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Fermentation of washing waters of Spanish-style green olive processing
Process Biochemistry 36 (2001) 797– 802 www.elsevier.com/locate/procbio
Fermentation of washing waters of Spanish-style green olive processing Antonio de Castro, Manuel Brenes * Food Biotechnology Department, Instituto de la Grasa, CSIC, A6da. Padre Garcı´a Tejero 4, 41012 Se6illa, Spain Received 16 May 2000; accepted 3 December 2000
Abstract Washing waters of Spanish-style green olive processing are heavily contaminated waste streams that represent an important environmental problem that needs to be solved. These liquids are only generated for 2 months a year and a storage period is needed if high-value substances are to be extracted from them. Fermentation of the washing waters under acidic conditions was studied throughout a year. A lactic acid fermentation took place in vessels with either no initial correction of pH, or pH corrected to 4. When the pH was initially lowered to 3, only yeasts grew and a significant concentration of ethanol was generated. The concentration of phenolic compounds decreased slightly during the fermentation process, particularly hydroxytyrosol, which was found in high concentration in these waste waters. If this orthodiphenol is to be recovered from washing waters, time is required to hydrolyse the elenolic acid glucoside. The glucoside was hydrolysed under the acidic conditions of the washing waters, and the reaction was affected by temperature and pH. Thus, fermentation of washing waters under certain conditions may give rise to solutions with no off-odours and a high concentration of lactic acid and hydroxytyrosol, which are economically interesting products. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Table olive; Washing water; Hydroxytyrosol; Lactic acid; Fermentation
1. Introduction The worldwide production of table olives has been calculated to be 1 million t, half of which corresponds to Spanish-style green table olives. The procedure for preparing this type of table olives consists of treating the fruits with dilute NaOH solution to hydrolyse their natural bitterness (oleuropein), followed by one or two water washes to remove the excess alkali and, finally, a spontaneous lactic acid fermentation in brine for several months [1]. The washing solutions are heavily contaminated and contain a high content of sugars and phenolic compounds, particularly hydroxytyrosol (3,4dihydroxyphenyl ethanol) [2]. These solutions are not accepted in municipal sewers and two alternatives have been reported to diminish their volume: (i) elimination of the washing step [3]; and (ii) reuse of the solutions * Corresponding author. Tel.: +34-954690850; 954691262. E-mail address: [email protected] (M. Brenes).
fax:
+34-
after neutralising the residual alkali [4]. Neither method has been applied successfully on an industrial scale. It would be of interest to obtain high-value products from these washing waters, such as organic acids (lactic acid) and polyphenols, particularly hydroxytyrosol — an orthodiphenol with high antioxidant activity [5] — and nutritional properties [6]. In fact, hydroxytyrosol is also a possible product from olive oil mill waste waters [7]. The washing waters are only generated for 2 months a year and, therefore, must be stored until extraction of the phenolic substances. Storage in acid conditions is preferable, since it has been reported that hydroxytyrosol does not change at low pH during fermentation of Spanish-style green table olives [8]. In addition, the other part of the oleuropein molecule generated during NaOH treatment, the elenolic acid glucoside, is hydrolysed under these acidic conditions [9]. Temperature could be an important factor in this reaction. The elenolic acid glucoside is a component of the phenolic extract of washing waters and may interfere during the process of hydroxytyrosol extraction.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 8 0 - 6
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A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
The objectives of this work were to investigate different fermentation procedures to prevent the oxidation of hydroxytyrosol, to obtain the maximum quantity of lactic acid, and to avoid the appearance of off-odours. The evolution of elenolic acid glucoside and elenolic acid during storage at different temperatures in acid conditions was also studied.
2. Materials and methods
2.1. Fermentation procedures Olives of the Manzanilla cultivar were treated with 0.5 M NaOH solution for 7 h. The alkaline solution was poured off and the fruits washed in water for 12 h. Washing water (50 l) was placed in each of eight PVC cylindrical vessels and stored at ambient temperature for 1 year. Acid (6N HCl) was added to four of the vessels to adjust the initial pH of the washing water (ca 11) to 4 (two vessels) and 3 (two vessels). HCl was employed in another two vessels to lower the pH to 3 and sodium metabisulfite (0.4 g/l) was added. The remaining two vessels, without added acid, were used as controls. Special care was taken to reduce to a minimum the contact between the washing waters and the atmosphere and anaerobic conditions were used during experiments.
