Influence of fermentation temperature on semi-continuous acetification for wine vinegar production

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1 Eur Food Res Technol (2001) 213:62 66 DOI /s ORIGINAL PAPER Giuseppe Fregapane Hipólito Rubio-Fernández María Desamparados Salvador Influence of fermentation temperature on semi-continuous acetification for wine vinegar production Received: 29 January 2001 / Published online: 18 May 2001 Springer-Verlag 2001 Abstract This paper describes semi-continuous acetic acid fermentations for wine vinegar production performed isothermally and using temperature gradients in order to determine the influence of temperature on this industrial biotransformation. Acetifications were conduced in 6- to 100-l reactors. Overall process productivity improved about 15 20% using a temperature gradient. An initial temperature of 32 C was employed until the product concentration reached g/l/h. Thereafter, the temperature was gradually reduced to 30 C until the end of the production cycle. Under these operating conditions, the 100-l acetator yielded an overall productivity of 1.84±0.03 g/l/h, a maximum acetification of 2.10±0.02 g/l/h, a 94.3±0.4% process yield, and a final acetic acid concentration of 116.0±0.9 g/l. Keywords Wine vinegar Acetic acid fermentation Fermentation temperature Introduction Wine vinegar possesses good sensory qualities and is a highly appreciated component of the diet in Mediterranean countries [1, 2]. This product is particularly important in the Castilla-La Mancha region of Spain, which produces more than 60% of all Spanish wine vinegar. In 1998 the figure was about 28 million liters (10% acetic acid). Nowadays, industrial wine vinegar is mainly produced by submerged culture in semi-continuous processes [3]. Although temperature is one of the variables that affects fermentation, little is known about its influence on the acetification process. In the 1950s a temperature of 30 C was established as the most suitable for industrial production of vinegar G. Fregapane ( ) H. Rubio-Fernández M.D. Salvador University of Castilla-La Mancha, Faculty of Chemistry, Department of Food Technology, Avda Camilo José Cela 10, Ciudad Real. Spain gfregapane@qata-cr.uclm.es (11 12% acetic acid). This is the temperature currently employed by the industry [3, 4]. Temperatures above 30 C tend to increase damage to bacteria due to the concentration of acetic acid in the fermentation medium. A much later study also confirmed 30 C as the optimum temperature for the maximum growth of Acetobacter aceti in discontinuous culture [5]. Only a few other investigations have been published in the last decades concerning this aspect of acetification technology. A two-stage acetification method was developed by Kunimatsu et al. in 1981 [6] and more recently by Higashide et al. (1994) [7] in an attempt to produce vinegar with a higher concentration of acetic acid. At the start of fermentation, the temperature was kept at about 30 C until the acetic acid concentration reached 12 15%, and then the temperature was gradually decreased to C to achieve higher acetic acid concentrations (greater than 18%) [6, 7]. A genetically modified Acetobacter strain was obtained by Fukaya et al. in 1989 [8] and a thermotolerant acetic acid bacterium was isolated in 1997 by Saeki et al. [9]. These could be used for vinegar fermentation at C; however the final acid concentration reached was less than 70 g/l and process productivity was too low for industrial purposes. The aim of the present work was to investigate the effect of temperature on acetic acid fermentation for wine vinegar production. To this end semi-continuous acetifications were conduced isothermally and with a temperature gradient in 6- to 100-l reactors. Materials and methods Strain. An industrial culture obtained from a local vinegar factory (Vinagres Parras, S.A., Torrijos, Toledo, Spain) was used. It was maintained on a liquid medium (30:70 wine/vinegar mixture) and stored in 20% (v/v) glycerol at 50 C. Growth medium. A commercial white table wine of the Airen variety was used as the growth medium. It was obtained directly from a local producer and had the following composition: 13 (%, v/v) alcohol, corresponding to 103 g/l ethanol; 4.7 g/l total acidity as tartaric acid; 0.8 g/l volatile acidity as acetic acid; ph 3.4; 0.5 g/l

