Limonin consumption at acidic pH values and absence of aeration by Rhodococcus fascians cells in batch and immobilized continuous systems

ELSEVIER

Limonin consumption at acidic pH values and absence of aeration by Rhodococcus jkscians cells in batch and immobilized continuous systems Manuel Cainovas, Lidia Garcia-Cases,

and Jo& Luis Iborra

Department of Biochemistry and Molecular University of Murcia, Murcia, Spain

Biology B and Immunology,

Faculty of Chemistry,

Limonin consumption from synthetic citrus juices at their usual pH values (4.0-5.0) in the absence of aeration was per$ormed with Rhodococcus fascians cells in batch and continuous immobilized systems. Limonin and its metabolites at deferent pH values (4.0 and 5.0) under nonaerated conditions within the synthetic citrus juice without R. fascians cells was established. Limonin and monolactone D of limonoate were the main components at pH 5.0 whereas at pH 4.0, limonin was the only possible bacterial substrate. Although R. fascians is stated as an aerobic organism, at the acidic pH of the synthetic citrus juices (pH 4.0) and without aeration of the medium, it was able to consume limonin (nearly 70% after 150 h) at a higher rate than at pH 7.0 (nearly 40% after 160 h) in batch systems. On the other hand, R. fascians cells were immobilized within polyurethane foam pads in a continuous reactor and fed on a synthetic citrus juice at a dilution rate of 2.4 IO-’ hh’. It reached an 85% conversion of limonin after 200 h. This level was under the human detection threshold. With respect to immobilization, the so-called specific rate of immobilization of R. fascians in polyurethane foam pads was calculated (Ki = 0.016 hh’). After 150 h, nearly no free cells were within the culture medium and bacteria were already immobilized. The optical density of the reactor bulk liquid showed that nearly no free biomass was present. Finally, from the sugars and citric acid contained within the synthetic citrus juice, only fructose was slightly consumed. 0 I998 Elsevier Science Inc.

Keywords:

Limonin;

Rhodococcus fascians; continuous

reactor; immobilized

Introduction pressing citrus fruits, the nonbitter limonoate-A-ring lactone contained in the citrus tissues passes into the liquid phase where it is converted to limonin in a process accelerated by the acidic pH of the juice and also by heat.‘.* Limonin is known as the main bitter component of citrus juices. The removal of limonin has been studied by several authors’-” and substantial progress has been made on its bacterial degradation. So far, four pathways for metabolizing limonin have been stated; the deoxylimonoids,4-6 the 17-dehydrolimonoids,6 the 7-ol-hydroxylimonoids,7s and finally the trans- 19-hydroxylimonoate pathway.* The latter When

Address reprint requests to Dr. J. L. Iborra, University of Murcia, Faculty of Chemistry/Department of Biochem. 8 Mol. Bio. B and Immunology, P.O. Box 4021, E-30100, Murcia, Spain Received 1 I December 1996; revised 3 June 1997; accepted 1 July 1997

Enzyme and Microbial Technology 22:ll l-l 16, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

cells

three pathways are found in Rhodococcus fascians whereas other microorganisms possess only one of the abovementioned pathways to degrade limonin. Among the organisms isolated so far, R. fascians is the only one that produces constitutive enzymes for the metabolism of limonoids.’ The rest of the bacteria studied requires the presence of a limonoid inducer within the growth media to produce cells capable of metabolizing limonoids. The most-studied pathway is that of the 17-dehydrolimonoids where the enzyme limonoate dehydrogenase plays the main role. This enzyme needs for its catalytic action the presence of cofactor NAD+ and sulfydryl groups. Its activity is optimum at pH 8.0-9.0. At the characteristic pH values of citrus juices (3.8-5), this enzyme is unstable and shows very low activity.4 If cells were to be used, protein molecules would be protected from the external pH. Located in their natural microenvironment, they would be able to develop their optimun catalytic activity. Nowadays, only data on the R. fascians limonin metabolism at the optimum