Washing water with the initial pH adjusted to 3 was also put into 330 ml jars which were sealed and stored at 15, 30, 40 and 50°C in thermostatic chambers. Samples were taken at regular intervals and experiments were run in duplicate.
2.2. Chemical analyses Sugars (glucose, fructose, sucrose and mannitol), alcohol (ethanol), organic acids (lactic, acetic and propionic), and polyphenols were analyzed by HPLC as described elsewhere [9]. The colour of washing waters was determined as the difference of absorbance at 440 and 700 nm (A440 − A700).
2.3. Microbiological analyses Samples from the different vessels and their decimal dilutions were plated using a Spiral System model DS (Interscience, Saint Nom La Breteche, France). Enterobacteriaceae were counted on Crystal-violet neutralred bile glucose agar (Merck), lactic acid bacteria on MRS agar (Oxoid), with and without 0.02% sodium azide (Sigma), and yeasts on OGYE agar (Oxoid). Plates were incubated at 32°C for 48 or 72 h. Samples were also inoculated into Differential Reinforced Clostridial Broth (Merck) and incubated at 37°C up to 14 days.
3. Results and discussion
Fig. 1. Evolution of glucose, fructose and mannitol in washing waters during fermentation. Pooled standard deviations for glucose, fructose and mannitol were 5.6, 3.2 and 4.3, respectively.
Despite the antibacterial activity of olive polyphenols [10,11], spontaneous fermentation of carbohydrates occurred throughout storage of the washing waters. In treatments with initial pH either uncorrected or adjusted to 4 the concentration of the glucose and fructose diminished very rapidly during the first 10–20 days (Fig. 1). At the same time, mannitol accumulated, a phenomenon which could be related to the fermentation route of fructose [12]. Mannitol is not well-assimilated by microorganisms usually found in olive brines [13] and, as can be observed in Fig. 1, a residual level of this compound was found even after 1 year of storage. In treatments with the initial pH lowered to 3, the consumption of glucose and fructose was slower than in the other treatments assayed. Nevertheless, the concentration of these carbohydrates was very low after 50– 100 days. Changes in carbohydrates were in parallel with growth of different microorganisms. Thus, Enterobacteriaceae were detected in treatments with no initial correction of pH only during the first weeks of storage. A maximum of 4.0× 108 CFU/ml was reached after 5 days, after which the population started to decline and these microorganisms were not detected after 19 days.
A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
Fig. 2. Lactic acid bacteria population in washing waters during fermentation. Pooled standard deviation for lactic acid bacteria was 1.1. Lactic acid bacteria were not detected when the initial pH was adjusted to 3.
Fig. 3. Yeast population in washing waters during storage. Pooled standard deviation for yeasts was 0.6.
Fig. 2 shows the evolution of lactic acid bacteria throughout the year of storage for treatments with
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initial pH uncorrected or adjusted to 4; in those treatments in which the pH was initially lowered to 3 these microorganisms were not detected. The population of lactic acid bacteria was always higher in control treatments (no correction of pH) than in those with pH adjusted to 4. It must be stressed that the addition of HCl to the olive brines at the beginning of the fermentation process may delay growth of lactic acid bacteria [3]. However, maximum levels of ca 108 CFU/ml were reached after 10–30 days, corresponding to the high consumption rate of glucose and fructose in this period for treatments with initial pH higher than 4 (Fig. 1). From this maximum, the population started to decline throughout the year. Unlike the lactobacilli, yeasts were detected in all the vessels (Fig. 3), although their population was generally higher in treatments with initial pH corrected to 3. An initial delay was also detected when SO2 was added to the washing water. Viable spores of clostridia were detected in a few instances from almost all treatments during storage and were always present in samples from one vessel of the control treatment which started to develop abnormal odour. Lactic acid was generated only in those treatments with initial pH higher than 4 (Table 1), corresponding to lactic acid bacteria growth in these treatments (Fig. 2). The highest concentration was reached in treatments with the initial pH adjusted to 4, in spite of the fact that the Lactobacillus population in these treatments was lower than in those with no correction of the initial pH (Fig. 2). It needs to be stressed that fermentation was spontaneous and there was no characterization of the types of Lactobacillus in each vessel. Moreover, the level of lactic acid produced during fermentation was higher than that found during fermentation of Spanishstyle green table olive processing [14]. Acetic acid was detected in the washing waters at the beginning of the storage in concentrations lower than 16 mM. This could proceed from the NaOH treatment
Table 1 Changes of some physico–chemical characteristics during fermentation of washing waters of the Spanish-style green olive processing Storage method Control
pH 3
pH 4
pH 3 and SO2
Parameter
1 day
365 days
1 day
365 days
1 day
365 days
1 day
365 days
Lactic acid (mM) Acetic acid (mM) Ethanol (mM) pH Colour (A440−A700)
ND a 16(1) 26(2) 10.6 0.67(0.13)
119(33) b 169(2) 146(23) 4.3 0.74(0.04)
ND 12(2) 20(3) 3.0 0.48(0.02)
ND 19(1) 451(62) 2.7 0.58(0.06)
ND 15(1) 23(1) 4.0 0.70(0.06)
154(8) 135(18) 236(29) 3.4 0.73(0.03)
ND 11(1) 17(2) 3.0 0.35(0.04)
ND 29(4) 520(59) 2.8 0.42(0.05)
a b
Not detected. Standard deviation.
