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A novel culture medium for Oenococcus oeni malolactic starter production
LWT - Food Science and Technology 64 (2015) 25e31
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A novel culture medium for Oenococcus oeni malolactic starter production Carmen Berbegal, Yaiza Benavent-Gil, Isabel Pardo, Sergi Ferrer* Enolab, ERI BioTecMed/IViSoCa, University of Valencia, Dr. Moliner 50, Burjassot, Valencia 46100, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2015 Received in revised form 12 April 2015 Accepted 13 May 2015 Available online 22 May 2015
Malolactic fermentation is essential in wine quality. One of the strategies used to control this fermentation involves the inoculation of selected lactic acid bacteria, mainly Oenococcus oeni. Laboratory media usually produce large amounts of biomass, but with little or no adaptability to wine. We propose a culture medium to grow and pre-adapt O. oeni cells, and the steps to scale-up production. To achieve this objective, 27 different media were tested. All contained grape must and wine, and nutritional supplements in order to benefit bacterial growth. Those media contained different ethanol levels, pH values, and grape must concentrations. The optimized culture medium named Oenococcus Production Medium (OPM) contained diluted commercial 4X concentrate white must (1:6), ASv ¼ 4%, and a pH of 3.8. The total time to obtain 80 L of an O. oeni liquid starter culture from the stock culture at the laboratory, with a final population of CFU ¼ 1 109 mL1, was 22 d. The starter culture was efficiently scaled-up, and preserved at 4 C, 20 C, or freeze-dried. This new culture medium also allowed adaptation of bacteria to the wine conditions, consuming all malic acid (3 g/L) in 7 d with an inoculum of CFU ¼ 1 106 mL1. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Oenococcus oeni Malolactic fermentation Malolactic starter culture Scale-up Production medium
1. Introduction The main value of malolactic fermentation (MLF) in vinification is biological de-acidification, which results from the transformation of L-malic acid into L-lactic acid by lactic acid bacteria (LAB), mainly by Oenococcus oeni (Lonvaud-Funel, 1999; Versari, Parpinello, & Cattaneo, 1999; Wibowo, Eschenbruch, Davis, Fleet, & Lee, 1985). MLF induces an increase in pH, contributes to microbiological stability, and changes wine taste (Davis, Wibowo, Fleet, & Lee, 1988; Kunkee, 1991; Maicas, Gil, Pardo, & Ferrer, 1999). MLF can be produced by two strategies: spontaneously or by inoculation with starter cultures. Traditionally, spontaneous and indigenous LAB carried out a natural MLF process. But this MLF method can take weeks or months and is uncontrolled (Zhang & Lovitt, 2006). The induction of MLF by using a starter culture ensures faster fermentation, reduces potential spoilage by other LAB, and allows the improvement of wine quality when using selected bacterial strains ~ a, Patchett, Liu, & Pilone, 2001; Pozo-Bayo n et al., (Mira de Ordun
Abbreviations: AUC, area under the curve; LAB, lactic acid bacteria; MLF, malolactic fermentation; OPM, Oenococcus production medium. * Corresponding author. Tel.: þ34 963 544 518. E-mail address: [email protected] (S. Ferrer). http://dx.doi.org/10.1016/j.lwt.2015.05.020 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
2005). Nevertheless, successful inoculation of the starter into wine depends not only on the suitable strain of O. oeni, but on the preparation and use of the cultures. For the production of a suitably large biomass, selected strains of O. oeni are grown under conditions that permit rapid growth and result in a high cell yields, but these conditions are very different to those present in wine. Consequently, when the starter culture is inoculated directly into wine, it loses much viability (Henick-Kling, 1988). O. oeni is usually grown in the laboratory in complex culture media such as MRS (De Man, ~ iga, Ferrer, & Pardo, 1994), or Rogosa, & Sharpe, 1960), MLO (Zún AGB (Dicks & van Vuuren, 1990). Most of them frequently contain grape juice (Davis, Wibowo, Eschenbruch, Lee, & Fleet, 1985) or apple juice (Champagne, Gardner, & Lafond, 1989). We prefer to use tomato juice instead, as it is a source of pantothenic acid, commonly used as a growth factor for wine bacteria (Amachi, Imamoto, & Yoshizumi, 1971; Richter, Vlad, & Unden, 2001; Terrade & Mira de ~ a, 2009). The culture media are generally supplemented Ordun with other nutrients like yeast extract, peptone, and Tween 80, to increase biomass production (Champagne, Gardner, & Doyon, 1989; Guerrini, Bastianini, Granchi, & Vincenzini, 2002; Krieger, Hammes, & Henick-Kling, 1990; Kunkee, 1974; Pilone & Kunkee, 1970). The growth of O. oeni has been also investigated in single sugars and their mixtures. The best growth was obtained with sugar mixtures (glucoseefructose) rather than growth on a single sugar (Maicas,
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lez-Cabo, Ferrer, & Pardo, 1999; Ferrer, & Pardo, 2002; Maicas, Gonza Zhang & Lovitt, 2005a). Other components like manganese or yeast mannoproteins and nutrient requirements have been studied € nig, (Stamer, Albury, & Pederson, 1964; Theobald, Pfeiffer, & Ko 2005). Results revealed that the essential bacterial nutrients were strain-specific and O. oeni strains showed a large number of auxotrophies (Diez, Guadalupe, Ayestar an, & Ruiz-Larrea, 2010; Terrade ~ a, 2009; Terrade, Noel, Couillaud, & De Mira & Mira de Ordun ~ a, 2009; Theobald et al., 2005). Hayman and Monk (1982) Ordun evaluated the effect of adding wine to a medium for the production of O. oeni biomass, and found that a content of 40e80% wine in the medium improved LAB survival and malolactic activity. Nevertheless, further studies are necessary to find a medium for biomass production with a cheap easy recipe that allows any O. oeni strain to grow and adapt before being inoculated into wine. The objectives of this work were to develop a liquid O. oeni production medium (OPM) that would permit high levels of biomass production, but also an adequate pre-adaptation to wine conditions, and evaluate cell viability and malolactic activity of the MLF starter culture in wine. Preservation conditions for the liquid starter culture were also studied. 2. Material and methods 2.1. Microorganisms O. oeni strains E5003, E5067, E5259, and E5245 were taken from the Enolab culture collection of the University of Valencia (Spain). All strains had been previously isolated from spontaneous mid-MLF in Tempranillo wines from the Ribera del Duero region (Spain). 2.2. Growth in biomass production media ~ iga, Pardo, & O. oeni strains were pre-grown in MLO broth (Zún Ferrer, 1993) to early stationary phase. Cells were then centrifuged (rotational speed of 8000 min1, 10 min), washed and transferred to the 27 culture media (Table 1) at a final concentration of CFU ¼ 1 106 mL1. The base of all these media contained: yeast extract (5 g/L), commercial tomato juice with no preservatives (23 mL/L), Tween 80 (0.5 mL/L), L-malic acid (3 g/L) and concentrate reconstituted white wine (glucose 0.13 g/L, fructose 0.14 g/L, L-malic acid 0.22 g/L, free SO2 1 mg/L, ethanol ASv ¼ 0%) from Agrovin S.A. (Spain) (400 mL/L). This basal medium was supplemented with 4X concentrated white grape must (glucose 360 g/L, fructose 360 g/L, Lmalic acid 5.3 g/L, free SO2 1.4 mg/L) from Agrovin S.A. (Spain), which was diluted 4, 6 and 8 times (576.5 mL/L) and the pH was adjusted to 3.8, 4 and 4.5. Media were sterilized by autoclave at 115 C, 30 min. After sterilization, ethanol degree was adjusted at ASv ¼ 4%, ASv ¼ 6% and ASv ¼ 8%. All growth studies were carried out in duplicate, in 10 mL screw cap tubes and incubated at 28 C, 7 d. Growth was monitored by measuring optical density at 600 nm (OD600) with a spectrophotometer (CECIL, CE 373). The bacterial growth curve was transformed into a value of area under the curve (AUC) (Gagnon & Peterson, 1998). 2.3. Malolactic fermentation in red wine The cultures were inoculated into red wine at a final concentration of CFU ¼ 1 106 mL1, and MLF was monitorized for 15 d. All fermentations were carried out in duplicate. The red wine was made at the laboratory using a red Tempranillo grape must, inoculated with S. cerevisiae Viniferm B4 (Agrovin S.A.) at a final concentration of CFU ¼ 2 106 mL1, and was incubated at 28 C, 10 d, until the AF was finished. When the sugar concentration was lower than 1 g/L, the ethanol was adjusted to ASv ¼ 11%, ASv ¼ 12%,
Table 1 Combination of diluted 4X grape must (1/4, 1/6 or 1/8), ethanol content (ASv ¼ 4%, ASv ¼ 6% or ASv ¼ 8%) and pH level (3.8, 4 or 4.5) resulted in 27 different media for the O. oeni biomass production. Medium
Diluted must
Ethanol %
pH
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/6 1/6 1/6 1/6 1/6 1/6 1/6 1/6 1/6 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8
4 4 4 6 6 6 8 8 8 4 4 4 6 6 6 8 8 8 4 4 4 6 6 6 8 8 8
3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5