0141-0229/98/$19.00 PII SOl41-0229(97)00137-3

Papers

0

Figure 1 Formation of limonin (L) from disodium limonoate (L-*) or monosodium of limonoate in the presence of oxygen (after Arberas-Mendiguren)16

pH for growth (slight alkali and neutral, pH 7-8) is available. Its metabolism at the characteristic pH values of citrus juices has not been studied yet. On the other hand, R. fuscians is classified as an aerobic microorganism;’ its growth on limonin in the absence of aeration has not been studied; therefore, with respect to the debittering process by biological means, the major inconveniences of citrus juices are both the acidic pH and the requirement for an absence of aeration conditions so as to not change their organoleptic characteristics. We recently reported about work on the evolution of the equilibrium of limonin and its species (Figure I) in the culture medium of R. fuscians at the citrus juice characteristic pH values.” In the present work, we report on the behavior of R. fuscians concerning its growth and consumption of limonin from a synthetic citrus juice under low pH and in the absence of aeration (as the processing of citrus juices requires) in a batch reactor. Since the equilibrium of limonin and its species might vary depending on pH (Figure 1) and the absence of aeration, we also present the possible substrates R. fascians can find within the synthetic citrus juice under the processing conditions. Previous studies have used solid supports for immobilization such as polyacrylamide gel’ ’ and k-carrageenan ‘4,‘8 but the first reagent is not allowed for human use and the second has not shown 112

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limonoate

(LH-),

monolactone

A (LA) or D (LDH)

enough stability to work at the typical pH values of the citrus juices for long periods of time.” On the contrary, polyurethane foam has been used for plenty of cell immobilization studies due to its high structural stability and biomass retention capability as well as its harmlessness to cells;12 therefore, the behavior of a continuous reactor with R. fascians cells freely suspended and entrapped in polyurethane foam pads was also studied at debittering limonincontaining synthetic juices.

Materials and methods Materials Limonin was extracted from lemon seeds according to Pifferi er al.” and was purified as described by Martinez-Madrid et al.‘” Rhodococcus fuscians NRRL- 15096 was supplied by the Spanish

Type Culture Collection (University of Valencia, Spain). All remaining reagents were analytical grade from Merck (Darmstadt. Germany) and Aldrich (Milwaukee, WI) and used without further purification.

Culture medium The culture was maintained in a medium containing per liter (w/v): K,HPO,, 0.5 g; KH,PO,, 0.5 g; NH&I, 0.2 g: FeCI,, 0.001 g;

1

Limonin

consumption

by Rhodococcus

fascians: Canovas

et al.

nutrient broth, 2.0 g; fructose, 4.0 g; adjusted to pH 7.0 or 4.0 with HCl and inoculated with 2% of a 48-h-old culture. Incubation was performed at 25°C on an orbital shaker (150 rpm). The synthetic juices consisted of glucose (23 g l-l), fructose (23 g 1-l). sucrose ($6 g l-l), and citric acid (10 g 1-l) added to the salts mentioned above. Limonin and thiamine were added at concentrations of 30 and 1 mg 1-‘, respectively, and the pH of the solution was adjusted to 4.0 with 0.1 N NaOH.

Batch and continuous

reactor for limonin removal

A reactor system (Braum model B., Germany) with a 2-l vessel thermostated at 25°C was filled with 1.0 1 of the abovementioned synthetic medium at pH 7.0 or 4.0 depending on the experiment. Nutrient broth was substituted by thiamine (0.01 g 1-l) and fructose by limonin (30 mg I-‘). Limonin in the culture medium was solubilized by the method of Maier and Margilett” at a final pH of 12 and then brought to the respective pH in use with concentrated HCI. Studies performed at different pHs were set at 0% air saturation depending on the experiments.

Analytical

100 -

methods

Limonin was quantified by HPLC at 207 nm with a LC-6 Shimadzu equipped with a RP-Cl8 Nova Pack (Millipore, I5 cm long, 3.9 inner diameter, 4 mm particle size. and 6 mm pore size), a SPD-M6A Shimadzu LJV-Vis diode-array detector, and an isocratic mobile phase acetonitrile:HzO (45:55) at 0.8 ml min- ’ flow rate. Limonin and its species (Figure I) were eluted isocratitally with a mobile phase made of acetonitrile:H,O (45:55) and 20 mM tetrabutylammonium pH 3.5 in a ratio of 57:43 (v/v) and 0.7 ml min-’ flow rate. This method was first proposed by ArberasMendiguren. ” The separation of limonin and its species is based upon their electric charge. Identification is through their characteristic UV-absorption spectra (range 200-300 nm). The concentration of the limonin species is expressed as a percentage of the total peak area. The analyses of sugars, o-glucose, D-fructose, sucrose, and citric acid were performed by enzymatic tests (Boerhinger Mannheim, Mannheim, Germany).