A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
800
Table 2 Changes of phenolic compounds during fermentation of washing waters of the Spanish-style green olive processing Storage method Control
pH 3
pH 4
pH 3 and SO2
Compound
1 day
365 days
1 day
365 days
1 day
365 days
1 day
365 days
Hydroxytyrosol (mM) Tyrosol (mM) Caffeic acid (mM) p-coumaric acid (mM)
31.4(1.3) a 3.2(0.1) 0.4(0.1) 0.5(0.1)
29.3(2.1) 2.7(0.1) ND b ND
22.9(4.0) 2.4(0.4) 0.3(0.1) 0.3(0.1)
22.3(3.3) 3.0(0.2) ND ND
31.6(1.6) 3.0(0.2) 0.3(0.1) 0.5(0.1)
30.4(1.5) 3.0(0.2) ND ND
24.4(3.2) 2.4(0.2) 0.3(0.1) 0.4(0.1)
24.2(3.2) 2.0(0.4) ND ND
a b
Standard deviation. Not detected.
of the olives before the washing step, as shown in a previous work [14]. The concentration of the acid increased slightly in those treatments with the initial pH adjusted to 3, and reached a value of 140 – 170 mM in treatments with initial pH higher than 4. This acid may have been generated by either heterofermentative lactic acid bacteria or Enterobacteriaceae in the case of the control treatments. Another main end product was ethanol. The concentration of this compound increased in all treatments, although particularly in those with the initial pH lowered to 3 and in a higher proportion when sodium metabisulfite was used. It is obvious that yeasts were the microorganisms responsible for the high amount of ethanol formed in these treatments (Fig. 3). Acids and ethanol production, particularly lactic acid, decreased the initial pH of vessels, although the degree depended on the type of treatment (Table 1). Thus, in the control treatment this parameter diminished from 10.6 on the first day of storage to 4.3 after 365 days. The decrease was 0.6 and 0.2 – 0.3 units for treatments with initial pH adjusted to 4 and 3, respectively. The colour of the solutions (A440 −A700) slightly increased in all the treatments (Table 1). This effect could be attributed to the oxidation of hydroxytyrosol which decreased in concentration throughout storage (Table 2). There was also a noteworthly decrease in the initial concentration of hydroxytyrosol (1 day storage) when the washing waters were acidified to pH 3. There is no apparent explanation for this phenomenon other than it could be related to the precipitate that was formed. Nevertheless, there was a high concentration of hydroxytyrosol in the washing waters, and a fermentation process occurred in spite of the inhibitory effect of olive phenolics against lactic acid bacteria [10]. The decrease in the concentration with acidification was also observed for tyrosol, although the concentration of this compound in the washing waters was very much lower than that of hydroxytyrosol. Another two phenolic compounds detected at the beginning of storage were
caffeic acid and p-coumaric acid, although at very low concentrations, and after 1 year they were not detected (Table 2). As mentioned before, oleuropein is the main phenolic compound in the flesh of olives with surface changing colour, and this compound is hydrolysed during NaOH treatment into hydroxytyrosol and elenolic acid glucoside [8]. Thus, peaks corresponding to hydroxytyrosol and elenolic acid glucoside were the two largest in the three-dimensional chromatogram, with UV absorption maxima at 280 and 240 nm respectively. Therefore, if hydroxytyrosol is to be obtained from the washing wasters, the phenolic extract must not contain elenolic acid glucoside. Thus, elenolic acid glucoside and eleno-
Fig. 4. Changes in elenolic acid and elenolic acid glucoside in washing waters during fermentation. Pooled standard deviations for elenolic acid and elenolic acid glucoside were 1.7 and 1.4, respectively.