ASv ¼ 13% and ASv ¼ 14%, 3.0 g/L of malic were added, and the pH was adjusted to 3.5. Then, it was sterilized by filtering through 0.22 mm pore filter and stored at 15 C until use. 2.4. Viable cell counts Cell viability in red wine was studied by plate counting. The volume of 0.1 mL of decimal serial dilutions in sterile saline solution ~ iga et al., 1993) were spread in duplicate on MLO agar plates (Zún and were incubated at 28 C for 7 d and then the colonies were counted. 2.5. Culture preservation Freeze-drying was performed after growing the bacteria until the end of the exponential phase in 50 mL of OPM medium. Cells were recovered by centrifugation at rotational speed of 6000 min1, 15 min in a Heraeus Multifuge 1 S-R centrifuge. Then, cells were washed twice with glutamic acid 0.98%, recovered with the same above centrifugation conditions, and resuspended in 2 mL of 0.98% glutamic acid. The bacterial solution was distributed in aliquots of 400 mL per tube. Tubes were frozen at 20 C, 1 h. Freeze-dying was performed at 60 C for 18 h under vacuum (Virtis Sentry). Tubes were vacuum sealed and stored at 4 C under dark. Refrigeration at 4 C and freezing at 20 C were performed after growing O. oeni until the exponential phase in OPM medium and later transferring aliquots to the respective temperature. Bacterial cultures were preserved and checked for viability up to 250 d. 2.6. Analytical methods Glucose, fructose, malic acid and ethanol were quantified by high-pressure liquid chromatography (HPLC) (Agilent series 1200) with an isocratic pump (Agilent G1310A) following the procedure described by Frayne (1986) with minor modifications. The mobile phase consisted of a solution of 0.75 mL of 85% H3PO4 per litre of
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deionized water with a flow rate of 0.7 mL/min. An Agilent G1322A degasser was employed. Samples (5 mL) were injected automatically (Agilent G1367B). The components were separated using an Aminex HPX-87H precolumn (Bio-Rad) coupled with two ion exclusion columns of 300 mm by 7.8 mm Aminex HPX-87H (BioRad) thermostatically controlled at 65 C (Agilent G1316A). Compounds were detected by a G1314B variable-wavelength detector (Agilent) set to 210 nm and a refractive index detector (Agilent G1362A) in series. Elution time was 45 min. External calibration was performed. All samples were centrifuged (rotational speed of 8000 min1, 10 min) and filtered through a membrane filter with a mean pore size of 0.22 mm before use. Quantification was performed by measuring peak height compared to external standards. 2.7. Scale-up of biomass production The scale up started with a culture of CFU ¼ 1 106 mL1 of O. oeni in 50 mL of liquid OPM medium incubated statically at 28 C. When the bacterial population reached the maximum biomass yield, the 50 mL were inoculated into 0.5 L of OPM sterile medium, and were incubated at 28 C until the bacteria population reached the maximum biomass yield. Then, the O. oeni culture was inoculated into the 10 L fermenter with 8 L of OPM sterile medium and was incubated stirred at room temperature. The 0.5 L and the 10 L fermenter were built using Pyrex bottles with a screw cap GL 45 with three ports GL 14 thread (Duran). In the 0.5 L fermenter, one of these ports was closed with a screw cap GL 14, the second one was connected to a set for pressure compensation (Duran) to permit fermentation gases to vent out, and the third one was connected with a screw Cap GL 14 for hose connection (Duran) with a joint silicone diameter 0.6 mm and a silicone tube of 3 mm 6 mm. This silicone tube was joined to one of the ports in the PVDF Luer lock 3 way valve (Cole Palmer) of the 10 L fermenter. In the 10 L fermenter, one of these three ports was connected to a pressure compensation set (Duran) to enable fermentation gases to vent out, and the other two were connected with a screw cap GL 14 for hose connection (Duran) to two silicone tubes. One of these tubes was used to inoculate the starter from the 0.5 L fermenter, and the other one was used to collect samples with a 50 mL Luer lock syringe. The industrial biomass production process was carried out inoculating the 10 L of the O. oeni culture into a fermenter with 80 L of OPM sterile medium (Bioprocess technology Bio-pro 100 L). The inoculated culture was incubated at 28 C until the bacteria population reached CFU ¼ 1 109 mL1. A microbiological analysis of the three scale-up steps was carried out to certify the correct sterilization of the culture medium. After the sterilization of 50 mL and 0.5 L fermenter of OPM medium by autoclave, 115 C 30 min, and the 10 L and 100 L fermenters, 115 C 45 min, 0.1 mL sample from each ~ iga fermenter were plated on MRS (De Man et al., 1960), MLO (Zún et al., 1993) and YPD (Landete, Ferrer, & Pardo, 2007) media. The plates were incubated at 28 C for 7 d. 2.8. Statistical analysis Statistical significance of differences between averages of duplicate measurements were evaluated by performing one-way ANOVA at a confidence level of p ¼ 0.05 and Tukey's Studentized Range Test. 3. Results and discussion 3.1. Growth in biomass production media O. oeni strain E5003 was selected to carry out the assays because it presented interesting technological features as malolactic starter
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(Berbegal, 2014). The culture medium designed to grow bacteria contained mainly grape must and wine in order to adapt cells to the harsh conditions of wine. White wine and must were used because the sterilization of red must and wine caused precipitates, and also turbidity made it difficult to monitor bacterial growth properly. Commercial wine concentrate and grape must were used to standardize the culture medium. When the white concentrate must was diluted 4 times (media 1e9), the growth was lower than when the must was diluted 6 (media 10e18) or 8 (media 19e27) times (Fig. 1). Between must diluted 6 or 8 times differences were smaller, although there was slightly better growth with the must diluted 6 times. The best growth was obtained with ASv ¼ 4% (1e3, 10e12, 19e21), and the worst results were found with ASv ¼ 8% (7e9, 16e18, 25e27). In all cases, growth with a pH of 4.5 resulted in the highest value of AUC followed by growth with a pH of 4, and the lowest value was obtained with a pH of 3.8. It was observed that at lower pH, more time was needed to reach to the same OD600 nm. The best results were found, as expected, when the must was diluted 6 times with the lowest ethanol content (ASv ¼ 4%) and with the highest pH (4.5) (media 12), and the worst growth was observed with the must diluted 4 times, the highest ethanol content (ASv ¼ 8%) and 3.8 pH (medium 7) (Fig. 1). Results were analyzed using an ANOVA test. This analysis revealed a significant effect (p < 0.05) for must, ethanol, pH and for the interactions musteethanol and mustepH. Through the Tukey post hoc test, the media with the lowest AUC were rejected (1, 2, 4, 5, 7, 8, 16, 17, and 25). All these media obtained an AUC below 2, and their pH levels were 3.8 or 4. The cultures from the 18 remaining media were selected and their malolactic activity in wine was studied. HPLC analysis showed that the must concentrate contained around 360 g/L of glucose and 360 g/L of fructose. When must concentrate was diluted 4 times bacterial growth was lower than when the must was diluted 1/6 or 1/8 probably because of the osmotic pressure. Composition of OPM medium was based on previous studies of growth, metabolism and biomass production of O. oeni. Champagne, Gardner, and Lafond (1989) studied the production of O. oeni biomass in apple juice media and grape juice media. Their results showed that there were variations between strains in their ability to grow, and the optimum pH was also strain dependent, varying from 4.5 to 4.8. Yeast extract supplementation was the most beneficial for growth. In our study, 5 g/L of yeast extract was added to the media and a range of pH was studied but always below 4.8 because this value was too high, compared to the wine pH. Also, malic acid was added to stimulate LAB growth and MLF activity (Terrade et al., 2009). The studies of Zhang and Lovitt (2005a) and lez-Cabo, et al. (1999) Maicas et al. (2002) and Maicas, Gonza showed that better growth of O. oeni was obtained in the presence of sugar mixtures (glucose:fructose, 1:1) in the culture medium compared to glucose alone. The optimal growth of O. oeni was at pH 4.5, with the maximum biomass yield and specific growth rate. In the present work, the highest biomass also was obtained when the bacterium was grown at pH 4.5 and the medium contained a sugar mixture of glucose:fructose (1:1). 3.2. Malolactic activity in red wine All the cultures from media with a value of AUC above 2 (Fig. 1) were inoculated in red wine with ASv ¼ 12% and pH 3.5. Some differences were found in malolactic activity, and 5 cultures (10, 13, 19, 22, and 26) consumed all the malic acid in 6 d (Fig. 2). These differences in malolactic activity depended mainly on the pH of the medium where the bacteria were grown before. The cultures that were grown in a medium with a pH of 4.5 resulted in a slower
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Fig. 1. Values of O. oeni E5003 area under the curve (AUC) in the 27 different culture media ( media is described in Table 1.
