butanol/Novozym 435/SC CHF3 system at different pressures, the temperature and pressure effect on lipasecatalyzed esterification was studied in the pressure range between 7.5 MPa and 30 MPa at 35 ◦ C and 55 ◦ C. 3.2. Esterification of LA In recent years, SCFs are used as media for biosynthesis due to their advantages compared to conventional organic solvents. Therefore, esterification of n-butanol with d,l-LA, catalyzed by immobilized lipase B from C. antarctica, was performed in different SCFs. The effect of the two most important reaction parameters, which affect the reaction performance, temperature and pressure, on the conversion of LA was studied. The most significant findings are summarized below. Lipase-catalyzed esterification of n-butanol with d,l-LA was previously performed in SC C2 H6 and SC CO2 . The comparison of the results was done with esterification, performed in SC CHF3 , at 35 ◦ C and 20 MPa. Fig. 2 shows the comparison between conversion for esterification of n-butanol with d,l-LA obtained when the three above mentioned SCFs were used. Knez et al. [31] describes our previous research work, which was upgraded with new reaction medium (SC CHF3 ). Lipase-catalyzed esterification of n-butanol with d,l-LA performed in SC CHF3 gave much higher conversion after 26 h at temperatures 35 ◦ C and 55 ◦ C and pressures between 7.5 MPa and 30 MPa in the comparison to the same reaction where SC CO2 was used as the reaction medium. When SC CHF3 was used as the reaction medium the highest conversion of LA (63.28%) was obtained, while the lowest conversion of LA was performed in SC C2 H6 (38.86%).
Fig. 2. The conversion of LA as a function of time of reaction performance at 35 ◦ C and 20 MPa in SC C2 H6 , SC CO2 and SC CHF3 . Reaction conditions: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm.
Furthermore, the study of the pressure influence on the total conversion of LA, yield (gBL /(gE ·h)) and initial rate (((gBL /gsample )/gE )/h) in SC CHF3 was performed at two different temperatures, modulating the pressure in the range from 7.5 MPa to 30 MPa at 55 ◦ C and 35 ◦ C. Influence of temperature and pressure on the conversion of LA is shown in Fig. 3, which clearly shows that the conversion is pressure and temperature dependant. The effect of the pressure and temperature on the yield after 26 h of reaction performance and initial rate in d,l-LA/n-butanol/Novozym 435/SC CHF3 system is shown in Fig. 4. At 55 ◦ C, by increasing the pressure from 10 MPa to 20 MPa significant increase in the
Fig. 3. Effect of pressure on the conversion of LA after 26 h of the reaction performance using SC CHF3 and SC CO2 as reaction medium. Reaction conditions: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm.
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Fig. 4. Effect of pressure and temperature on the yield and initial rate after 26 h of the reaction performance using SC CHF3 as reaction medium. Reaction conditions: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm.
conversion of LA (from 65.3% to 87.5%) and in yield (from 7.7 gBL /(gE ·h) to 10.3 gBL /(gE ·h)) were obtained. The reaction mixture in this pressure region turned from three phase system to two phase system. Figs. 3 and 4 also show that the conversion and yield slightly increased when increasing the pressure from 7.5 MPa to 10 MPa and from 20 MPa to 30 MPa, which was due to the fact that an increase in pressure leads to enhanced solvent density, improving its solvating power in the reaction bulk [40]. Compared to the reaction, performed in SC CO2 [31], much higher conversion after 26 h of reaction was achieved when SC CHF3 was used at 55 ◦ C and pressure between 7.5 MPa and 30 MPa, because of different properties in density, polarity and dipole moment of SC CHF3 and SC CO2 . A maximum yield of 10.4 gBL /(gE ·h) after 26 h of reaction performance was attained at 55 ◦ C and 30 MPa (Fig. 4). At 7.5 MPa and 10 MPa at 35 ◦ C for both SCFs and at 55 ◦ C in SC CO2 a slight decrease in the conversion of LA was observed. Furthermore, the increase in temperature from 35 ◦ C to 55 ◦ C enhances the enzyme activity for reaction performance in SC CHF3 and SC CO2 . The increase in initial rate with higer