A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
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and the concentration of hydroxytyrosol decreased slightly. An acid hydrolysis of elenolic acid glucoside and elenolic acid occurred during storage. Thus, after a year of storage, washing waters were available containing a phenolic extract with a high concentration of the antioxidant hydroxytyrosol.
Acknowledgements Part of the research project, ALI-97-0646, was supported by the Spanish Government through CICYT
References Fig. 5. Plot of the Arrhenius equation relating temperature and rate of elenolic acid glucoside disappearance in washing waters acidified at pH 3. (r 2 =0.98).
lic acid changes during storage were followed (Fig. 4). The hydrolysis of elenolic acid glucoside was probably chemical, as it was more rapid with decreasing pH. It has been reported that lactic acid bacteria isolated from table olive fermentation may hydrolyse the glycosidic bond of oleuropein due to the action of b-glucosidase [15,16]. However, in these experiments elenolic acid glucoside was hydrolyzed even in treatments with no detected lactic acid bacteria. In fact, the hydrolysis of this compound during Spanish-style green table olive fermentation is a chemical process [9]. The disappearance of elenolic acid glucoside from the washing waters was slow; the same process originated elenolic acid, although this acid also hydrolysed as a consequence of the acid conditions. The hydrolysis rate of elenolic acid glucoside may be fitted to first order kinetics [9], and this reaction was affected to a large extent by temperature. A plot of 1/T against Ln k according to the Arrhenius equation is shown in Fig. 5. The activation energy was 0.142 KJ/mol. For comparative purposes, it must be stressed that hydrolysis lasted 1 and 8 months at 40 and 20°C respectively, without significant changes in hydroxytyrosol content.
4. Conclusions The results of this study indicate that washing waters from the Spanish-style green table olive process may be successfully fermented. A spontaneous fermentation occurred during storage which could be controlled by adjusting the initial pH. Lactic acid bacteria grew when the initial pH was higher than 4, and a significant concentration of lactic acid was formed. Under acid conditions, the colour of solutions increased slightly
[1] Borbolla JM, Rejano L. On the preparation of Sevillan style olives. The washing of fruits treated with lye. Grasas Aceites 1978;29:281– 91. [2] Sa´nchez AH, Garcı´a P, Rejano L, Brenes M, Garrido A. The effects of acidification and temperature during washing of Spanish-style green olives on the fermentation process. J Agric Food Chem 1995;68:197– 202. [3] Gonza´lez F, Rejano L, Dura´n MC, Sa´nchez F, Garcı´a P, Castro A, Garrido A. Preparation of Spanish green olives without washing waters. Solution to the problems caused by HCl additions and effects of treatments with weak lye solutions. Grasas Aceites 1984;35:155– 9. [4] Rejano L, Castro A, Gonza´lez F, Dura´n MC, Sa´nchez AH, Montan˜o A, Garcı´a P, Sa´nchez F, Garrido A. Influence of several treatments with hydrochloric acid on the elaboration of Spanish-style green olives. Grasas Aceites 1986;37:19– 24. [5] Baldioli M, Servili M, Perreti G, Montedoro G. Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. J Am Oil Chem Soc 1996;73:1589– 93. [6] Manna C, Galletti P, Cucciolla V, Moltedo O, Leone A, Zappia V. The protective effect of the olive oil polyphenol (3,4-dihidroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in caco-2-cells. J Nutr 1997;127:286– 92. [7] Capasso R, Evidente A, Avolio S, Solla F. A highly convenient synthesis of hydroxytyrosol and its recovery from agricultural waste waters. J Agric Food Chem 1999;47:1745– 8. [8] Brenes M, Rejano L, Garcı´a P, Sa´nchez AH, Garrido A. Biochemical changes in phenolic compounds during Spanish-style green olive processing. J Agric Food Chem 1995;43:2702–6. [9] Brenes M, Castro