must diluted 1/4,
must diluted 1/6,
must diluted 1/8). The code number of the
Fig. 2. Malic acid consumption (%) in red wine after 6 d of incubation of the O. oeni strain E5003 previously grown in the different culture media ( must diluted 1/4, 1/6, must diluted 1/8). The code number of the media is described in Table 1.
consumption of malic acid in red wine. The best results of activity in wine were found when O. oeni strain E5003 was previously grown in a medium with a pH of 3.8. This can be correlated to the fact that this pH was more similar to the wine pH (3.5), so the cells were more adapted to it. Out of these 5 media, 4 of them (10, 13, 19 and 22) had been grown in a pH of 3.8, proving the importance of the pH as a growth factor in the culture media for MLF in wine later on. The culture with the highest biomass in the culture medium was selected for further studies. Medium 10 had the highest value of area under the curve (2.44 AUC) (Fig. 1) so this medium was chosen to be the OPM. That was the medium with the must diluted 6 times, with an ASv ¼ 4% and a pH of 3.8 (Table 2). HPLC analysis of this medium showed around 40 g/L of glucose, 40 g/L of fructose, and 3.5 g/L of malic acid. The OPM medium contains must and wine and pH is 3.8, so growth conditions are similar to wine conditions, cells are adapted to wine and do not need successive transfers before inoculation. The starter culture kept high cell viability and malolactic activity when it was inoculated in wine.
Table 2 Composition of OPM medium. Constituent/conditions
Concentration
Yeast extract Commercial tomato juice (with no preservatives) Tween 80 L-Malic acid White wine White grape must Ethanol pH Growth temperature
5 g/L 23 mL/L 0.5 mL/L 3 g/L 400 mL/L 536.5 mL/L 4% 3.8 28 C
must diluted
3.3. Scale-up of biomass production Microbiological analysis of the 4 scaled-up steps was performed to certify the correct sterilization of the OPM culture medium. The results showed that sterilization of all media was adequate, with no growth appearing in any control media (CFU < 1 102 mL1 in all plate media). Scale up started with a culture of CFU ¼ 1 106 mL1 O. oeni E5003 in 50 mL of OPM medium. The bacterial population reached CFU ¼ 1 109 mL1 after 6 d incubation. Then, the 50 mL were inoculated into 0.5 L of OPM sterile medium (Fig. 3a). The inoculated fermenter was incubated at 28 C and the bacterial population reached CFU ¼ 1 109 mL1 after 4 d of incubation (Fig. 3b). The O. oeni culture was inoculated into the 10 L fermenter with 8 L of OPM sterile medium and became a final concentration of CFU ¼ 1.3 109 mL1 after 6 d of incubation (Fig. 3c). The 8 L of O. oeni culture were inoculated into the 80 L of OPM medium, and incubated at 28 C to a final concentration of CFU ¼ 1 109 mL1. The culture reached this cell concentration in 6 d (Fig. 3d). The total time of the production process to obtain 80 L of O. oeni liquid starter culture, from the stock culture with a final population of CFU ¼ 1 109 mL1, were 22 d. Many authors have studied the medium composition required to achieve maximum biomass production and to improve the malolactic activity of O. oeni in wine (Champagne, Gardner, & Doyon, 1989; Champagne, Gardner, & Lafond, 1989; Henick-Kling, 1988; Krieger, Hammes, & Henick-Kling, 1992; Zhang & Lovitt, 2005b). A high biomass is needed to assure MLF by the inoculated bacteria, but a high biomass yield usually means that these cells are not well adapted to harsh wine conditions. On the contrary, good adaptation and acclimation of bacteria to wine involves poor
C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31
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Fig. 3. Growth of O. oeni strain E5003 (CFU ¼ mL1) in OPM medium in the different fermenters during the scale-up: a) 50 mL, b) 0.5 L, c) 8 L, d) 80 L.
growth and a low biomass yield. Besides, nowadays cells are regularly collected and freeze-dried after growth, in order to preserve them and permit convenient use in cellars. These concentration and preservation processes can involve loss of viability, or imply a change in the adaptation abilities of the cells to the wine. The production of a liquid starter overcomes these two last drawbacks, and it has been demonstrated that it is possible to produce well-adapted bacteria with a good yield. A liquid active malolactic starter can be obtained in a short time, and this process is easily scalable. The whole process was tested with other O. oeni strains from Enolab collection with technological features (E5067, E5259, and E5245), to be used as malolactic starters. In all 3 cases, a final population between CFU ¼ 8.9 108 and 1.3 109 mL-1 was achieved in the 80 L fermenter. Total time of the production process to obtain the O. oeni liquid starters, from the stock culture was also 22 d for every strain. Therefore, the medium designed could be effectively used to produce biomass of different O. oeni strains.
Preservation by refrigeration at 4 C was better than freezedrying for a short period of time, up to 50 d, but it was worse than freezing at 20 C. Therefore, for preservation from 2 to 4 months, refrigeration was the best option because cell viability was CFU ¼ 2.5 107 mL1 after 124 d. Storage at 4 C of the liquid starter is feasible for most industrial productions and uses. It provides a good viability for periods up to 3 or 4 months, and it is cheaper and easier to store and distribute than freeze-dried or frozen cultures. Many authors have studied the effect of different preservation methods, mainly freeze-drying, of O. oeni and its influence on MLF and survival in wine (G-Alegria et al., 2004; Huaa, WenYing, Huaa, Zhong Chao, & AiLian, 2009; Maicas, Pardo, & Ferrer, 2000; Zhao & Zhang, 2005, 2009). Maicas et al. (2000) studied the effects of freezing at 20 C in glycerol, by volume, of 40% and freeze-drying of O. oeni upon induction of MLF in red wine. Their results in freezedrying experiments were similar to those described for storage at 20 C, but the ability to induce MLF in wine depended on the medium where the cells were previously grown before being stored frozen or freeze-dried. These results are in agreement with ours.