2 63 residual sugars, glucose, and fructose; and 70 ppm total SO 2, which was removed by bubbling through air. Semi-continuous fermentation. A 6-l and a 100-l non-commercial bubble column reactor were used as described elsewhere [10]. After the acetification cycle was complete, about 40% of the fermented broth (1.6 or 28 l for the 6- and 100-l acetator, respectively) was discharged as end product vinegar, at an acetic acid concentration close to 115 g/l. The same volume of fresh wine was then loaded into the fermentor using a peristaltic pump (flow 1 or 20 l/h), and a new production cycle was begun. Initial fermentation conditions for the wine/vinegar mixture (40:60), were 47±2 g/l ethanol and 70±2 g/l acetic acid. The temperature of the fermenting broth was maintained (±0.5 C) by circulating cool water through an internal stainless steel coil. Oxygen probes (Syland Scientific Gmbh, Heppenheim, Germany) inserted in the vessel and in the recirculation line were used to monitor dissolved oxygen concentration as an easy means of following the course of fermentation over time. In all fermentations aeration was 0.25 min 1 as vvm (air flow /reactor working volume ratio). Five to seven fermentations were carried out for each experiment in the 6-l laboratory fermentor, and three to four in the 100-l acetator. Polynomial regression (second order) was used to fit fermentation curves (instantaneous acetification vs actual acetic acid concentration; r >0.98) shown in Figs. 1 and 3. Isothermal and temperature gradient fermentations. In order to study the influence of the fermentation temperature on semi-continuous acetification for wine vinegar production, isothermal and temperature gradient fermentations were carried out. Isothermal fermentations were performed at two degree intervals in a temperature range of C. Temperature gradients were used to reduce the decline in the acetification during the second half of the fermentation process. These experiments were conduced under two sets of conditions. In the first case the fermentation temperature was maintained at 32 C until an acetic acid concentration of 95 g/l was reached, after which the temperature was gradually reduced to 30 C in about 2 h and held there until the end of the process. In the second case, a two-step temperature gradient was employed using an initial temperature of 34 C which was gradually reduced to 32 C and then to 30 C when the concentration of acetic acid in the fermentation medium reached 80 and 95 g/l, respectively. Assays. Samples of a few milliliters were taken every 1 3 h from the fermentors during the acetification, and acetic acid, ethanol, ph, and optical density were determined. Acetic acid was measured by titration with phenolphthalein as an indicator. It was assumed that all broth acidity was due to acetic acid. Ethanol was enzymatically assayed by kit No (Boehringer Manheim, Barcelona, Spain). The biomass was monitored by measuring the absorbance within a linear range at 575 nm. A previously prepared calibration curve was used to convert the absorbance readings into biomass dry weight. The yield of the process was calculated as in the vinegar industry, that is as the quotient of the final and initial total concentration, where total concentration is defined by the sum of the alcohol (% v/v or ) and acetic acid (g/100 ml) concentrations: 1 alcohol provides g/100 ml of acetic acid in this type of fermentation. Statistical analysis. Statistical analysis was performed using the SPSS 9.0 statistical software (SPSS Inc., Chicago, USA). Results Fig. 1 Influence of fermentation temperature on the instantaneous acetification in semi-continuous acetifications (n 5) performed in the 6-l reactor. Experimental points were omitted to increase clarity; fitted curves are displayed Reproducibility of main acetification parameters (i.e., acetic acid productivity and final acetic acid concentration) was good (coefficient of variation often <6%) and mean values, standard deviation, and statistical significance (one-way ANOVA and Duncan s comparison test) are shown in the tables and throughout the text. Isothermal fermentation At 32 and 34 C acetification s were higher than at 30 C (the temperature currently used by the industry) during the stage of the fermentation up to an acetic acid concentration of g/l (Fig. 1). However, a reduction in the acetification was observed during the final stage of the process. Overall acetic acid productivity (AcHp) improved significantly at higher temperatures, in the range C, from 1.21±0.07 (mean±sd) to 1.69±0.08 g/l/h, respectively (p<0.001; Table 1). However, at temperatures over 34 C process productivity decreased: 1.26±0.05 g/l/h at 36 C (p<0.001 compared to productivity at 34 C) and insufficient growth to start an acetification cycle was observed at 38 C. The same behavior was observed for the maximum acetification (AcHp max ), in which the highest mean value was 2.2 g/l/h at 32 C. The process time decreased with productivity, from 36 h at 26 C to 25 h at 32 C (p<0.001), although the time at 36 C was 31 h. No lag phase was observed in the given acetification conditions. The pro-