A. Transformation of oleuropein and its hydrolysis products during Spanish-style green olive processing. J Sci Food Agric 1998;77:353– 8. [10] Fleming HP, Walter WM, Etchells JL. Antimicrobial properties of oleuropein and products of its hydrolysis from green olives. Appl Microbiol 1973;26:865– 9. [11] Sayadi S, Allouche N, Jaoua M, Aloui F. Detrimental effects of high molecular-mass polyphenols on olive mill wastewater biotreatment. Process Biochem 2000;35:725– 35. [12] Chen KH, McFeeters RF, Fleming HP. Fermentation characteristics of heterolactic acid bacteria in green bean juice. J Food Sci 1983;48:962– 6. [13] Garrido A, Garcı´a P, Montan˜o A, Brenes M, Dura´n MC. Biochemical changes during the preservation stage of ripe olive processing. Die Nahrung 1993;37:583– 91. [14] Montan˜o A, Sa´nchez AH, Castro A. Controlled fermentation of Spanish-style green olives. J Food Sci 1993;58:842– 4. [15] Ciafardini G, Marsilio V, Lanza B, Pozzi N. Hydrolysis of
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oleuropein by Lactobacillus plantarum strains associated with olive fermentation. Appl Environ Microbiol 1994;60:4142– 7. [16] Marsilio V, Lanza B. Characterization of an oleuropein degrad-
.
ing strain of Lactobacillus plantarum. Combined effects of compounds present in olive fermenting brines (phenols, glucose and NaCl) on bacterial activity. J Sci Food Agric 1998;76:520–4.
Fermentation of washing waters of Spanish-style green olive processing Antonio de Castro, Manuel Brenes * Food Biotechnology Department, Instituto de la Grasa, CSIC, A6da. Padre Garcı´a Tejero 4, 41012 Se6illa, Spain Received 16 May 2000; accepted 3 December 2000
Abstract Washing waters of Spanish-style green olive processing are heavily contaminated waste streams that represent an important environmental problem that needs to be solved. These liquids are only generated for 2 months a year and a storage period is needed if high-value substances are to be extracted from them. Fermentation of the washing waters under acidic conditions was studied throughout a year. A lactic acid fermentation took place in vessels with either no initial correction of pH, or pH corrected to 4. When the pH was initially lowered to 3, only yeasts grew and a significant concentration of ethanol was generated. The concentration of phenolic compounds decreased slightly during the fermentation process, particularly hydroxytyrosol, which was found in high concentration in these waste waters. If this orthodiphenol is to be recovered from washing waters, time is required to hydrolyse the elenolic acid glucoside. The glucoside was hydrolysed under the acidic conditions of the washing waters, and the reaction was affected by temperature and pH. Thus, fermentation of washing waters under certain conditions may give rise to solutions with no off-odours and a high concentration of lactic acid and hydroxytyrosol, which are economically interesting products. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Table olive; Washing water; Hydroxytyrosol; Lactic acid; Fermentation
1. Introduction The worldwide production of table olives has been calculated to be 1 million t, half of which corresponds to Spanish-style green table olives. The procedure for preparing this type of table olives consists of treating the fruits with dilute NaOH solution to hydrolyse their natural bitterness (oleuropein), followed by one or two water washes to remove the excess alkali and, finally, a spontaneous lactic acid fermentation in brine for several months [1]. The washing solutions are heavily contaminated and contain a high content of sugars and phenolic compounds, particularly hydroxytyrosol (3,4dihydroxyphenyl ethanol) [2]. These solutions are not accepted in municipal sewers and two alternatives have been reported to diminish their volume: (i) elimination of the washing step [3]; and (ii) reuse of the solutions * Corresponding author. Tel.: +34-954690850; 954691262. E-mail address: [email protected] (M. Brenes).