3.4. Malolactic starter culture preservation Maintenance of the liquid starter culture O. oeni strain E5003 produced in OPM medium was studied using freezing at 20 C and refrigeration at 4 C. These methods were compared to freeze-drying, the most common technique for preserving MLF starter culture. Culture preservation after freeze-drying showed that cell viability was stabilized and maintained for 8 months. At this point, this method presented the best viability results, so this preservation method would be the most adequate for long-term storage (Fig. 4). Alternatively, freezing at 20 C presented a very low drop in cell viability throughout the first month. This technique would be the most adequate for culture preservation for 30 d, as during this time the freezing method presents the best results, preserving the bacterial population over 51.31% (CFU ¼ 7 108 mL1). After that period LAB viability decreased drastically (Fig. 4).
Fig. 4. Cell viability of O. oeni strain E5003 (CFU ¼ mL1) preserved by refrigeration at 4 C (C), freezing at 20 C (-) and freeze-drying (:) during 250 d.
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Fig. 5. Viable cells (CFU ¼ mL1) and malic acid consumption (%) of O. oeni strain E5003 previously grown in OPM medium in red wine with ASv ¼ 11% (C), ASv ¼ 12% (-), ASv ¼ 13% (:) and ASv ¼ 14% (;).
3.5. Malolactic activity and cell viability in red wine Fig. 5 shows the malolactic activity and cell viability of the starter culture produced in the 100 L fermenter when it was inoculated in red wine with ASv ¼ 11%, ASv ¼ 12%, ASv ¼ 13% and ASv ¼ 14% in a final concentration of CFU ¼ 1 106 mL1. When wine had an ASv ¼ 11% or ASv ¼ 12%, the number of viable cells increased promptly and depleted all malic acid (3 g/L) in 7 d. With an ASv ¼ 13%, the O. oeni culture grew slower but malic acid was consumed in 14 d. During the time of the assay, MLF was not accomplished for wines with ASv ¼ 14%, however, the viability of the bacteria remained stable during this period, which allowed the bacteria to grow later and perform MLF correctly (data not shown). If a particular winery has difficult or unusual wines, with high ethanol content, low pH, etc., grape must and wine from the OPM medium can be replaced by grape must and wine from the winery. Thus, the O. oeni strain that is going to be inoculated in the winery will be more adapted to their specific wine conditions. 4. Conclusions A liquid culture medium has been designed (OPM) to grow O. oeni and obtain high levels of biomass, and also adequate adaptation to the wine conditions. Composition of the biomass production medium permits to reach a population of O. oeni of CFU ¼ 1 109 mL1 in 6 d. The process to obtain industrial levels of O. oeni biomass has been scaled up using 80 L of the OPM medium. The bacterial population reaches CFU ¼ 1 109 mL1 in 22 d from the laboratory stock. For a long storage period of the O. oeni strain E5003 starter culture, freeze-drying is the best option; nevertheless, for up to 3 months of preservation, storage at 4 C is the best choice, and for a 1 month storage period, freezing at 20 C is the best option. This new culture medium also allowed the bacteria to adapt to the wine conditions, consuming all the wine malic acid in 7 d with an inoculum of CFU ¼ 1 106 mL1. Acknowledgements This work was supported by the project CENIT-2008 1002, V segles Universitat-Empresa grant of the University of Valencia, and Agrovin S.A. English text revised by the English language reviewer Beverly Johnson. References Amachi, T., Imamoto, S., & Yoshizumi, H. (1971). A growth factor for malo-lactic fermentation bacteria. II. Structure and synthesis of a novel pantothenic acid
derivative isolated from tomato juice. Agricultural and Biological Chemistry, 35, 1222e1230. Berbegal, C. (2014). Novel liquid starter cultures for malolactic fermentation in wine (Ph.D. thesis) (pp. 81e91). Valencia, Spain: Dep. of Microbiology and Ecology, University of Valencia. Champagne, C. P., Gardner, N., & Doyon, G. (1989). Production of Leuconostoc oenos biomass under pH control. Applied of Environmental Microbiology, 55, 2488e2492. Champagne, C. P., Gardner, N., & Lafond, A. (1989). Production of Leuconostoc oenos in apple juice media. Lebensmittel-Wissenschaft & Technologie, 22, 376e381. Davis, C. R., Wibowo, D., Eschenbruch, R., Lee, T. H., & Fleet, G. H. (1985). Practical implications of malolactic fermentation: a review. American Journal of Enology and Viticulture, 36, 290e301. Davis, C. R., Wibowo, D., Fleet, G. H., & Lee, T. H. (1988). Properties of wine lactic acid bacteria: their potential enological significance. American Journal of Enology and Viticulture, 39, 137e142. De Man, J., Rogosa, M., & Sharpe, M. (1960). A medium for the cultivation of Lactobacilli. Journal of Applied Microbiology, 23, 130e135. Dicks, L. M. T., & van Vuuren, H. J. J. (1990). Differentiation of Leuconostoc species by nicotinamide adenine dinucleotide-dependent D-lactic dehydrogenase profiles. FEMS Microbiology Letters, 67, 9e14. Diez, L., Guadalupe, Z., Ayestar an, B., & Ruiz-Larrea, F. (2010). Effect of yeast mannoproteins and grape polysaccharides on the growth of wine lactic acid and acetic acid bacteria. Journal of Agricultural and Food Chemistry, 58, 7731e7739. Frayne, R. F. (1986). Direct analysis of the major organic components in grape must and wine using high performance liquid chromatography. American Journal of Enology and Viticulture, 37, 281e287. G-Alegria, E., Lopez, I., Ruiz, J. I., Saenz, J., Fernandez, E., Zarazaga, M., et al. (2004). High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiology Letters, 230, 53e61. Gagnon, R., & Peterson, J. (1998). Estimation of confidence intervals for area under the curve from destructively obtained pharmacokinetic data. Journal of Pharmacokinetics and Biopharmaceutics, 26, 87e102. Guerrini, S., Bastianini, A., Granchi, L., & Vincenzini, M. (2002). Effect of oleic acid on Oenococcus oeni strains and malolactic fermentation in wine. Current Microbiology, 44, 5e9. Hayman, D. C., & Monk, P. R. (1982). Starter culture preparation for the induction of malolactic fermentation in wine. Food Technology Australia, 34, 14e18. Henick-Kling, T. (1988). Improving malolactic fermentation. In R. E. Smart, R. Thoronton, S. Rodroguez, & J. Young (Eds.), International symposium on cool climate viticulture & enology (pp. 238e242). Auckland, New Zealand. Huaa, L., WenYing, Z., Huaa, W., Zhong Chao, L., & AiLian, W. (2009). Influence of culture pH on freeze-drying viability of Oenococcus oeni and its relationship with fatty acid composition. Food and Bioproducts Processing, 8756e8761. Krieger, S. A., Hammes, W. P., & Henick-Kling, T. (1990). Management of malolactic fermentation using starter cultures (pp. 45e50). Vineyard & Winery Management. Nov/Dec. Krieger, S. A., Hammes, W. P., & Henick-Kling, T. (1992). Effect of medium composition on growth rate, growth yield and malolactic activity of Leuconostoc oenos LoZH1-t7-1. Food Microbiology, 9, 1e11. Kunkee, R. E. (1974). Malo-lactic fermentation and winemaking. In A. D. Webb (Ed.), Chemistry of winemaking (pp. 151e170). Washington, DC: American Chemical Society. Kunkee, R. E. (1991). Some roles of malic acid in the malolactic fermentation in wine making. FEMS Microbiology Letters, 88, 55e72. Landete, J. M., Ferrer, S., & Pardo, I. (2007). Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine. Food Control, 18, 1569e1574. Lonvaud-Funel, A. (1999). Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie van Leeuwenhoek, 76, 317e331.