3 64 Table 1 Semi-continuous acetifications (n 5) performed isothermally at different temperatures in a 6-l laboratory reactor Temperature ( C) AcH p (g/l/h) 1.21±0.07 a 1.50±0.08 b 1.52±0.06 b 1.69±0.08 c 1.68±0.03 c 1.26±0.05 a AcH p max (g/l/h) 1.61±0.09 a 2.00±0.10 b 2.15±0.07 c 2.19±0.06 c 2.06±0.07 b,c 1.60±0.04 a Process time (h) 36.2±1.6 a 29.2±1.4 b,c 28.5±1.2 c 24.7±0.3 d 25.6±0.9 d 31.1±0.5 b Yield (%) 90.9±1.1 a 91.6±0.6 a 90.9±0.8 a 91.1±0.3 a 90.6±0.9 a 88.5±0.5 b final AcH (g/l) 113.8±1.2 a 113.6±1.4 a,b 113.4±1.4 a,b 112.5±1.3 a,b 111.5±0.9 b,c 109.9±0.9 c final EtOH (g/l) 3.8±1.5 a,c 4.8±0.7 a,b,c 4.2±0.8 a,b,c 4.0±0.5 a,c 4.7±0.6 a,b,c 5.5±0.8 b Biomass/EtOH met (mg/g) 2.33±0.22 a,b 2.18±0.44 a,b,c 2.17±0.40 a,b,c 1.98±0.42 a,b,c 1.76±0.14 c 1.16±0.01 d EtOH met, metabolized ethanol a,b,c,d Values with different letters are statistically different (p 0.001) Table 2 Semi-continuous acetifications (n 5) performed with a temperature gradient in the 6-l laboratory reactor Temperature gradient AcH p (g/l/h) 1.83± ±0.04 p 30 <0.001 <0.001 p AcH p max (g/l/h) 2.43± ±0.06 p 30 < p Process time (h) 24.0± ±0.7 p 30 <0.001 <0.001 p Yield (%) 91.6± ±0.1 p p final AcH (g/l) 114.0± ±0.4 p p final EtOH (g/l) 4.4± ±0.2 p p Biomass/EtOH met (mg/g) 1.66± ±0.20 p p Fig. 2 Arrhenius plot for the initial acetification of isothermal fermentations carried out in the temperature range C in the 6-l reactor EtOH met, metabolized ethanol; p 30, p-values of the statistical difference with respect to the 30 C isothermal fermentation (from Table 1) p 32 30, statistical difference p-value with the temperature gradient fermentation cess yield was close to 91% for all experimental temperatures, except at 36 C, where it was significantly lower (89%). In the temperature range between 26 and 34 C, the final concentration of acetic acid was about g/l and the residual ethanol at the end of fermentation was g/l, corresponding to ethanol (vol.%). The biomass-subst yield coefficient based on metabolized ethanol (Biomass/EtOH met [mg/g]) decreased with temperature from 2.33 mg/g at 26 C to 1.76 mg/g at 34 C (p=0.013). The initial acetification of these isothermal fermentations followed an Arrhenius-type pattern in the range C, with a calculated activation energy of J/mol (Fig. 2). Temperature gradient fermentation As reported in Table 2, overall acetic acid productivity was about 20% greater using a temperature gradient (1.83±0.05 g/l/h at C) as compared to constant temperature fermentation of 30 C (1.50±0.06 g/l/h; p<0.001). The final acetic acid concentration was similar (113.6±1.4 and 114.0±1.0 g/l at 30 and C, respectively; p=0.357) and the process yield was slightly higher (90.9±0.8 and 91.6±0.3, respectively; p=0.045). Only minor differences, which were not statistically significant with the exception of maximum acetification, were observed between the two temperature gradients.