fax:
+34-
after neutralising the residual alkali [4]. Neither method has been applied successfully on an industrial scale. It would be of interest to obtain high-value products from these washing waters, such as organic acids (lactic acid) and polyphenols, particularly hydroxytyrosol — an orthodiphenol with high antioxidant activity [5] — and nutritional properties [6]. In fact, hydroxytyrosol is also a possible product from olive oil mill waste waters [7]. The washing waters are only generated for 2 months a year and, therefore, must be stored until extraction of the phenolic substances. Storage in acid conditions is preferable, since it has been reported that hydroxytyrosol does not change at low pH during fermentation of Spanish-style green table olives [8]. In addition, the other part of the oleuropein molecule generated during NaOH treatment, the elenolic acid glucoside, is hydrolysed under these acidic conditions [9]. Temperature could be an important factor in this reaction. The elenolic acid glucoside is a component of the phenolic extract of washing waters and may interfere during the process of hydroxytyrosol extraction.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 8 0 - 6
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A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
The objectives of this work were to investigate different fermentation procedures to prevent the oxidation of hydroxytyrosol, to obtain the maximum quantity of lactic acid, and to avoid the appearance of off-odours. The evolution of elenolic acid glucoside and elenolic acid during storage at different temperatures in acid conditions was also studied.
2. Materials and methods
2.1. Fermentation procedures Olives of the Manzanilla cultivar were treated with 0.5 M NaOH solution for 7 h. The alkaline solution was poured off and the fruits washed in water for 12 h. Washing water (50 l) was placed in each of eight PVC cylindrical vessels and stored at ambient temperature for 1 year. Acid (6N HCl) was added to four of the vessels to adjust the initial pH of the washing water (ca 11) to 4 (two vessels) and 3 (two vessels). HCl was employed in another two vessels to lower the pH to 3 and sodium metabisulfite (0.4 g/l) was added. The remaining two vessels, without added acid, were used as controls. Special care was taken to reduce to a minimum the contact between the washing waters and the atmosphere and anaerobic conditions were used during experiments.
Washing water with the initial pH adjusted to 3 was also put into 330 ml jars which were sealed and stored at 15, 30, 40 and 50°C in thermostatic chambers. Samples were taken at regular intervals and experiments were run in duplicate.
2.2. Chemical analyses Sugars (glucose, fructose, sucrose and mannitol), alcohol (ethanol), organic acids (lactic, acetic and propionic), and polyphenols were analyzed by HPLC as described elsewhere [9]. The colour of washing waters was determined as the difference of absorbance at 440 and 700 nm (A440 − A700).
2.3. Microbiological analyses Samples from the different vessels and their decimal dilutions were plated using a Spiral System model DS (Interscience, Saint Nom La Breteche, France). Enterobacteriaceae were counted on Crystal-violet neutralred bile glucose agar (Merck), lactic acid bacteria on MRS agar (Oxoid), with and without 0.02% sodium azide (Sigma), and yeasts on OGYE agar (Oxoid). Plates were incubated at 32°C for 48 or 72 h. Samples were also inoculated into Differential Reinforced Clostridial Broth (Merck) and incubated at 37°C up to 14 days.
3. Results and discussion
Fig. 1. Evolution of glucose, fructose and mannitol in washing waters during fermentation. Pooled standard deviations for glucose, fructose and mannitol were 5.6, 3.2 and 4.3, respectively.
Despite the antibacterial activity of olive polyphenols [10,11], spontaneous fermentation of carbohydrates occurred throughout storage of the washing waters. In treatments with initial pH either uncorrected or adjusted to 4 the concentration of the glucose and fructose diminished very rapidly during the first 10–20 days (Fig. 1). At the same time, mannitol accumulated, a phenomenon which could be related to the fermentation route of fructose [12]. Mannitol is not well-assimilated by microorganisms usually found in olive brines [13] and, as can be observed in Fig. 1, a residual level of this compound was found even after 1 year of storage. In treatments with the initial pH lowered to 3, the consumption of glucose and fructose was slower than in the other treatments assayed. Nevertheless, the concentration of these carbohydrates was very low after 50– 100 days. Changes in carbohydrates were in parallel with growth of different microorganisms. Thus, Enterobacteriaceae were detected in treatments with no initial correction of pH only during the first weeks of storage. A maximum of 4.0× 108 CFU/ml was reached after 5 days, after which the population started to decline and these microorganisms were not detected after 19 days.
A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
Fig. 2. Lactic acid bacteria population in washing waters during fermentation. Pooled standard deviation for lactic acid bacteria was 1.1. Lactic acid bacteria were not detected when the initial pH was adjusted to 3.
