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Isolation of Acetic Acid Bacteria and Preparation of Starter Culture for Apple Cider Vinegar Fermentation

Advances in Microbiology, 2019, 9, 556-569
ISSN Online: 2165-3410
ISSN Print: 2165-3402
10.4236/aim.2019.96034 Jun. 30, 2019 556 Advances in Microbiology
Isolation of Acetic Acid Bacteria and
Preparation of Starter Culture for Apple Cider
Vinegar Fermentation
Bernadette Mathew, Shaily Agrawal, Nandita Nashikkar, Sunita Bundale*, Avinash Upadhyay
Hislop School of Biotechnology, Hislop College, Nagpur, Maharashtra, India
are commonly used as food condiments and preservatives. Apple
cider vinegar (ACV) is also used in the Ayurvedic pharmaceutical industry
because of its medicinal properties. Since specific
ally selected starter cultures
for commercial vinegar production are not readily available, apple juice sup-
plemented with sugar is commonly inoculated with a microbiologically unde-
fined culture obtained from the previous batch of ACV. The present work
ses on the isolation of yeasts and acetic acid bacteria from ACV and the
preparation of a starter culture. ACV was produced in a bench scale bioreac-
tor using a traditional fermentation process wherein an acetic acid concentra-
tion of 3.8% was obtained after three weeks. Several acetic acid bacteria
(AAB) were isolated from ACV using selective media. Microscopy revealed
the cultures to be gram negative to gram variable short rods. The growth pat-
tern of the isolates on differenti
al media and biochemical tests suggested the
presence of
Ten potent isolates were
selected for starter culture preparation. Two consortia were formulated with
five AAB isolates in each along with a yeast isolate and used for ACV produc-
tion, wherein an acetic acid concentration of 4.2% - 4.9% was obtained in 10 -
12 days. Thus,
these two starter cultures with locally isolated AAB can be
used for the commercial production of apple cider vinegar.
, Apple Cider Vinegar, Starter Culture, Acetic
1. Introduction
Vinegar, from the French
vin aigre
, meaning “sour wine,” can be made from
How to cite this paper:
S., Nashikkar, N., Bundale,
S. and
, A. (2019)
Isolation of Acetic
Acid Bacteria and Preparation of Starter
Culture for Apple Cider Vinegar Ferment
Advances in Microbiology
, 556-569.
May 5, 2019
June 27, 2019
June 30, 2019
Copyright © 201
9 by author(s) and
Research Publishing Inc.
This work is licensed under the Creative
Commons Attribution International
License (CC BY
Open Access
B. Mathew et al.
10.4236/aim.2019.96034 557 Advances in Microbiology
almost any fermentable carbohydrate source, including wines and ciders, mo-
lasses, dates, grains, honey, maple syrup, starchy vegetables, whey and fruits [1].
It is a widely used food preservative and condiment. According to FDA (Food
and Drug Administration of the United States), it contains 4% acetic acid that is
produced from sugary or alcoholic materials through fermentation along with
varying amounts of fixed fruit acids, salts, coloring materials and some volatile
products such as esters and phenolics which impart characteristic aroma and
flavor [2]. Apple cider vinegar (ACV), one of the most popular vinegars, is well
known for its medicinal properties and general health benefits. It contains trace
minerals and vitamins-A, C, E and several different forms of vitamin-B includ-
ing beta carotene and bioflavonoid that are needed for cell function. It’s anti-
oxidant, anti glycemic, anti hypertensive, antibacterial, antifungal and anti
tumor properties that are also well established [3]. Further, ACV is extensively
used as the base in Ayurvedic preparations termed “asavas”.
Traditionally, fruit juices have been used for domestic as well as industrial
production of vinegar in a two-step processthe fermentative production of al-
cohol from the fruit sugars by yeasts, mainly
Saccharomyces ellipsoideus
Saccharomyces cerevisiae
followed by the oxidation of the ethanol and residual
or added sugars to acetic acid by AAB [4]. These are obligate aerobic Gram
negative or Gram variable, ellipsoidal to rod-shaped, straight or slightly curved,
0.6 - 0.8 μm × 1.0 - 0.4 μm, occurring singly, in pairs or chains. Pleomorphic
forms occur which may be spherical, elongated, swollen, club shaped, curved or
filamentous. They are able to oxidize substrates such as glucose, ethanol, lactate
or glycerol to acetic acid. On the basis of their abilities to over oxidize acetate or
lactate and the positions of their flagella, these bacteria are conventionally cate-
gorized into two major genera
In liquid me-
forms a film or pellicle made of cellulose. The AAB and yeasts
present in the fermentation broth get entangled in the cellulosic pellicle to form
a mat-like structure called the “mother of vinegar” [5].
Alcohol and wine vinegars including apple cider vinegar are most often pro-
duced in submerged bioreactors, which supply the bacteria with a constant in-
flow of oxygen and enable an efficient production process [6]. The oxidation is
started by adding the “mother of vinegar” obtained from previous vinegar to the
fresh fermentation. This is a microbiologically undefined culture and very few
in-depth studies are available about the AAB and other microbes in such seed
cultures, which leads to variation in the product quality. Moreover, most
processes require at least three weeks to produce an adequate concentration of
acetic acid. Hence, there is a pressing need for easily available “starter cultures”
so that product quality is maintained and fermentation time is cut short [7].
The present work focuses on the bench scale production of ACV using a tra-
ditional apple recipe for isolation of AAB and yeast strains and formulation and
assessment of mixed starter cultures made up of yeast and high acetic acid
producing isolates in view of rapid and maximum production of acetic acid.