4 65 Table 3 Semi-continuous acetifications (n 3) performed under different temperature conditions in the 100-l reactor Temperature ( C) AcH p (g/l/h) 1.62± ± ±0.03 p p AcH p max (g/l/h) 2.05± ± ±0.02 p p Process time (h) 29.0± ± ±0.5 p <0.001 p Yield (%) 94.5± ± ±0.4 p p final AcH (g/l) 115.2± ± ±0.9 p p final EtOH (g/l) 6.3± ± ±0.5 p p Biomass/EtOH met 1.41± ± ±0.15 (mg/g) p 30 <0.001 <0.001 p EtOH met, metabolized ethanol p 30, p-values of the statistical difference with respect to the 30 C isothermal fermentation p 32, statistical difference p-value with the 32 C fermentation Isothermal (at 30 and 32 C) and temperature gradient (32 30 C) acetifications were also conduced in a 100-l pilot acetator. Results of these experiments confirmed the behavior observed with the 6-l reactor and are reported in Table 3 and Fig. 3. Overall acetic acid productivity increased about 15% from 1.62±0.06 under isothermal fermentation conditions at 30 C to 1.84±0.03 g/l/h using C (p=0.001) temperature gradient acetification with the process time reduced from 29 h at 30 C to 24.5 h at C (p<0.001). No statistically significant differences were observed for the yield or the final concentration of acetic acid. The observed reduction in the acetification during the second stage of the fermentation process was lower using a temperature gradient than with isothermal fermentation at 32 C (Fig. 3). Discussion These results indicate that at fermentation temperatures higher than 30 C (the temperature currently used by industry) and up to 34 C, acetification s were higher during the first stage of acetification, up to an acetic acid concentration of about g/l. Despite a decrease in the acetification during the second stage of fermentation, acetification processes performed at 32 and 34 C increased overall productivity by 10%, from 1.52±0.06 to Fig. 3 Influence of the temperature on semi-continuous acetifications (n 3) performed under different fermentation temperature conditions in the 100-l acetator: 30 ( ), 32 ( ), and C ( ) temperature conditionsn 1.69±0.08 g/l/h at 30 and 32 C, respectively (p<0.001), with practically the same process yield and final acetic acid concentration (p=0.668 and 0.220, respectively). Fermentation s were slightly better at 32 C than at 34 C. At 36 C the initial acetification decreased, probably because the acetic acid bacteria are more sensitive to the product of biotransformation at higher temperature. Although reactor makers recommend a fermentation temperature of 30 C, several vinegar factories are currently conducting acetification at 32 C. Under isothermal fermentation conditions at 30 C, our 100-l acetator achieved an overall productivity similar to that of the Frings acetator ( g/l/h) [11] and greater productivity than other fermentors currently used in Spanish wine vinegar factories ( g/l/h) [12]. The process yield and the final product concentration were also similar to those obtained in industry. Based on our isothermal fermentation results, we conducted acetifications using a temperature gradient in an attempt to improve further process productivity. Under these operating conditions, overall process productivity increased 15 20%. The 100-l acetator yielded an overall productivity of 1.84±0.03 g/l/h, a maximum acetification of 2.10±0.02 g/l/h, a 94.3±0.4% process yield and 116.0±0.9 g/l final acetic acid. Temperature gradients in industrial acetification for wine vinegar production are very easy to implement and require very low plant or reactor modifications. The vin-

5 66 egar industry would benefit from application of these fermentation temperature conditions by improved overall process productivity and reduced expenses incurred in cooling the fermenting vinegar. While the 2 C temperature increase during the first stage of the proposed acetification process might increase oxidation and loss of flavor compounds important in the quality of wine vinegar, the use of a temperature gradient will reduce the fermentation time. Therefore, it would be of interest to determine the sensory quality of the finished product. For these results to be generally applicable, it is necessary to verify that the temperature-related behavior is similar to that in other industrial strains (currently under investigation). However, there is partial confirmation from the fact that several vinegar factories use a temperature of 32 C. Furthermore, this study is one of the few that have considered the effect of this variable on the acetification process and it is the first to suggest that a simple temperature gradient could significantly improve acetification performance. Acknowledgements The authors would like to thank the following wine vinegar factories for the valuable information provided: Vinagres Parras, S.A. (Toledo), Vulpi, S.L. (Toledo), El Encierro, S.L.(La Rioja), Mueloliva, S.A. (Córdoba), and Thor, S.A. (Madrid). References 1. Carnacini A, Gerbi V (1992) Vitic Enol Sci 47: Gonzalez-Viñas MA, Salvador MD, Cabezudo MD (1996) J Sensory Studies 11: Adams MR (1985) Vinegar. In: Wood BJB (ed) Microbiology of fermented foods. Elsevier Applied Science Publishers, New York, pp Allgeier RJ, Hildebrandt FM (1960) Adv Appl Microbiol 11: De Ory I, Romero LE, Cantero D (1998) Appl Microbiol Biotechnol 48: Kunimatsu Y, Okumura H, Masai H, Yamada K, Yamada M (1981) Production of vinegar with high acetic acid concentration. U.S. Pat 4,282, Higashide T, Okumura H, Kawamura Y, Hismatsu M, Yamada T (1994) Nippon Shokuhin Kogyo Gakkaishi 41: Fukaya M, Tagami H, Tayama K, Okumura H, Kawamura Y, Beppu T (1989) Agric Biol Chem 53: Saeki A, Theeragool G, Matsushita K, Toyama H, Lotomg N, Adachi O (1997) Biosci Biotech Biochem 61: Fregapane G, Rubio-Fernandez H, Nieto J, Salvador MD (1999) Biotechnol Bioeng 63: Sokollek SJ, Hammes WP (1997) System Appl Microbiol 20: Llaguno C, Polo MC (1991) El vinagre de vino. Consejo Superior de Investigaciones Cientificas, Madrid

Wine Vinegar Production Using a Noncommercial 100-Litre Bubble Column Reactor Equipped with a Novel Type of Dynamic Sparger

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