Banana Vinegars Production Using Thermotolerant Acetobacter pasteurianus Isolated From Ivorian Palm Wine

Vinegar or sour wine is a product of alcoholic and subsequent acetous fermentation of sugary precursors. Among acetic acid producing bacteria, only few genera (Acetobacter and Gluconobacter) are used in vinegar industry. In this paper, we intended to produce vinegar at 37 °C using two Acetobacter pasteurianus strains (S3 and S32). These species were isolated from palm (Elaeis guineensis) wine and presented potentialities for industrial vinegar production at 37 °C. Successive fermentations were carried up and semi-continuous acetous fermentation was performed to increase acid production. Concentrated bananas (Musa ssp.) juice (11°Brix) was fermented using Saccharomyces cerevisae within 7 days, yielding 6.4% alcohol. After fermentation, 60 and 58 g/L acetic acid were produced in vinegars obtained using S3 and S32 stains respectively in 34 days and 5 flow cycles. Malic and acetic acids were the most substantial acids produced in alcoholic juice with 5 631.473 and 2 833.055 mg/L respectively. Among the eight organic acids responsible for vinegars total acidity, acetic acid was major compound with 23 459.416 and 21 268.407 mg/L for S3 and S32 strains respectively. Alcohol and acetic acid fermentation efficiency were 90.9% and 85.39 87.63% respectively. All the results above showed that S3 and S32 strains revealed great potentialities for successful industrial vinegar production from overripe banana.