C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31 Maicas, S., Ferrer, S., & Pardo, I. (2002). NAD(P)H regeneration is the key for heterolactic fermentation of hexoses in Oenococcus oeni. Microbiology, 148, 325e332. Maicas, S., Gil, J. V., Pardo, I., & Ferrer, S. (1999). Improvement of volatile composition of wines by controlled addition of malolactic bacteria. Food Research International, 32, 491e496. Maicas, S., Gonz alez-Cabo, P., Ferrer, S., & Pardo, I. (1999). Production of Oenococcus oeni biomass to induce malolactic fermentation in wine by control of pH and substrate addition. Biotechnology Letters, 21, 349e353. Maicas, S., Pardo, I., & Ferrer, S. (2000). The effects of freezing and freeze-drying of Oenococcus oeni upon induction of malolactic fermentation in red wine. International Journal of Food Science and Technology, 35, 75e79. ~ a, R., Patchett, M. L., Liu, S. Q., & Pilone, G. J. (2001). Growth and Mira de Ordun arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations. Applied and Environmental Microbiology, 67, 1657e1662. Pilone, G. J., & Kunkee, R. E. (1970). Carbonic acid from decarboxylation by “malic” enzyme in lactic acid bacteria. Journal of Bacteriology, 103, 404e409. n, M. A., G-Alegría, E., Polo, M. C., Tenorio, C., Martín-Alvarez, Pozo-Bayo P. J., Calvo de la Banda, et al. (2005). Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures. Journal of Agricultural and Food Chemistry, 53, 8729e8735. Richter, H., Vlad, D., & Unden, G. (2001). Significance of pantothenate for glucose fermentation by Oenococcus oeni and for suppression of the erythritol and acetate production. Archives of Microbiology, 175(1), 26e31. Stamer, J. R., Albury, M. N., & Pederson, C. S. (1964). Substitution of manganese for tomato juice in the cultivation of lactic acid bacteria. Applied Microbiology, 12, 165e168. ~ a, R. (2009). Determination of the essential nutrient Terrade, N., & Mira de Ordun requirements of wine-related bacteria from the genera Oenococcus and Lactobacillus. International Journal of Food Microbiology, 133, 8e13.
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~ a, R. (2009). A new chemically Terrade, N., Noel, R., Couillaud, R., & De Mira Ordun defined medium for wine lactic acid bacteria. Food Research International, 42, 363e367. €nig, H. (2005). Manganese-dependent growth of Theobald, S., Pfeiffer, P., & Ko Oenococci. Journal of Wine Research, 16, 171e178. Versari, A., Parpinello, G. P., & Cattaneo, M. (1999). Leuconostoc oenos and malolactic fermentation in wine: a review. Journal of Industrial Microbiology and Biotechnology, 23, 447e455. Wibowo, D., Eschenbruch, R., Davis, C. R., Fleet, G. H., & Lee, T. H. (1985). Occurrence and growth of lactic acid bacteria in wine: a review. American Journal of Enology and Viticulture, 36, 302e313. Zhang, D., & Lovitt, R. W. (2005a). Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures. Journal of Applied Microbiology, 99, 565e572. Zhang, D., & Lovitt, R. W. (2005b). Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture. Applied Microbiology and Biotechnology, 69, 658e664. Zhang, D., & Lovitt, R. W. (2006). Strategies for enhanced malolactic fermentation in wine and cider maturation. Journal of Chemical Technology and Biotechnology, 81, 1130e1140. Zhao, G., & Zhang, G. (2005). Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying. Journal of Applied Microbiology, 99, 333e338. Zhao, G., & Zhang, G. (2009). Influences of protectants, rehydration media and storage on the viability of freeze-dried Oenococcus oeni for malolactic fermentation. World Journal of Microbiology and Biotechnology, 25, 1801e1806. ~ iga, M., Ferrer, S., & Pardo, I. (1994). A selective medium for the isolation of Zún malolactic mutants of Leuconostoc oenos. Letters in Applied Microbiology, 19, 451e453. ~ iga, M., Pardo, I., & Ferrer, S. (1993). An improved medium for distinguishing Zún between homofermentative and heterofermentative lactic acid bacteria. International Journal of Food Microbiology, 18, 37e42.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
A novel culture medium for Oenococcus oeni malolactic starter production Carmen Berbegal, Yaiza Benavent-Gil, Isabel Pardo, Sergi Ferrer* Enolab, ERI BioTecMed/IViSoCa, University of Valencia, Dr. Moliner 50, Burjassot, Valencia 46100, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2015 Received in revised form 12 April 2015 Accepted 13 May 2015 Available online 22 May 2015
Malolactic fermentation is essential in wine quality. One of the strategies used to control this fermentation involves the inoculation of selected lactic acid bacteria, mainly Oenococcus oeni. Laboratory media usually produce large amounts of biomass, but with little or no adaptability to wine. We propose a culture medium to grow and pre-adapt O. oeni cells, and the steps to scale-up production. To achieve this objective, 27 different media were tested. All contained grape must and wine, and nutritional supplements in order to benefit bacterial growth. Those media contained different ethanol levels, pH values, and grape must concentrations. The optimized culture medium named Oenococcus Production Medium (OPM) contained diluted commercial 4X concentrate white must (1:6), ASv ¼ 4%, and a pH of 3.8. The total time to obtain 80 L of an O. oeni liquid starter culture from the stock culture at the laboratory, with a final population of CFU ¼ 1 109 mL1, was 22 d. The starter culture was efficiently scaled-up, and preserved at 4 C, 20 C, or freeze-dried. This new culture medium also allowed adaptation of bacteria to the wine conditions, consuming all malic acid (3 g/L) in 7 d with an inoculum of CFU ¼ 1 106 mL1. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Oenococcus oeni Malolactic fermentation Malolactic starter culture Scale-up Production medium
1. Introduction The main value of malolactic fermentation (MLF) in vinification is biological de-acidification, which results from the transformation of L-malic acid into L-lactic acid by lactic acid bacteria (LAB), mainly by Oenococcus oeni (Lonvaud-Funel, 1999; Versari, Parpinello, & Cattaneo, 1999; Wibowo, Eschenbruch, Davis, Fleet, & Lee, 1985). MLF induces an increase in pH, contributes to microbiological stability, and changes wine taste (Davis, Wibowo, Fleet, & Lee, 1988; Kunkee, 1991; Maicas, Gil, Pardo, & Ferrer, 1999). MLF can be produced by two strategies: spontaneously or by inoculation with starter cultures. Traditionally, spontaneous and indigenous LAB carried out a natural MLF process. But this MLF method can take weeks or months and is uncontrolled (Zhang & Lovitt, 2006). The induction of MLF by using a starter culture ensures faster fermentation, reduces potential spoilage by other LAB, and allows the improvement of wine quality when using selected bacterial strains ~ a, Patchett, Liu, & Pilone, 2001; Pozo-Bayo n et al., (Mira de Ordun
Abbreviations: AUC, area under the curve; LAB, lactic acid bacteria; MLF, malolactic fermentation; OPM, Oenococcus production medium. * Corresponding author. Tel.: þ34 963 544 518. E-mail address: [email protected] (S. Ferrer). http://dx.doi.org/10.1016/j.lwt.2015.05.020 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