Immobilization

in polyurethane

foam

Cubes of a polyurethane foam matrix with an average pore size of 0.75 mm and 30 pores cm.-’ (Polyol International BV, Switzerland) were prepared. After being washed thoroughly with ethanol. cut into 2 X 2 X 2 cm pads and autoclaved in a 50 mM sodium phosphate buffer pH 6.5, 125 cubes I .O l- ’ culture medium were used for cell immobilization.

Free and immobilized

cell determination

Free biomass was determined by turbidimetry at 600 nm. Quantification of immobilized cells within the polyurethane foam pads was achieved after cell liberation through vigorous shaking in the presence of 0. I N NaOH using the method for free biomass.

Results and discussion Transformation of limonin and limonin species with pH under the absence of aeration After pressing citrus fruits, the nonbitter limonoate A-ring lactone contained in the citrus tissues passes into the liquid phase where it is converted to limonin in a process accelerated by the acid pH of the juice and also by heat.‘,’ Different experiments were performed in which limonin

Time (hours) Figure 2 Evolution of limonin (A), monolactone A (W), monolactone D (*I, and limonoate (0) at pH 5.0 (A) and pH 4.0 (B) in the absence of aeration and without R. fascians cells

was solubilized as expressed in MATERIALS AND METHODS within the growth medium (mineral salts and thiamine) without R. fascians cells. The pH was then adjusted to the desired value and the mixture was left under anaerobic conditions so as to observe limonin and its species evolution at the conditions of citrus juice processing. Figure 2 shows the evolution of limonin and its species at pH 5.0 and 4.0 (citrus juice pH range) in the absence of oxygen. At pH 5.0, the main components in the growth medium were limonin and limonoate-D-ring lactone whereas the species limonoate and limonoate-A-ring lactone were quickly transformed to limonin (Figure I); however, at this pH value in presence of enough air in the culture media, the main limonin metabolite was limonoate-D-ring lactone the entire length of the experiment.‘” On the contrary at pH 4.0, limonoate and limonoate-A-ring lactone disappeared after 100 h. The latter was transformed to limonin after 4 days; however, previous experiments at this pH in the presence of oxygen within the growth media showed that the transformation to limonin was much lower than in the absence of oxygen.‘O With these results in mind. it can be concluded that aeration of the culture medium has considerable importance in the proportion of limonin metabolites. The absence of aeration favored the formation of limonin when pH was acid enough; thus. at the pH values of the citrus juices and after some time, the main metabolite was limonin. R. fuxians would therefore have this substrate as the main component of the culture medium in case it was present. This fact is important since the relative amount of the adequate substrate formed can control the rate of overall limonin removal during R. fusciam citrus juice limonin consumption. In previous studies,” it has been shown that limonin is the best limonin metabolite as substrate for R. ,fusciuns cells.

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metabolite pathway that works better in the absence of aeration at acidic pH values. Probably metabolizing limonoate (main component at pH 7.0, Figure 1) through the pathway of the 17-dehydrolimonoids is hampered under these conditions while other metabolic pathways would be able to use limonin as substrate at acidic pH values. Although R. fuscians has been reported as an aerobic microorganism, at pH 4.0 in the absence of aeration, it was able to consume limonin. Probably, traces of air were enough to grow on limonin as the sole source of carbon and energy.

Continuous

0’

0

I

I

I

’

