Acetobacter Strains Isolated During the Acetification of Blueberry

Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Acetobacter strains isolated during the acetification of blueberry (Vaccinium corymbosum L.) wine C. Hidalgo1, D. Garcıa1, J. Romero2, A. Mas1, M.J. Torija1 and E. Mateo1 1 Biotecnologia Enologica, Dept. Bioquımica i Biotecnologia, Facultat d’Enologia, Universitat Rovira i Virgili, Tarragona, Spain 2 Laboratorio de Biotecnologıa, INTA, Universidad de Chile, Santiago de Chile, Chile Significance and Impact of the Study: In this study, the Acetic Acid Bacteria population involved in blueberry vinegar production using Schutzenbach€ method is first reported. Inoculation of an Acetobact- er cerevisiae strain speeded up and conducted the process compared to spontaneous process. However, two genotypes of Acetobacter pasteurianus were also isolated in both processes (inoculated and spontaneous). These Acet. pasteurianus isolates can be exploited for studies of inoculation in vinegar production from blueberry or other raw material using Schutzenbach€ method or other vinegar production methods. Keywords Abstract Acetobacter, highbush blueberries, Schutzenbach€ method, starter culture, Highbush blueberries (Vaccinium corymbosum L.) are known to have positive vinegar. health benefits. The production of blueberry vinegar is one method to preserve this seasonal fruit and allow extended consumption. In this study, blueberry Correspondence wine acetification was performed with naturally occurring micro-organisms Estibaliz Mateo, Biotecnologia Enologica, and with an inoculated Acetobacter cerevisiae strain. Acetifications were carried Dept. Bioquımica i Biotecnologia, Facultat out in triplicate using the Schutzenbach€ method. The successful spontaneous d’Enologia, Universitat Rovira i Virgili, C/ MarcelÁlı Domingo s/n. 43007 Tarragona, processes took up to 66% more time than the processes involving inoculation. Spain. The isolation of acetic acid bacteria (AAB) and the analysis of these AAB using E-mail: [email protected] molecular methods allowed the identification of the main genotypes responsible of the blueberry acetification. Although the Acet. cerevisiae strain 2013/0224: received 5 February 2013, revised was the predominant strain isolated from the inoculated process samples, 14 May 2013 and accepted 14 May 2013 Acetobacter pasteurianus was isolated from samples for both processes and was the only species present in the spontaneous acetification samples. To the best doi:10.1111/lam.12104 of our knowledge, this is the first report describing the identification and variability of AAB isolated during blueberry acetification. The isolated Acet. pasteurianus strains could be used for large-scale blueberry vinegar production or as a starter culture in studies of other vinegar production methods. 2009). In some of these studies, molecular techniques Introduction have been used to identify the AAB species and geno- Acetic acid bacteria (AAB) are important micro-organ- types present in these niches. For instance, Acetobacter isms in the food and biotechnological industries because malorum was reported as the responsible from straw- of their ability to oxidize many types of sugar and alco- berry vinegar production by traditional method (Hidalgo hols (De Ley et al. 1984). The production of vinegar is et al. 2013) and Acetobacter pasteurianus was the domi- one of the most important industrial processes in which nant species identified in cereal vinegar elaborated by these bacteria are involved. solid-state fermentation (Wu et al. 2010). In the single Vinegars derived from different raw materials, such as study carried out on AAB from blueberries, several dif- grapes, cereals, onions, persimmons, berries and whey, ferent AAB genera were identified using biochemical have been produced and studied (Solieri and Giudici tests (Acetobacter, Gluconobacter, Asaia, Gluconacetobacter Letters in Applied Microbiology 57, 227--232 © 2013 The Society for Applied Microbiology 227 Acetobacter strains from blueberry wine acetification C. Hidalgo et al. and Swaminathania) from different varieties of blueberry the submerged method (Saeki et al. 1997; Sokollek and (Gerard et al. 2010). Hammes 1997; Hidalgo et al. 2010b), and no studies have Highbush blueberries (Vaccinium corymbosum L.) are a tested such starters with the Schutzenbach€ method. rich source of dietary antioxidants (Gu et al. 2004; See- Therefore, more inoculation studies should be carried out ram 2006; Borges et al. 2010) that have multiple benefi- in order to better understand the AAB population cial biological effects. This fruit is native to North dynamic and the efficiency of the inoculated strains. America, and the largest blueberry industries in the world The aim of this work was to study the AAB population are in the United States