2005). Nevertheless, successful inoculation of the starter into wine depends not only on the suitable strain of O. oeni, but on the preparation and use of the cultures. For the production of a suitably large biomass, selected strains of O. oeni are grown under conditions that permit rapid growth and result in a high cell yields, but these conditions are very different to those present in wine. Consequently, when the starter culture is inoculated directly into wine, it loses much viability (Henick-Kling, 1988). O. oeni is usually grown in the laboratory in complex culture media such as MRS (De Man, ~ iga, Ferrer, & Pardo, 1994), or Rogosa, & Sharpe, 1960), MLO (Zún AGB (Dicks & van Vuuren, 1990). Most of them frequently contain grape juice (Davis, Wibowo, Eschenbruch, Lee, & Fleet, 1985) or apple juice (Champagne, Gardner, & Lafond, 1989). We prefer to use tomato juice instead, as it is a source of pantothenic acid, commonly used as a growth factor for wine bacteria (Amachi, Imamoto, & Yoshizumi, 1971; Richter, Vlad, & Unden, 2001; Terrade & Mira de ~ a, 2009). The culture media are generally supplemented Ordun with other nutrients like yeast extract, peptone, and Tween 80, to increase biomass production (Champagne, Gardner, & Doyon, 1989; Guerrini, Bastianini, Granchi, & Vincenzini, 2002; Krieger, Hammes, & Henick-Kling, 1990; Kunkee, 1974; Pilone & Kunkee, 1970). The growth of O. oeni has been also investigated in single sugars and their mixtures. The best growth was obtained with sugar mixtures (glucoseefructose) rather than growth on a single sugar (Maicas,
26
C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31
lez-Cabo, Ferrer, & Pardo, 1999; Ferrer, & Pardo, 2002; Maicas, Gonza Zhang & Lovitt, 2005a). Other components like manganese or yeast mannoproteins and nutrient requirements have been studied € nig, (Stamer, Albury, & Pederson, 1964; Theobald, Pfeiffer, & Ko 2005). Results revealed that the essential bacterial nutrients were strain-specific and O. oeni strains showed a large number of auxotrophies (Diez, Guadalupe, Ayestar an, & Ruiz-Larrea, 2010; Terrade ~ a, 2009; Terrade, Noel, Couillaud, & De Mira & Mira de Ordun ~ a, 2009; Theobald et al., 2005). Hayman and Monk (1982) Ordun evaluated the effect of adding wine to a medium for the production of O. oeni biomass, and found that a content of 40e80% wine in the medium improved LAB survival and malolactic activity. Nevertheless, further studies are necessary to find a medium for biomass production with a cheap easy recipe that allows any O. oeni strain to grow and adapt before being inoculated into wine. The objectives of this work were to develop a liquid O. oeni production medium (OPM) that would permit high levels of biomass production, but also an adequate pre-adaptation to wine conditions, and evaluate cell viability and malolactic activity of the MLF starter culture in wine. Preservation conditions for the liquid starter culture were also studied. 2. Material and methods 2.1. Microorganisms O. oeni strains E5003, E5067, E5259, and E5245 were taken from the Enolab culture collection of the University of Valencia (Spain). All strains had been previously isolated from spontaneous mid-MLF in Tempranillo wines from the Ribera del Duero region (Spain). 2.2. Growth in biomass production media ~ iga, Pardo, & O. oeni strains were pre-grown in MLO broth (Zún Ferrer, 1993) to early stationary phase. Cells were then centrifuged (rotational speed of 8000 min1, 10 min), washed and transferred to the 27 culture media (Table 1) at a final concentration of CFU ¼ 1 106 mL1. The base of all these media contained: yeast extract (5 g/L), commercial tomato juice with no preservatives (23 mL/L), Tween 80 (0.5 mL/L), L-malic acid (3 g/L) and concentrate reconstituted white wine (glucose 0.13 g/L, fructose 0.14 g/L, L-malic acid 0.22 g/L, free SO2 1 mg/L, ethanol ASv ¼ 0%) from Agrovin S.A. (Spain) (400 mL/L). This basal medium was supplemented with 4X concentrated white grape must (glucose 360 g/L, fructose 360 g/L, Lmalic acid 5.3 g/L, free SO2 1.4 mg/L) from Agrovin S.A. (Spain), which was diluted 4, 6 and 8 times (576.5 mL/L) and the pH was adjusted to 3.8, 4 and 4.5. Media were sterilized by autoclave at 115 C, 30 min. After sterilization, ethanol degree was adjusted at ASv ¼ 4%, ASv ¼ 6% and ASv ¼ 8%. All growth studies were carried out in duplicate, in 10 mL screw cap tubes and incubated at 28 C, 7 d. Growth was monitored by measuring optical density at 600 nm (OD600) with a spectrophotometer (CECIL, CE 373). The bacterial growth curve was transformed into a value of area under the curve (AUC) (Gagnon & Peterson, 1998). 2.3. Malolactic fermentation in red wine The cultures were inoculated into red wine at a final concentration of CFU ¼ 1 106 mL1, and MLF was monitorized for 15 d. All fermentations were carried out in duplicate. The red wine was made at the laboratory using a red Tempranillo grape must, inoculated with S. cerevisiae Viniferm B4 (Agrovin S.A.) at a final concentration of CFU ¼ 2 106 mL1, and was incubated at 28 C, 10 d, until the AF was finished. When the sugar concentration was lower than 1 g/L, the ethanol was adjusted to ASv ¼ 11%, ASv ¼ 12%,
Table 1 Combination of diluted 4X grape must (1/4, 1/6 or 1/8), ethanol content (ASv ¼ 4%, ASv ¼ 6% or ASv ¼ 8%) and pH level (3.8, 4 or 4.5) resulted in 27 different media for the O. oeni biomass production. Medium
Diluted must
Ethanol %
pH
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/6 1/6 1/6 1/6 1/6 1/6 1/6 1/6 1/6 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8
4 4 4 6 6 6 8 8 8 4 4 4 6 6 6 8 8 8 4 4 4 6 6 6 8 8 8
3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5 3.8 4 4.5
ASv ¼ 13% and ASv ¼ 14%, 3.0 g/L of malic were added, and the pH was adjusted to 3.5. Then, it was sterilized by filtering through 0.22 mm pore filter and stored at 15 C until use. 2.4. Viable cell counts Cell viability in red wine was studied by plate counting. The volume of 0.1 mL of decimal serial dilutions in sterile saline solution ~ iga et al., 1993) were spread in duplicate on MLO agar plates (Zún and were incubated at 28 C for 7 d and then the colonies were counted. 2.5. Culture preservation Freeze-drying was performed after growing the bacteria until the end of the exponential phase in 50 mL of OPM medium. Cells were recovered by centrifugation at rotational speed of 6000 min1, 15 min in a Heraeus Multifuge 1 S-R centrifuge. Then, cells were washed twice with glutamic acid 0.98%, recovered with the same above centrifugation conditions, and resuspended in 2 mL of 0.98% glutamic acid. The bacterial solution was distributed in aliquots of 400 mL per tube. Tubes were frozen at 20 C, 1 h. Freeze-dying was performed at 60 C for 18 h under vacuum (Virtis Sentry). Tubes were vacuum sealed and stored at 4 C under dark. Refrigeration at 4 C and freezing at 20 C were performed after growing O. oeni until the exponential phase in OPM medium and later transferring aliquots to the respective temperature. Bacterial cultures were preserved and checked for viability up to 250 d. 2.6. Analytical methods Glucose, fructose, malic acid and ethanol were quantified by high-pressure liquid chromatography (HPLC) (Agilent series 1200) with an isocratic pump (Agilent G1310A) following the procedure described by Frayne (1986) with minor modifications. The mobile phase consisted of a solution of 0.75 mL of 85% H3PO4 per litre of
C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31