Fig. 3. Yeast population in washing waters during storage. Pooled standard deviation for yeasts was 0.6.
Fig. 2 shows the evolution of lactic acid bacteria throughout the year of storage for treatments with
799
initial pH uncorrected or adjusted to 4; in those treatments in which the pH was initially lowered to 3 these microorganisms were not detected. The population of lactic acid bacteria was always higher in control treatments (no correction of pH) than in those with pH adjusted to 4. It must be stressed that the addition of HCl to the olive brines at the beginning of the fermentation process may delay growth of lactic acid bacteria [3]. However, maximum levels of ca 108 CFU/ml were reached after 10–30 days, corresponding to the high consumption rate of glucose and fructose in this period for treatments with initial pH higher than 4 (Fig. 1). From this maximum, the population started to decline throughout the year. Unlike the lactobacilli, yeasts were detected in all the vessels (Fig. 3), although their population was generally higher in treatments with initial pH corrected to 3. An initial delay was also detected when SO2 was added to the washing water. Viable spores of clostridia were detected in a few instances from almost all treatments during storage and were always present in samples from one vessel of the control treatment which started to develop abnormal odour. Lactic acid was generated only in those treatments with initial pH higher than 4 (Table 1), corresponding to lactic acid bacteria growth in these treatments (Fig. 2). The highest concentration was reached in treatments with the initial pH adjusted to 4, in spite of the fact that the Lactobacillus population in these treatments was lower than in those with no correction of the initial pH (Fig. 2). It needs to be stressed that fermentation was spontaneous and there was no characterization of the types of Lactobacillus in each vessel. Moreover, the level of lactic acid produced during fermentation was higher than that found during fermentation of Spanishstyle green table olive processing [14]. Acetic acid was detected in the washing waters at the beginning of the storage in concentrations lower than 16 mM. This could proceed from the NaOH treatment
Table 1 Changes of some physico–chemical characteristics during fermentation of washing waters of the Spanish-style green olive processing Storage method Control
pH 3
pH 4
pH 3 and SO2
Parameter
1 day
365 days
1 day
365 days
1 day
365 days
1 day
365 days
Lactic acid (mM) Acetic acid (mM) Ethanol (mM) pH Colour (A440−A700)
ND a 16(1) 26(2) 10.6 0.67(0.13)
119(33) b 169(2) 146(23) 4.3 0.74(0.04)
ND 12(2) 20(3) 3.0 0.48(0.02)
ND 19(1) 451(62) 2.7 0.58(0.06)
ND 15(1) 23(1) 4.0 0.70(0.06)
154(8) 135(18) 236(29) 3.4 0.73(0.03)
ND 11(1) 17(2) 3.0 0.35(0.04)
ND 29(4) 520(59) 2.8 0.42(0.05)
a b
Not detected. Standard deviation.
A. de Castro, M. Brenes / Process Biochemistry 36 (2001) 797–802
800
Table 2 Changes of phenolic compounds during fermentation of washing waters of the Spanish-style green olive processing Storage method Control
pH 3
pH 4
pH 3 and SO2
Compound
1 day
365 days
1 day
365 days
1 day
365 days
1 day
365 days
Hydroxytyrosol (mM) Tyrosol (mM) Caffeic acid (mM) p-coumaric acid (mM)
31.4(1.3) a 3.2(0.1) 0.4(0.1) 0.5(0.1)
29.3(2.1) 2.7(0.1) ND b ND
22.9(4.0) 2.4(0.4) 0.3(0.1) 0.3(0.1)
22.3(3.3) 3.0(0.2) ND ND
31.6(1.6) 3.0(0.2) 0.3(0.1) 0.5(0.1)
30.4(1.5) 3.0(0.2) ND ND
24.4(3.2) 2.4(0.2) 0.3(0.1) 0.4(0.1)
24.2(3.2) 2.0(0.4) ND ND
a b
Standard deviation. Not detected.