40

80

120

160

reactor operation

In order to know the continuous conversion of limonin at typical conditions of citrus juice processing (pH 4.0 in the absence of aeration), studies were performed on the evolution of biomass and limonin consumption in a continuous reactor. The reactor was loaded with a synthetic citrus juice at pH 4.0 and 2.4 ml hh’ flow rate (dilution rate, D = 0.06 dd’). This low starting flow rate was used since the maximum specific growth rate for R. fusciuns at pH 4.0 was 0.35 dd ’ . The biomass in the reactor was determined throughout the procedure. Results are shown in Table 1. A slight increase in biomass was observed after 10 h. This latter increase lasted up to the 35 h of the experiment followed by a decrease in optical density until the end of operation. With regard to the limonin consumption, its level reached a value of 11 mg 1-l within the system, with a limonin consumption of 1.1 mg day-’ and a conversion of nearly 70% (Table 1). Although, the dilution rate was lower than the specific growth rate, biomass decreased from an initial OD of 10.5 U to 7.9. Previous studies have shown that at pH values equal to 4.0, limonin was consumed although the cell population remained nearly constant,‘* thus reflecting a certain uncoupling between cell growth and substrate consumption. This disconnection is favorable for substrate metabolism. Besides, the decrease in biomass (as OD values showed) might be as a result of cell size decrease rather than a decrease in cell number: nevertheless, the system was shut down after 120 h of operation. Conversion was a little less than in the batch reactor at pH 4.0 in the absence of aeration conditions. These results showed that it was possible to use R. fusciuns to consume limonin from synthetic citrus juices in a continuous system, but possible washout would occur at even very low dilution rates. With regard to the effect of the cell metabolism on the concentration of sugars, it has been stated that R. fulsciuns

‘0

Time (h) Figure 3 Biomass (0) and limonin evolution (0) at pH 7.0 (A) and pH 4.0 (B) in the absence of aeration in batch systems

EfSect of pH and the absence of aeration on limonin comsumption and cell growth As previously stated, citrus juice debittering requires the absence of air to avoid changes in organoleptic properties;‘7 furthermore, in the previous paragraph, it has been shown that citrus juice pH values control the relative amount of limonin and its metabolites and therefore the possible limonin removal. Thus, the effect of pH in the absence of aeration on limonin consumption and cell growth was studied in batch systems. Figure 3 represents typical growth curves for R. fusciuns suspension cultures at pH 7.0 and 4.0, respectively, with limonin as carbon and energy source in the absence of aeration. At pH 7.0 after 80 h, an increase in biomass of 1.2 OD U took place. Limonin reaching a level of 18mgl-’ after 160 h. On the contrary, Figure 3B shows results at pH 4.0 where the adaptation time was shorter than at pH 7.0 and the increment of biomass much higher; furthermore, in the absence of aeration, the concentration of limonin after 160 h was lower than 9 mg 1-l. The concentration is considered to be under the human bitterness threshold. It is possible that R. fusciuns possesses a limonin

Table 1 Biomass changes (OD), limonin conversion, and glucose, fructose, and citric acid evolution during continuous operation pH 4.0 in the absence of aeration using synthetic citrus juices Time (h)

OD (600 nm)

Cs limonin (%)

0 17 35 80 115

10.51 10.80 11.45 8.33 7.93

0 65.46 67.08 67.01 67.60

Assays were performed

114

Glucose (g I-‘) 24.16 24.01 24.10 23.80 23.32

in triplicate

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1

k rt 5 ‘2

0.10 0.14 0.18 0.20 0.15

Fructose (g I-‘) 23.87 23.57 23.08 23.66 23.09

2 2 2 k 2

0.12 0.15 0.20 0.12 0.19

at

Citric acid (g I-‘) 1.50 10.48 10.58 10.47 10.48

2 i 2 2 t

0.15 0.11 0.10 0.15 0.13

Limonin

consumption

by Rhodococcus

fascians: Canovas et al.

Table 2 Free biomass Changes (OD), limonin conversion, and glucose, fructose, and citric acid evolution in a continuous cells immobilized within polyurethane foam pads at pH 4.0 in the absence of aeration Time (h)

OD (600 nm)

Cs limonin (%)

0 17 64 113 185

3.24 2.50 1.00 0.61 0.01

0 88.43 85.95 83.93 85.84

Assays were performed

Glucose (g I-‘) 23.00 27.03 31.85 34.54 41.03

reactor

0.12 0.14 0.12 0.20 0.15

23.00 22.42 22.41 20.36 19.90

-t 2 5 2 5

0.10 0.15 0.13 0.12 0.10

Citric acid (g I-‘) 11.00 10.85 11.05 11.00 10.90

+ 2 k i ?