deionized water with a flow rate of 0.7 mL/min. An Agilent G1322A degasser was employed. Samples (5 mL) were injected automatically (Agilent G1367B). The components were separated using an Aminex HPX-87H precolumn (Bio-Rad) coupled with two ion exclusion columns of 300 mm by 7.8 mm Aminex HPX-87H (BioRad) thermostatically controlled at 65 C (Agilent G1316A). Compounds were detected by a G1314B variable-wavelength detector (Agilent) set to 210 nm and a refractive index detector (Agilent G1362A) in series. Elution time was 45 min. External calibration was performed. All samples were centrifuged (rotational speed of 8000 min1, 10 min) and filtered through a membrane filter with a mean pore size of 0.22 mm before use. Quantification was performed by measuring peak height compared to external standards. 2.7. Scale-up of biomass production The scale up started with a culture of CFU ¼ 1 106 mL1 of O. oeni in 50 mL of liquid OPM medium incubated statically at 28 C. When the bacterial population reached the maximum biomass yield, the 50 mL were inoculated into 0.5 L of OPM sterile medium, and were incubated at 28 C until the bacteria population reached the maximum biomass yield. Then, the O. oeni culture was inoculated into the 10 L fermenter with 8 L of OPM sterile medium and was incubated stirred at room temperature. The 0.5 L and the 10 L fermenter were built using Pyrex bottles with a screw cap GL 45 with three ports GL 14 thread (Duran). In the 0.5 L fermenter, one of these ports was closed with a screw cap GL 14, the second one was connected to a set for pressure compensation (Duran) to permit fermentation gases to vent out, and the third one was connected with a screw Cap GL 14 for hose connection (Duran) with a joint silicone diameter 0.6 mm and a silicone tube of 3 mm 6 mm. This silicone tube was joined to one of the ports in the PVDF Luer lock 3 way valve (Cole Palmer) of the 10 L fermenter. In the 10 L fermenter, one of these three ports was connected to a pressure compensation set (Duran) to enable fermentation gases to vent out, and the other two were connected with a screw cap GL 14 for hose connection (Duran) to two silicone tubes. One of these tubes was used to inoculate the starter from the 0.5 L fermenter, and the other one was used to collect samples with a 50 mL Luer lock syringe. The industrial biomass production process was carried out inoculating the 10 L of the O. oeni culture into a fermenter with 80 L of OPM sterile medium (Bioprocess technology Bio-pro 100 L). The inoculated culture was incubated at 28 C until the bacteria population reached CFU ¼ 1 109 mL1. A microbiological analysis of the three scale-up steps was carried out to certify the correct sterilization of the culture medium. After the sterilization of 50 mL and 0.5 L fermenter of OPM medium by autoclave, 115 C 30 min, and the 10 L and 100 L fermenters, 115 C 45 min, 0.1 mL sample from each ~ iga fermenter were plated on MRS (De Man et al., 1960), MLO (Zún et al., 1993) and YPD (Landete, Ferrer, & Pardo, 2007) media. The plates were incubated at 28 C for 7 d. 2.8. Statistical analysis Statistical significance of differences between averages of duplicate measurements were evaluated by performing one-way ANOVA at a confidence level of p ¼ 0.05 and Tukey's Studentized Range Test. 3. Results and discussion 3.1. Growth in biomass production media O. oeni strain E5003 was selected to carry out the assays because it presented interesting technological features as malolactic starter
27
(Berbegal, 2014). The culture medium designed to grow bacteria contained mainly grape must and wine in order to adapt cells to the harsh conditions of wine. White wine and must were used because the sterilization of red must and wine caused precipitates, and also turbidity made it difficult to monitor bacterial growth properly. Commercial wine concentrate and grape must were used to standardize the culture medium. When the white concentrate must was diluted 4 times (media 1e9), the growth was lower than when the must was diluted 6 (media 10e18) or 8 (media 19e27) times (Fig. 1). Between must diluted 6 or 8 times differences were smaller, although there was slightly better growth with the must diluted 6 times. The best growth was obtained with ASv ¼ 4% (1e3, 10e12, 19e21), and the worst results were found with ASv ¼ 8% (7e9, 16e18, 25e27). In all cases, growth with a pH of 4.5 resulted in the highest value of AUC followed by growth with a pH of 4, and the lowest value was obtained with a pH of 3.8. It was observed that at lower pH, more time was needed to reach to the same OD600 nm. The best results were found, as expected, when the must was diluted 6 times with the lowest ethanol content (ASv ¼ 4%) and with the highest pH (4.5) (media 12), and the worst growth was observed with the must diluted 4 times, the highest ethanol content (ASv ¼ 8%) and 3.8 pH (medium 7) (Fig. 1). Results were analyzed using an ANOVA test. This analysis revealed a significant effect (p < 0.05) for must, ethanol, pH and for the interactions musteethanol and mustepH. Through the Tukey post hoc test, the media with the lowest AUC were rejected (1, 2, 4, 5, 7, 8, 16, 17, and 25). All these media obtained an AUC below 2, and their pH levels were 3.8 or 4. The cultures from the 18 remaining media were selected and their malolactic activity in wine was studied. HPLC analysis showed that the must concentrate contained around 360 g/L of glucose and 360 g/L of fructose. When must concentrate was diluted 4 times bacterial growth was lower than when the must was diluted 1/6 or 1/8 probably because of the osmotic pressure. Composition of OPM medium was based on previous studies of growth, metabolism and biomass production of O. oeni. Champagne, Gardner, and Lafond (1989) studied the production of O. oeni biomass in apple juice media and grape juice media. Their results showed that there were variations between strains in their ability to grow, and the optimum pH was also strain dependent, varying from 4.5 to 4.8. Yeast extract supplementation was the most beneficial for growth. In our study, 5 g/L of yeast extract was added to the media and a range of pH was studied but always below 4.8 because this value was too high, compared to the wine pH. Also, malic acid was added to stimulate LAB growth and MLF activity (Terrade et al., 2009). The studies of Zhang and Lovitt (2005a) and lez-Cabo, et al. (1999) Maicas et al. (2002) and Maicas, Gonza showed that better growth of O. oeni was obtained in the presence of sugar mixtures (glucose:fructose, 1:1) in the culture medium compared to glucose alone. The optimal growth of O. oeni was at pH 4.5, with the maximum biomass yield and specific growth rate. In the present work, the highest biomass also was obtained when the bacterium was grown at pH 4.5 and the medium contained a sugar mixture of glucose:fructose (1:1). 3.2. Malolactic activity in red wine All the cultures from media with a value of AUC above 2 (Fig. 1) were inoculated in red wine with ASv ¼ 12% and pH 3.5. Some differences were found in malolactic activity, and 5 cultures (10, 13, 19, 22, and 26) consumed all the malic acid in 6 d (Fig. 2). These differences in malolactic activity depended mainly on the pH of the medium where the bacteria were grown before. The cultures that were grown in a medium with a pH of 4.5 resulted in a slower
28
C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31
Fig. 1. Values of O. oeni E5003 area under the curve (AUC) in the 27 different culture media ( media is described in Table 1.
must diluted 1/4,
must diluted 1/6,
must diluted 1/8). The code number of the
Fig. 2. Malic acid consumption (%) in red wine after 6 d of incubation of the O. oeni strain E5003 previously grown in the different culture media ( must diluted 1/4, 1/6, must diluted 1/8). The code number of the media is described in Table 1.
consumption of malic acid in red wine. The best results of activity in wine were found when O. oeni strain E5003 was previously grown in a medium with a pH of 3.8. This can be correlated to the fact that this pH was more similar to the wine pH (3.5), so the cells were more adapted to it. Out of these 5 media, 4 of them (10, 13, 19 and 22) had been grown in a pH of 3.8, proving the importance of the pH as a growth factor in the culture media for MLF in wine later on. The culture with the highest biomass in the culture medium was selected for further studies. Medium 10 had the highest value of area under the curve (2.44 AUC) (Fig. 1) so this medium was chosen to be the OPM. That was the medium with the must diluted 6 times, with an ASv ¼ 4% and a pH of 3.8 (Table 2). HPLC analysis of this medium showed around 40 g/L of glucose, 40 g/L of fructose, and 3.5 g/L of malic acid. The OPM medium contains must and wine and pH is 3.8, so growth conditions are similar to wine conditions, cells are adapted to wine and do not need successive transfers before inoculation. The starter culture kept high cell viability and malolactic activity when it was inoculated in wine.