and Chile. Currently, Chile has a during the production of blueberry vinegar by the dominant position in the Southern Hemisphere, and Schutzenbach€ method. The acetifications were carried out blueberry cultivation is an important economic activity in spontaneously and after inoculation with an Acetobacter this country (Brazelton 2011). cerevisiae strain that was previously isolated from grapes As with any activity related to the production of fresh (Prieto et al. 2007). fruit, there are substandard fruit, seasonal surpluses and fruit waste generated during the cultivation, and these Results and discussion materials could be used to produce fruit vinegar. In the case of blueberries, the production of vinegar could be a Acetification kinetics good option to preserve the healthy properties of this fruit. The kinetics of the spontaneous and inoculated processes There are two well-defined methods used to produce was followed by measuring the increase in acidity over time vinegar: traditional or surface processes and submerged (Fig. 1). The blueberry wine was produced spontaneously methods. The primary differences between these two at room temperature and it had an initial alcohol concen- methods are related to the time needed to complete the tration of 7Á3% (v/v), acetic acid concentration of 0Á6% acetification process and to the quality of the final prod- (w/v) and a pH of 3Á15. During vinegar production, no uct. In the traditional method, the time required to variation in the pH was observed. The three spontaneous obtain the expected level of acetic acid is longer than in acetifications had similar kinetics, reaching maximum the submerged method due to the strong aeration mecha- acidity [5Á5% (w/v)] after 28 days. However, the three nism used in the latter method to meet the oxygen samples inoculated with Acet. cerevisiae reached higher demand of the AAB. In traditional method, AAB grows acidity values [6Á6to6Á9% (w/v)], and the highest acidity on the air–liquid interface obtaining oxygen from atmo- levels were reached sooner than the highest levels for the spheric air, which slows down the acetification but allows spontaneous process, but the exact time needed to reach gaining metabolic by-products from AAB that increase the highest level of acidity varied among the triplicates the sensorial complexity of the vinegar. Therefore, as (9, 17 and 24 days). consequence of the production system, the elaboration In this study, the Schutzenbach€ method was used time is lengthier but the quality of the final product is successfully to produce blueberry vinegar. This system significantly higher in the traditional method. was designed to achieve acetification rates that are faster Alternative systems for the production of vinegar have than those obtained with traditional methods (Llaguno been designed to achieve faster rates of production than those for traditional methods while preserving the quality of the final product to the greatest extent possible. These 8 alternative methods, among which is the Schutzenbach€ 7 method, are focused on the use of inert materials, such as 6 – bacterial supports, to mimic the air liquid interface 5 created in the traditional method to allow direct contact 4 with atmospheric air. The Schutzenbach€ method uses wood shavings as a bacterial support material, and to 3 increase the oxygen accessibility, the acetifying liquid is 2 pumped through the wood shavings, achieving relatively Titratable acicity % (w/v) 1 high acetification rates (Llaguno 1991). 0 The use of well-defined starter cultures in vinegar elab- 0 5 10 15 20 25 30 Time (days) oration to date has been limited or nonexistent. Few studies have tested the use of a selected AAB culture as a Figure 1 Kinetics of acetification represented by the titratable acidity. starter for the production of vinegar by the traditional Average of spontaneous acetifications (--□--) and individual inoculated method (Gullo et al. 2009; Hidalgo et al. 2010b, 2013) or acetifications [replicates 1 (♦), 2 (▲) and 3 (●)]. 228 Letters in Applied Microbiology 57, 227--232 © 2013 The Society for Applied Microbiology C. Hidalgo et al. Acetobacter strains from blueberry wine acetification Table 1 Enumeration of acetic acid bacteria by plating (culturability, [bp] Ladder Ac0 Ap1 Ap2 CFU mlÀ1) and microscopy (total cells, Cell mlÀ1) during blueberry acetification processes 10380 À1 À1 Type of process Samples Cell ml CFU ml 5000 Spontaneous Initial 8Á00 Æ 0Á15E+06 2Á50 Æ 0Á21E+06 2000 acetification Mid 1Á42 Æ 0Á17E+07 4Á57 Æ 0Á32E+06 1000 Final 7Á64 Æ 0Á89E+07 1Á83 Æ 0Á68E+04 Inoculated Initial 3Á68 Æ 0Á85E+08 1Á30 Æ 0Á10E+08 700 acetification Mid 3Á39 Æ 0Á47E+08 3Á60 Æ 0Á70E+06 Á Æ Á + Á Æ Á + Final 4 89 0 83E 08 8 70 0 85E 04 500 300 1991). The elaboration of vinegar from grape

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