temperature at certain pressure was detected. Before studying the lipase-catalyzed esterification of n-butyl lactate, the exact composition of the d,l-LA solution (53.3% (w/w) monomer (LA1), 28.7% (w/w) lactoyllactic acid (LA2), and 11.7% (w/w) water) was determined by HPLC and Karl Fisher titrator. Remaining 6.3% (w/w) represents lactide (LA3), the cyclic diester of LA. For the synthesis of n-butyl lactate monomeric form (LA1) was used. The composition of the d,l-LA in the reaction system was monitored for each reaction, because the composition of the d,lLA during reaction performance was changing. The content of LA1 and LA2 decreased during esterification, because they were used for the formation of the product n-butyl lactate. According to the results of HPLC analysis (Fig. 5) for enzymatic esterification of d,lLA in SC CHF3 at 55 ◦ C and 30 MPa the LA1 and LA2 content of the reaction mixture decreased, due to the formation of the product n-butyl lactate. The amount of LA3 remained practically constant during the reaction. Water content in reaction mixture at the beginning of the reaction was 3.54% (w/w). As can be seen in Fig. 5, water content increased during reaction, because water is a by-product of synthesis of n-butyl lactate. Pressure influence on the concentration of d,l-LA and n-butyl lactate (BL) was studied at two different temperatures (55 ◦ C and 35 ◦ C) and in the pressure range from 7.5 MPa to 30 MPa. Fig. 6 represents the concentration of d,l-LA and BL in the reaction mixture after 26 h in SC CHF3 at 55 ◦ C and pressure ranges from 7.5 MPa to
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Fig. 5. Composition of d,l-LA solution, water content and content of BL in the reaction mixture versus reaction time. Reaction conditions in SC CHF3 for BL: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm, 55 ◦ C, 30 MPa.
Fig. 6. Concentration of d,l-LA and BL in the reaction mixture after 26 h. Reaction conditions in SC CHF3 for BL: 1 mmol of d,l-LA and 3.6 mmol of n-butanol, 50 mg enzyme per mmol d,l-LA, 700 rpm, 55 ◦ C, 30 MPa.
30 MPa. While the concentration of BL increased from 13.95% (w/w) to 19.00% (w/w), the concentration of LA1 decreased for about 10% (w/w). At 7.5 MPa the concentration of LA1 was 19.55% (w/w) and with higher pressure decreased due to the enhanced product formation. At 20 MPa and 30 MPa the concentration of LA1 was found to be lower compared to the concentration of LA2. The reason for increased concentration of BL is that the LA2 (open chain dimmer of d,l-LA) is able to decompose to monomers and form n-butyl lactate. Concentration of LA2 was the same for 7.5 MPa and 10 MPa. By increasing the pressure from 10 MPa to 30 MPa a decrease in concentration of LA2 from 16.94% (w/w) to 11.90% (w/w) was oserved, which could be due to the fact that an increase in pressure leads to decomposition of LA2 to monomers. The concentration of LA3 remained approx. the same for all tested pressures (5.6%, w/w), and we assume that higher temperatures [41] are necessary for the decomposition of the LA3 cyclic di-ester to lactic acid monomers. 3.3. Antifungal and antibacterial activities In spite of the modern improvements in food hygiene and food production techniques, food safety is an increasing important public healt issue. Therefore, the reducing or inhibiting foodborne pathogens, possibly in combination with the existing methods is still needed [42]. Antifungal and antibacterial activities of d,l-LA and n-butyl lactate (standard and synthesized) were determined. Till now, the studies of antifungal and antibacterial activities on microorganisms (A. niger, T. viride, P. cyclopium, P. flourescens and B. cereus) were not reported. Therefore, presented results are unique.
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Table 2 Antimicrobial activities of selected fungi strains. Fungal strains
72 h Blankb
S. cerevisiae A. niger T. viride P. cyclopium
14 46 70 18
Percentage mycelial zone inhibitiona LA
n-Butyl lactate
Sample of BL
100 100 100 100
100 100 100 100
100 100 100 100
a Diameter of zones including diameter of disk 12 mm (average of three replicates). b Colony diameter of the mycelium on the physiological salt solution plate (mm).