of the olives before the washing step, as shown in a previous work [14]. The concentration of the acid increased slightly in those treatments with the initial pH adjusted to 3, and reached a value of 140 – 170 mM in treatments with initial pH higher than 4. This acid may have been generated by either heterofermentative lactic acid bacteria or Enterobacteriaceae in the case of the control treatments. Another main end product was ethanol. The concentration of this compound increased in all treatments, although particularly in those with the initial pH lowered to 3 and in a higher proportion when sodium metabisulfite was used. It is obvious that yeasts were the microorganisms responsible for the high amount of ethanol formed in these treatments (Fig. 3). Acids and ethanol production, particularly lactic acid, decreased the initial pH of vessels, although the degree depended on the type of treatment (Table 1). Thus, in the control treatment this parameter diminished from 10.6 on the first day of storage to 4.3 after 365 days. The decrease was 0.6 and 0.2 – 0.3 units for treatments with initial pH adjusted to 4 and 3, respectively. The colour of the solutions (A440 −A700) slightly increased in all the treatments (Table 1). This effect could be attributed to the oxidation of hydroxytyrosol which decreased in concentration throughout storage (Table 2). There was also a noteworthly decrease in the initial concentration of hydroxytyrosol (1 day storage) when the washing waters were acidified to pH 3. There is no apparent explanation for this phenomenon other than it could be related to the precipitate that was formed. Nevertheless, there was a high concentration of hydroxytyrosol in the washing waters, and a fermentation process occurred in spite of the inhibitory effect of olive phenolics against lactic acid bacteria [10]. The decrease in the concentration with acidification was also observed for tyrosol, although the concentration of this compound in the washing waters was very much lower than that of hydroxytyrosol. Another two phenolic compounds detected at the beginning of storage were
caffeic acid and p-coumaric acid, although at very low concentrations, and after 1 year they were not detected (Table 2). As mentioned before, oleuropein is the main phenolic compound in the flesh of olives with surface changing colour, and this compound is hydrolysed during NaOH treatment into hydroxytyrosol and elenolic acid glucoside [8]. Thus, peaks corresponding to hydroxytyrosol and elenolic acid glucoside were the two largest in the three-dimensional chromatogram, with UV absorption maxima at 280 and 240 nm respectively. Therefore, if hydroxytyrosol is to be obtained from the washing wasters, the phenolic extract must not contain elenolic acid glucoside. Thus, elenolic acid glucoside and eleno-
Fig. 4. Changes in elenolic acid and elenolic acid glucoside in washing waters during fermentation. Pooled standard deviations for elenolic acid and elenolic acid glucoside were 1.7 and 1.4, respectively.
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and the concentration of hydroxytyrosol decreased slightly. An acid hydrolysis of elenolic acid glucoside and elenolic acid occurred during storage. Thus, after a year of storage, washing waters were available containing a phenolic extract with a high concentration of the antioxidant hydroxytyrosol.
Acknowledgements Part of the research project, ALI-97-0646, was supported by the Spanish Government through CICYT
References Fig. 5. Plot of the Arrhenius equation relating temperature and rate of elenolic acid glucoside disappearance in washing waters acidified at pH 3. (r 2 =0.98).
lic acid changes during storage were followed (Fig. 4). The hydrolysis of elenolic acid glucoside was probably chemical, as it was more rapid with decreasing pH. It has been reported that lactic acid bacteria isolated from table olive fermentation may hydrolyse the glycosidic bond of oleuropein due to the action of b-glucosidase [15,16]. However, in these experiments elenolic acid glucoside was hydrolyzed even in treatments with no detected lactic acid bacteria. In fact, the hydrolysis of this compound during Spanish-style green table olive fermentation is a chemical process [9]. The disappearance of elenolic acid glucoside from the washing waters was slow; the same process originated elenolic acid, although this acid also hydrolysed as a consequence of the acid conditions. The hydrolysis rate of elenolic acid glucoside may be fitted to first order kinetics [9], and this reaction was affected to a large extent by temperature. A plot of 1/T against Ln k according to the Arrhenius equation is shown in Fig. 5. The activation energy was 0.142 KJ/mol. For comparative purposes, it must be stressed that hydrolysis lasted 1 and 8 months at 40 and 20°C respectively, without significant changes in hydroxytyrosol content.
4. Conclusions The results of this study indicate that washing waters from the Spanish-style green table olive process may be successfully fermented. A spontaneous fermentation occurred during storage which could be controlled by adjusting the initial pH. Lactic acid bacteria grew when the initial pH was higher than 4, and a significant concentration of lactic acid was formed. Under acid conditions, the colour of solutions increased slightly
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