Table 2 Composition of OPM medium. Constituent/conditions
Concentration
Yeast extract Commercial tomato juice (with no preservatives) Tween 80 L-Malic acid White wine White grape must Ethanol pH Growth temperature
5 g/L 23 mL/L 0.5 mL/L 3 g/L 400 mL/L 536.5 mL/L 4% 3.8 28 C
must diluted
3.3. Scale-up of biomass production Microbiological analysis of the 4 scaled-up steps was performed to certify the correct sterilization of the OPM culture medium. The results showed that sterilization of all media was adequate, with no growth appearing in any control media (CFU < 1 102 mL1 in all plate media). Scale up started with a culture of CFU ¼ 1 106 mL1 O. oeni E5003 in 50 mL of OPM medium. The bacterial population reached CFU ¼ 1 109 mL1 after 6 d incubation. Then, the 50 mL were inoculated into 0.5 L of OPM sterile medium (Fig. 3a). The inoculated fermenter was incubated at 28 C and the bacterial population reached CFU ¼ 1 109 mL1 after 4 d of incubation (Fig. 3b). The O. oeni culture was inoculated into the 10 L fermenter with 8 L of OPM sterile medium and became a final concentration of CFU ¼ 1.3 109 mL1 after 6 d of incubation (Fig. 3c). The 8 L of O. oeni culture were inoculated into the 80 L of OPM medium, and incubated at 28 C to a final concentration of CFU ¼ 1 109 mL1. The culture reached this cell concentration in 6 d (Fig. 3d). The total time of the production process to obtain 80 L of O. oeni liquid starter culture, from the stock culture with a final population of CFU ¼ 1 109 mL1, were 22 d. Many authors have studied the medium composition required to achieve maximum biomass production and to improve the malolactic activity of O. oeni in wine (Champagne, Gardner, & Doyon, 1989; Champagne, Gardner, & Lafond, 1989; Henick-Kling, 1988; Krieger, Hammes, & Henick-Kling, 1992; Zhang & Lovitt, 2005b). A high biomass is needed to assure MLF by the inoculated bacteria, but a high biomass yield usually means that these cells are not well adapted to harsh wine conditions. On the contrary, good adaptation and acclimation of bacteria to wine involves poor
C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31
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Fig. 3. Growth of O. oeni strain E5003 (CFU ¼ mL1) in OPM medium in the different fermenters during the scale-up: a) 50 mL, b) 0.5 L, c) 8 L, d) 80 L.
growth and a low biomass yield. Besides, nowadays cells are regularly collected and freeze-dried after growth, in order to preserve them and permit convenient use in cellars. These concentration and preservation processes can involve loss of viability, or imply a change in the adaptation abilities of the cells to the wine. The production of a liquid starter overcomes these two last drawbacks, and it has been demonstrated that it is possible to produce well-adapted bacteria with a good yield. A liquid active malolactic starter can be obtained in a short time, and this process is easily scalable. The whole process was tested with other O. oeni strains from Enolab collection with technological features (E5067, E5259, and E5245), to be used as malolactic starters. In all 3 cases, a final population between CFU ¼ 8.9 108 and 1.3 109 mL-1 was achieved in the 80 L fermenter. Total time of the production process to obtain the O. oeni liquid starters, from the stock culture was also 22 d for every strain. Therefore, the medium designed could be effectively used to produce biomass of different O. oeni strains.
Preservation by refrigeration at 4 C was better than freezedrying for a short period of time, up to 50 d, but it was worse than freezing at 20 C. Therefore, for preservation from 2 to 4 months, refrigeration was the best option because cell viability was CFU ¼ 2.5 107 mL1 after 124 d. Storage at 4 C of the liquid starter is feasible for most industrial productions and uses. It provides a good viability for periods up to 3 or 4 months, and it is cheaper and easier to store and distribute than freeze-dried or frozen cultures. Many authors have studied the effect of different preservation methods, mainly freeze-drying, of O. oeni and its influence on MLF and survival in wine (G-Alegria et al., 2004; Huaa, WenYing, Huaa, Zhong Chao, & AiLian, 2009; Maicas, Pardo, & Ferrer, 2000; Zhao & Zhang, 2005, 2009). Maicas et al. (2000) studied the effects of freezing at 20 C in glycerol, by volume, of 40% and freeze-drying of O. oeni upon induction of MLF in red wine. Their results in freezedrying experiments were similar to those described for storage at 20 C, but the ability to induce MLF in wine depended on the medium where the cells were previously grown before being stored frozen or freeze-dried. These results are in agreement with ours.
3.4. Malolactic starter culture preservation Maintenance of the liquid starter culture O. oeni strain E5003 produced in OPM medium was studied using freezing at 20 C and refrigeration at 4 C. These methods were compared to freeze-drying, the most common technique for preserving MLF starter culture. Culture preservation after freeze-drying showed that cell viability was stabilized and maintained for 8 months. At this point, this method presented the best viability results, so this preservation method would be the most adequate for long-term storage (Fig. 4). Alternatively, freezing at 20 C presented a very low drop in cell viability throughout the first month. This technique would be the most adequate for culture preservation for 30 d, as during this time the freezing method presents the best results, preserving the bacterial population over 51.31% (CFU ¼ 7 108 mL1). After that period LAB viability decreased drastically (Fig. 4).
Fig. 4. Cell viability of O. oeni strain E5003 (CFU ¼ mL1) preserved by refrigeration at 4 C (C), freezing at 20 C (-) and freeze-drying (:) during 250 d.
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C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31
Fig. 5. Viable cells (CFU ¼ mL1) and malic acid consumption (%) of O. oeni strain E5003 previously grown in OPM medium in red wine with ASv ¼ 11% (C), ASv ¼ 12% (-), ASv ¼ 13% (:) and ASv ¼ 14% (;).
3.5. Malolactic activity and cell viability in red wine Fig. 5 shows the malolactic activity and cell viability of the starter culture produced in the 100 L fermenter when it was inoculated in red wine with ASv ¼ 11%, ASv ¼ 12%, ASv ¼ 13% and ASv ¼ 14% in a final concentration of CFU ¼ 1 106 mL1. When wine had an ASv ¼ 11% or ASv ¼ 12%, the number of viable cells increased promptly and depleted all malic acid (3 g/L) in 7 d. With an ASv ¼ 13%, the O. oeni culture grew slower but malic acid was consumed in 14 d. During the time of the assay, MLF was not accomplished for wines with ASv ¼ 14%, however, the viability of the bacteria remained stable during this period, which allowed the bacteria to grow later and perform MLF correctly (data not shown). If a particular winery has difficult or unusual wines, with high ethanol content, low pH, etc., grape must and wine from the OPM medium can be replaced by grape must and wine from the winery. Thus, the O. oeni strain that is going to be inoculated in the winery will be more adapted to their specific wine conditions. 4. Conclusions A liquid culture medium has been designed (OPM) to grow O. oeni and obtain high levels of biomass, and also adequate adaptation to the wine conditions. Composition of the biomass production medium permits to reach a population of O. oeni of CFU ¼ 1 109 mL1 in 6 d. The process to obtain industrial levels of O. oeni biomass has been scaled up using 80 L of the OPM medium. The bacterial population reaches CFU ¼ 1 109 mL1 in 22 d from the laboratory stock. For a long storage period of the O. oeni strain E5003 starter culture, freeze-drying is the best option; nevertheless, for up to 3 months of preservation, storage at 4 C is the best choice, and for a 1 month storage period, freezing at 20 C is the best option. This new culture medium also allowed the bacteria to adapt to the wine conditions, consuming all the wine malic acid in 7 d with an inoculum of CFU ¼ 1 106 mL1. Acknowledgements This work was supported by the project CENIT-2008 1002, V segles Universitat-Empresa grant of the University of Valencia, and Agrovin S.A. English text revised by the English language reviewer Beverly Johnson. References Amachi, T., Imamoto, S., & Yoshizumi, H. (1971). A growth factor for malo-lactic fermentation bacteria. II. Structure and synthesis of a novel pantothenic acid
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