Lopes et al. [6] reported the microbicidal activity and the minimum lethal activity of butyl lactate on Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhimurium). E. coli and S. cerevisiae were investigated by both groups with a different protocol, where the method prepared by our group for antifungal activity was agar well-diffusion method and the method for antibacterial activity was disk diffusion method. S. cerevisiae is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like E. coli as the model prokaryote [43,44]. The agricultural pathogenic fungus A. niger causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food [45]. P. cyclopium is the main contaminant fungi detected in seeds harvested. Such contaminants are fearsome, since they affect the seeds before harvest time and may find optimal developing conditions when the seeds are stored, leading to alteration of the germination quality of these seeds [46]. E. coli is a Gram-negative bacterium, which is one of the most extensively studied micro-organisms because of its wide use as a model organism in research and microbial genetics and physiology as well as its use in industrial applications [44]. Although E. coli are normal inhabitants of the colons of all warm-blooded mammals, some pathogenic strains can cause urinary tract infections, diarrhea and also new born meningitis [44,47]. Another used a Gram-negative bacterium is P. fluorescens. This rod-shaped bacterium presents biocontrol properties and protecting the roots of some plant species against parasitic fungi [48,49]. B. cereus is a Gam-positive, rodshaped bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals [50]. Antifungal activities of d,l-LA and n-butyl lactate (standard and synthesized) against S. cerevisiae, A. niger, T. viride and P. cyclopium are presented in Table 2, where the results of the microbial growth, applying three parallel measurements, are given. The results of antifungal activity assay showed that all samples exhibited 100% inhibitory effects on the growth of three tested mycelial fungi. The results of antifungal activity suggest the use of d,l-LA and n-butyl lactate (standard and enzymatically synthesized) as an antimicrobial aditive in food processing to provide improve food safety.
Fig. 7. Growth inhibition of T. viride after 72 h growth: (A) physiological salt solution, (B) d,l-LA, (C) standard of n-butyl lactate and (D) synthesized n-butyl lactate.
Antifungal activities of d,l-LA and n-butyl lactate (standard and synthesized) against T. viride after 72 h growth are presented in Fig. 7. They showed 100% inhibitory effects on the growth of fungus T. viride in comparison with control of physiological salt solution. Antibacterial activity of substances was determined on the paper discs and expressed in terms of an inhibition zone. The diameter of the clear zone of inhibition around a paper disk agar is shown in Table 3, which summarizes antibacterial activities of d,l-LA and n-butyl lactate (standard and synthesized) in comparison with a physiological salt solution control after a 24 h and 48 h growth. Studies have suggested that d,l-LA [51] and n-butyl lactate (standard and synthesized) [6] have antibacterial activity against the tested bacteria. Comparisons of d,l-LA and n-butyl lactate showed that the clear zone around the paper disk on the plate agar was wider for the d,l-LA than for n-butyl lactate, demonstrating that the d,l-LA has higher antimicrobial activity than n-butyl lactate. Diameter of inhibition zones of enzymatically synthesized n-butyl lactate was determined to be in all cases slightly higher than those of commercial n-butyl lactate. The reason could be the fact that the samples contain some unreacted d,l-LA. Fig. 8 shows inhibition zones including discs of 6 mm in diameter for d,l-LA, standard of n-butyl lactate and synthesized n-butyl lactate against B. cereus after 24 h growth. The comparison of antimicrobial activity between standard (which is 97% pure and used any dilutions) and synthesized n-butyl lactate (used immediately after synthesis without any treatement and any dilutions) was done owing to confirmation of the similar antimicrobial properties of both n-butyl lactates. The aim was to examine how the n-butyl lactate itself affects the inhibition of the growth. d,l-LA shows the highest antifungal and antibacterial activities but the point of the research was to examine the
Table 3 Antimicrobial activities of selected bacterial strains. Bacterial strains
Inhibition zones diametera 24 h
E. coli P. flourescens B. cereus
48 h
Blankb
LA
n-Butyl lactate
Sample of BL
Blankb
LA
n-Butyl lactate
Sample of BL
– – –
43.0 ± 0.6 40.3 ± 2.5 45.3 ± 0.6
20.0 ± 1.0 19.0 ± 3.6 18.3 ± 1.2
23.3 ± 2.3 23.0 ± 3.6 23.0 ± 1.0
– – –
43.7 ± 0.6 40.0 ± 2.6 41.3 ± 3.2
22.0 ± 1.0 17.5 ± 2.2 10.7 ± 0.6
23.3 ± 2.3 21.3 ± 3.5 18.8 ± 1.4
Each value represents the mean S.D: (n = 3). –, no inhibition. a Diameter of inhibition zones including diameter of disk 6 mm. b Physiological salt solution (9 g/L NaCl).
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Fig. 8. Growth inhibition of B. cereus after 24 h growth: (1/1 and 1/2) physiological salt solution, (2/1 and 2/2) d,l-LA, (3/1 and 3/2) standard of n-butyl lactate and (4/1 and 4/2) synthesized n-butyl lactate.
antifungal and antibacterial activities of n-butyl lactate, since it could be used as a food additive, therefore its antimicrobial activity is of great importance.
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