TECHNOLOGICAL CHARACTERISTICS OF CALABRIAN ACETIC ACID BACTERIA
Andrea CARIDI, Rossana SIDARI
Faculty of Agriculture, Mediterranea University of Reggio Calabria, Gallina, Italy
Tel. +39.0965.682816/312330; Fax +39.0965.680727; e-mail
acaridi@unirc.it
INTRODUCTION
In Calabria, the homemade production of vinegars is widespread. They are made using acescent wines, diluted to 8 % v/v of ethanol and then acetified.
A preliminary survey has shown the occasional occurrence of high-quality samples of wine vinegar obtained using naturally selected acetic acid bacteria, due to the absence of industrial acetifiers in Calabria.
In recent years we have examined a total of 65 samples of acescent wines and homemade wine vinegars in order to isolate and identify the acetic acid bacteria present in the Calabrian ecosystems and, consequently, to evaluate their grade of biodiversity.
These studies have shown that the vast majority - over 90% - of more than 500 strains of Calabrian acetic acid bacteria are included in the species Acetobacter
pasteurianus.
The aim of the present work was to exploit the basic technological characteristics of the 35 best strains in order to identify starter cultures able to control the acetification process.
MATERIALS AND METHODS
Thirty-two strains of Acetobacter pasteurianus, two strains of Acetobacter
aceti and one strain of Gluconacetobacter hansenii were employed (Table
1).
They were taken from 530 strains, previously isolated from Calabrian acescent wines (42 samples) and homemade vinegars (23 samples), identified, and pre-selected on Glucose Yeast extract Calcium carbonate
Agar for high acetic acid production (Figure 1).
To collect the strains and to prepare their precultures wine agar was used made of white wine diluted with water in a ratio of one to two, adjusted to pH 5.5 using NaOH 5N and with the addition of agar 25 g/L; the medium was sterilised at 121°C for 15’ and then poured in Petri plates.
Micro-acetification trials were performed in duplicate using 500 mL-aerobic flasks (Figure 2) containing 200 mL of diluted white wine, together with 50 mg/L of thiamine and 300 mg/L of diammonium phosphate, all previously sterilized by filtration on cellulose membrane and finally inoculated with pre-culture of the strains.
The flasks were incubated at 30 °C for the acetification process and microbial growth was evaluated by measuring the optical density at 520nm. At the end of the acetification process, the vinegars were refrigerated at 4 °C for 24 hours and analysed for pH, titratable acidity, glycerol and total polyphenols.
RESULTS
The pH value of the base wine was 4.56. In the vinegars, this parameter had a mean value of 3.11, with a minimum of 3.06 for the strain of A.
pasteurianus 15 and a maximum of 3.28 for the strain of A. pasteurianus
145. Figure 3 reports these values grouped in three frequency classes. The three non A. pasteurianus strains fall into the followings frequency classes:
A. aceti 117 in the first (pH 3.10), A. aceti 230 (pH 3.11) and G. hansenii
435 (pH 3.12) in the second.
The titratable acidity content of the wine, expressed in g of acetic acid %, was 0.88. In the vinegars, this parameter had a mean value of 7.22, with a minimum of 5.23 for the strain of A. pasteurianus 145 and a maximum of 7.53 for the strain of A. pasteurianus 66. Figure 4 reports these values grouped in three frequency classes. The two strains of A. aceti 117 (7.25 g %) and 230 (7.10 g %) and the strain of G. hansenii 435 (7.36 g %) are included in the third frequency class.
Glycerol can influence the sensory characteristics of vinegars; during the acetification process it tends to partially decrease. The glycerol content of the wine was 4.05 g/L. In the vinegars this parameter had a mean value of 3.14, with a minimum of 2.40 for the strain of A. pasteurianus 40 and a maximum of 3.50 for the strain of A. pasteurianus 98. Figure 5 reports these values grouped in three frequency classes. The strain of A. aceti 230 is included in the second frequency class (2.90 g/L); A. aceti 117 (3.05 g/L) and G. hansenii 435 (3.40 g/L) are included in the third frequency class.
The total polyphenol content of the wine was 360 mg/L. In the vinegars this parameter had a mean value of 365, with a minimum of 305 for the strain of
A. pasteurianus 418 and a maximum of 395 for the strain of A. pasteurianus
323. Figure 6 reports these values grouped in three frequency classes. The strain of A. aceti 117 (345 mg/L) and the G. hansenii 435 (355 mg/L) are included in the second frequency class; A. aceti 230 (365 mg/L) is included in the third frequency class.
PERSPECTIVES
A remarkable biodiversity was observed among the strains.
This technological survey constitutes a basic step to exploit the bacterial biodiversity in the Calabrian ecosystems. Such biodiversity could be further explored with the ultimate goal of carrying out selection programmes of these strains.
Table 1. List of the 35 strains of Calabrian
acetic acid bacteria employed. Strain Identification 15 Acetobacter pasteurianus 40 Acetobacter pasteurianus 52 Acetobacter pasteurianus 66 Acetobacter pasteurianus 76 Acetobacter pasteurianus 98 Acetobacter pasteurianus 104 Acetobacter pasteurianus 117 Acetobacter aceti 129 Acetobacter pasteurianus 145 Acetobacter pasteurianus 158 Acetobacter pasteurianus 174 Acetobacter pasteurianus 181 Acetobacter pasteurianus 203 Acetobacter pasteurianus 210 Acetobacter pasteurianus 221 Acetobacter pasteurianus 230 Acetobacter aceti 244 Acetobacter pasteurianus 254 Acetobacter pasteurianus 262 Acetobacter pasteurianus 276 Acetobacter pasteurianus 281 Acetobacter pasteurianus 308 Acetobacter pasteurianus 323 Acetobacter pasteurianus 349 Acetobacter pasteurianus 351 Acetobacter pasteurianus 361 Acetobacter pasteurianus 376 Acetobacter pasteurianus 390 Acetobacter pasteurianus 402 Acetobacter pasteurianus 418 Acetobacter pasteurianus 435 Gluconacetobacter hansenii 448 Acetobacter pasteurianus 460 Acetobacter pasteurianus 537 Acetobacter pasteurianus
Figure 1. Pre-selection of the
acetic acid bacteria using
Glucose Yeast extract Calcium carbonate Agar
Figure 2. Micro-acetification trials
0 10 20 30 40 50 60 70 P e r c e nt a ge of s tr a ins 3.06 - 3.10 3.11 - 3.15 ≥ 3.16 Range of pH
Figure 3. Distribution of the 35 strains of Calabrian acetic acid bacteria in frequency classes for the pH value in vinegar (wine: 4.56)
0 10 20 30 40 50 60 70 80 90 100 P e r c e nt a ge of s tr a ins ≤ 6.00 6.01 - 7.00 7.01 - 7.53
Range of titratable acidity (g of acetic acid %)
Figure 4. Distribution of the 35 strains of Calabrian acetic acid bacteria in frequency classes for the titratable acidity content in
vinegar (wine: 0.88 g %) 0 10 20 30 40 50 60 70 80 P e r c e nt a ge of s tr a ins ≤ 2.50 2.51 - 3.00 3.01 - 3.50 Range of glycerol (g/L)
Figure 5. Distribution of the 35 strains of Calabrian acetic acid bacteria in frequency classes for the glycerol content in vinegar
(wine: 4.05 g/L) 0 10 20 30 40 50 60 P e r c e nt a ge of s tr a ins 305 - 334 335 - 364 365 - 395
Range of total polyphenols (mg/L)
Figure 6. Distribution of the 35 strains of Calabrian acetic acid bacteria in frequency classes for the total polyphenol content in