Introduction
Food deterioration can be due to a combination of various factors like light, oxygen, heat, humidity and/or contamination of all kind of microorganisms (Yusuf, Jibril, Misau, & Danjuma, 2012).Among these microorganisms, some having beneficial effects are widely used in Biotechnology for production of compound such as vinegar, spirit, wine and antibiotics.This ability allows processing and storage of agro-perishable foods to preserve or to valorize them over a long period of time.That is necessary for developping countries both economically and socially (Seyram, Ameyapoh, Karou, & De Souza, 2009).
Concentrated juice with 78.18% TS and 13.15% RS decreased in vinegars (7.25-8.36%and 0.79-0.57%,for vinegar with S3 and S32).The results of the evaluation of Brix during alcohol and acetic fermentations showed decreasing rates of dry contents.Banana juice (11°Brix) was finally 2 °Brix at the end of acetic fermentation HPLC analysis of acid contents in alcoholic and produced vinegars, eight major organic acids (Table 2): Tartaric, Malic, Ascorbic, Citric, Oxalic, Formica, Lactic and Acetic acids.In alcoholic juice, Malic and Acetic acids were the most important acids produced with 5 631.5 and 2 833.1 mg/L respectively.Acetic acid was the major acid produced in vinegars obtained from alcoholic juice with 23 459.4 mg/L and 21 268.4 mg/L for S3 and S32 strains respectively.Figure 6 shows chromatograms of 4 organic acids analyses by HPLC means in vinegars with method 2. It presents acetic acid as the major organic acid produced among detected acids (all detected acids concentrations are provided by   et al. (2011).These authors suggested that variability in alcohol production could be due to metabolic behaviour and adaptability of yeast to different sugar concentrations and fermentation conditions.
The semi-continuous acetification yielded 6% and 5.8% Acetic acid respectively with S3 and S32 vinegars.FE was 87.63% and 85.39% for S3 and S32.Ndoye et al. (2007) produced experimental vinegar in bioreactor by semi-continuous method at 35°C and obtained 8-9 % of acetic acid within 35 days.Their acetic acid yield was greater-than our productions because of the use of bioreactor where acetification parameters are carefully under control.Ameyapoh et al. (2010) and Yusuf et al. (2012) produced both 47 g/L from mango and banana at 30 °C within respectively 15 and 11days continuous fermentation.45 g/L Banana vinegar was also produced in continuous fermentation by Seyram et al. (2009) within 23-25 days at 30 °C while Kaur et al. (2011) used immobilized Acetobacter aceti cells to produce 47 g/L and 44 g/L total and volatile acidity by semi-continuous fermentation at 30 °C in 36 h.Comparatively to these productions, vinegars produced by the two thermotolerant Acetobacter pasteurianus were interesting considering their capacity of acetification at high temperature (37 °C).Gullo, De Vero and Giudici (2009) reported that A. pasteurianus strains have positive effects to start acetification processes from musts that not contain acetic acid initially.They are also able to promote the growth of other naturally occurring bacteria.According to FDA (Food and Drug Administration of the United States), vinegar is defined as a liquid containing 4% acetic acid corresponding to 4 g acetic acid per 100 mL of alcoholic fermented solution obtained from sugar solutions (Beheshti & Shafiee, 2009).Furthermore, Ndoye et al. (2006) stated that Acetobacter strains capable of producing 1.7 g/L acetic acid can be used as acetators in fermenting processes.From then on, these strains present potentialities to produce industrial vinegar.However, some experiments in bioreactors should be carried out to confirm this predisposition.
The highest acid concentrations in both vinegars were obtained with acetic acid (23459.4mg/L for S3 and 21268.4mg/L for S32).These results are confirmed by Walter (2005) who showed that acetic acid is the major acid produced in vinegars and constitute with organic acids like Citric acid, Tartric acid, Malic acid and Succinic acid, the total acid contents in vinegars.Yang and Choong (2001) also agree that all volatile short chains acids affect total acidity in acetic fermentation but acetic acid remains the main acid produced with Propionic acid and butyric acid in very low yields.
The low rate of dry matter (39.25%) is banana paste is in accordance with 23.96 to 42.63% dry matter in pulps obtained by Coulibaly, Nemlin and Kamenan (2007) from some banana varieties.Olivier et al. (2009) mentioned also 41.1% and Assemand, Camara, Kouamé, Konan and Kouamé (2012) reported 37.87% to 42.8% when characterizing some «Agnrin» banana varieties.These variations of dry matter contents in bananas can be explained by differences between ripening states of bananas.According to Assemand et al. (2012), the reduction of dry matter contents of bananas in ripening state is due to the increasing water rate that was helped by water osmotic migration.Otherwise, the decreasing rates observed all along vinegars making until vinegars (2.27% and 2.08 %) are attributable to transformation steps (pressure and centrifugation).inorder to clarify vinegars.
Concerning pH variations, Olivier et al. (2009) reported pH 5.6 as the highest value observable in bananas in ripening phase.Hailu, Workneh, and Belew (2012) observed also the same variations in ripening bananas .This was due to acidification brought by amylolytic enzymes and fruits water content that increased after degradation of polymeric carbon hydrates making their conversion easier into acids (Hsiao & Siebert, 1999).The decreasing pH values until 2.63 and 2.71 in vinegars were attributable to volatile acids brought in juices through alcoholic and acetic fermentations.
Total sugar (TS) and reducing sugar (RS) rates obtained were more or less comparable with those obtained by Limpaiboon, Kankaew and Wongwicharn (2011) and Lii, Chang, and Young (1982).These authors obtained respectively 22.16% and 33.6% TS.Moreover, Assemand et al. (2012) was reporting 29.97% TS and 8.66% RS.These various rates of sugar contents in bananas were justified by Kouame, Camara, and Dick (2010) by the difference between banana varieties compositions and the ripening system used.Otherwise, Forster, Rodríguez, and Romero (2002) affirmed that changes into bananas composition are linked to farming methods and to soils structures of regions.The decreasing sugar contents from concentrated juice to vinegars, proportional to Brix variations was due to the use of sugar by yeast and acetic bacteria for growing (Ameyapoh et al., 2010).

Conclusion
The present study successfully achieved vinegar production from banana.Alcoholic fermentation within one week with Saccharomyces cerevisae and acetous fermentation during 34 days using two Acetobacter pasteurianus strains isolated from Ivorian palm wine were performed.6.4% alcohol and 5.8-6 acetic acid degree vinegars were yielded with 90.9% and 85.39%-87.63%fermentation efficiency for alcoholic and acetous fermentations respectively.The semi-continuous fermentation method of banana wine was well experimented and consequently, the two acetic strains are really empowered for industrial vinegar production.
The mean value of pH observed in overripe bananas was 4.92.It turned into 5.11 in banana juice before decreasing until 2.63 and 2.71 respectively in vinegars obtained from S3 and S32.
was carried up used to increa juice were see id production n cycles (Figu reasing or con 50 mL to start on at 10 000 rp ry, Romero, & emi-continuou l rate; Ac = Ac nana juice wa were isolated d every day un p after a pre se vinegar pro ded with 1 mL was determine ure 2) were sta nstant acid valu t a new one.F pm, 25 °C for 1 & Cantero, 200 us operation pr cidity of juice s inoculated w from Elaeis g

Table 2 .
Organic acids contents of banana vinegars by HPLC analysis