0.15 0.10 0.18 0.15 0.25

in triplicate

does not consume sucrose.‘*i8 On the other hand, analyses of the sugars fructose and glucose and citric acid can be seen in Table I. R. fascians did not consume them to any great extent; thus, the main problem was the loss of biomass which led to variations in the amount of limonin during the operation time. The use of a continuous reactor with immobilized cells at low pH values would be interesting, since limonin would disappear from the medium without a concomitant increase in cell number, thereby avoiding the possibility of clogging the reactor by excess biomass and favoring cell retention within the solid support. We therefore, decided to immobilize R. fascians cells to solve the problem of biomass washout. Continuous

2 t 5 c +

Fructose (g I--‘)

reactor with

with immobilized

cells

The debittering process of citrus juices with immobilized cells requires solid supports with chemical and physical stability at low pH values for long periods of time. Polyurethane foam has been used in plenty of cell immobilization studies due to its high structural stability and biomass retention capability as well as its harmlessness for cells, I2 therefore, the reactor was loaded with 125 cubes of polyurethane foam (details in MATERIALS AND METHODS), 1.O 1 synthetic citrus juice, and 5% (v/w) of a 48-h-old culture. It was incubated at 25°C and run with a 2.4 ml min-’ flow rate of the same synthetic juice as before at pH 4.0 in the absence of aeration. The reactor working volume was maintained at 1.O 1 throughout the course of the experiment. One cube was found to immobilize approximately 4.4 mg cells. At this point, limonin conversion, the substrate consumption rate, and the free biomass were determined. As shown in Table 2, the fall in free biomass concentration followed a characteristic exponential curve. The initial phase could be represented by a straight line which allowed the calculation of the specific rate of immobilization for R. fascians in polyurethane foam at 0.016 h-‘. After 200 h, the amount of free biomass remained essentially unchanged (nearly no free biomass within the reactor bulk liquid) (Table 2). Manjon et al3 using a continuous reactor with R. fascians immobilized in kcarrageenan beads, tested limonin consumption at pH 4.0 in the presence of aeration and observed a release of biomass from the beads of 16.0 mg 1-l within the reactor bulk liquid whereas in this work using polyurethane as the solid support, nearly no free biomass was found. The fact that at pH 4.0 in the presence of aeration k-carrageenan beads were mechanically and structurally altered justifies the use of

polyurethane foam pads at these pH values in the absence of aeration conditions. Additionally, limonin consumption was constant most of the time (85-90% conversion) until reaching a concentration of 4-5 mg 1-l within the reactor effluent (concentration under human detection level). Studies performed with R. fascians immobilized in k-carrageenan beads with limonin as the carbon and energy sources at pH 7.0” showed that limonin consumption was slightly reduced when aeration was lowered; however, a decrease in limonin concentration was enough to debitter the reactor feed. On the other hand, the synthetic citrus juices contained fructose, glucose, sucrose, and citric acid in the concentration detailed in MATERIALS AND METHODS. As was expected, no variation in the citric acid concentration was observed whereas sucrose disappeared from the reactor at the same time that glucose increased and fructose decreased slowly (Table 2). It has been reported that citric acid is not consumed by R. fascians cells whereas fructose is an alternative substrate within citrus juices,‘* furthermore, sucrose seems to be hydrolyzed due to either the acid pH of the culture medium or the action of R. fascians or even both effects. Experiments devoted to ascertain this fact are being undertaken. The fact that fructose decreased means that there is a dilution rate compromise which removes limonin (bitter component) and is not capable of affecting the fructose level to a great extent. It would also be necessary to test the activity and stability of R. fascians immobilized within polyurethane foam pads when citrus essential oils are present since it has been reported that these citrus juice components were able to hinder the activity of immobilized Acinetobacter sp. due to their antibacterial activity.’

Conclusions R. fascians cells are able to metabolize limonin contained within synthetic citrus juice under the processing conditions of industrial operations (absence of aeration) in batch and immobilized within polyurethane foam pads in continuous systems. Sucrose as a component of the citrus juice seems to be hydrolyzed either by the bacterial cells or due to the pH values of the synthetic juices; however, both possibilities would only cause a certain increase in the sweet taste without changing other organoleptic properties of the juice. On the other hand, it is possible that R. fascians could take up and consume limonin through a pathway other than the

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Papers 17-dehydrolimonoids at the acidic pH of citrus juices in the absence of aeration. This fact opens a new field of research.

8.

Acknowledgments

9.

This work was supported by the “Spanish CICYT BI0930660-CO4-01 and BI096-1016-C02-01 projects.” L. Garcia-cases is a fellow of OTRI, Spain. Lemon seeds were supplied by J. A. Andreu of Riverbend, Spain. We also acknowledge this company help to support L. Garcia-Cases by the OTRI fellow.

IO.

11.

12.

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