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10.4236/aim.2019.96034 558 Advances in Microbiology
2. Materials and Methods
2.1. Chemicals and Reagents
All chemicals and solvents were of analytical grade and purchased from Merck,
2.2. Culture Media
Culture media were purchased from Hi-media, Mumbai, India. A few media
were modified as per the requirements. Several selective and differential media
such as Carr medium [8] with bromothymol blue as indicator, Frateur medium
[9], GYC medium containing glucose 4%, yeast extract 1%, CaCO3 1%, agar
1.5%, DSM agar [10] containing dextrose sorbitol and mannitol and MS agar, a
mannitol salts medium with phenol red as the indicator were used for the
enrichment, isolation and differentiation of AAB. Potato dextrose agar (PDA)
was used to isolate yeasts. A glucose (10%) yeast extract (5%) peptone (3%) me-
dium (GYP) was used for the production of ACV using the selected isolates and
the formulated starter cultures.
2.3. Production of ACV for Isolation of AAB
ACV was prepared using suitably modified reagent bottles as small bioreactors.
Holes were punched in the lids to allow the CO2 produced during fermentation
to escape. Seven experimental bioreactors were organized each containing 50 g
crushed apples and 2 g glucose in 60 ml water. Commercially available dried ac-
tive yeast powder containing lyophilized
Saccharomyces cerevisiae
or innocula
from different old unpasteurized wines were added to hasten alcoholic fermen-
tation. After 72 h the contents of the bottles were filtered through muslin cloth
and transferred to conical flasks. The flasks were thereafter incubated aerobically
on a shaker incubator or statically at 30˚C for four weeks.
2.4. Estimation of Acetic Acid
Aliquots were withdrawn regularly and acetic acid was estimated titrimerically
[11]. The acetic acid in vinegar represents 98% of acids [12]; the total acidity is
therefore also a measure for acetic acid concentration.
2.5. Isolation of Yeasts and AAB from ACV Bioreactors
Appropriately diluted samples and pellicle material from the flasks showing the
highest concentration of acetic acid were plated on Frateur medium containing
ethanol and CaCO3 for initial isolation of AAB. Isolated pure cultures were
maintained on GYP agar slants containing CaCO3 and sub cultured regularly.
PDA was used for the isolation of yeasts from the bioreactors at 48 h. A loop-
ful of the fermenting liquid was streaked on to PDA plates and incubated at
30˚C. Isolated pure yeast cultures were maintained on PDA slants and sub
cultured every three weeks.
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2.6. Microscopic and Biochemical Characterization of Yeast and
AAB Isolates
The selected AAB isolates were gram stained and the morphology observed. Mo-
tility was observed by the hanging drop method. Standard biochemical tests
generally used to identify AAB such as production of catalase and oxidase, ni-
trate reduction, and Voges Proskauer’s test was performed [13]. The utilization
of various carbohydrates such as glucose, trehalose, malonate, arabinose, man-
nitol, citrate, sucrose, and arginine was also studied. The yeast cultures were
simple stained and the morphology observed.
2.7. Differentiation between Acetobacter and Gluconobacter
Twelve selected AAB pure cultures were streaked onto differential media such as
DSM, MS and Carr medium to differentiate between
. Biochemical characterization, including carbohydrate utilization, along
with differences in growth pattern on the differential media was considered to
categorize the isolates as species of
2.8. Preparation of Mixed Starter Cultures
Two sets of starter cultures were prepared, designated SC-I and SC-II with five AAB
isolates comprising of both
cultures in each group.
The selected AAB isolates were inoculated in 5 ml each of GYP medium in
test tubes and incubated for 48 h at room temperature. SC-I consisted of one ml
of 48 h old broth cultures each of isolates A1, A5, G3, G8, A9 and SC-II con-
sisted of one ml of 48 h old broth cultures each of isolates A2, A4, A6, G7, G10.
The yeast isolate was cultivated in potato dextrose broth and 1ml of a 72 h yeast
culture was included in the starter cultures SC-I and SC-II.
2.9. ACV Production Using Prepared Starter Cultures
The pre-formulated starter cultures SC-I and SC-II were added to two different
250 ml Erlenmeyer flasks containing 100 ml sterile GYP medium containing 50 g
crushed apples. The inoculated flasks were stoppered with a rubber cork and in-
cubated statically for 48 h for ethanolic fermentation. The side arm tube facili-
tated the dispersal of liberated CO2. Thereafter, the flasks were placed on a rota-
ry shaker at 80 rpm at 28˚C to facilitate the aerobic acetification. After six days
some of the flasks were incubated statically to observe pellicle formation. All the
flasks were monitored for two weeks. Aliquots were withdrawn at 48hr intervals
and acetic acid was estimated.
3. Results
3.1. Production of ACV for Isolation of AAB
All the seven ACV bioreactors showed active bubbling of CO2 within 24 h indi-
cating a robust ethanolic fermentation (Figure 1). This fermented dry apple cid-
er after 48 h had a sweetish taste and an alcoholic odor.
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Figure 1. Production of ACV for isolation of AAB; robust ethanolic fermentation of ap-
ple juice using various innocula.
An acetic acid concentration above the required 4% was observed only after
six weeks and only in ACV bioreactors B and D. The acetic acid concentration in
the other bioreactors was found to be less than 1%. Hence bioreactors B and D
were used for the isolation of AAB. The ACV flask B incubated statically devel-
oped a typical pellicle produced by
Acetobacter spp
(Figure 2).
3.2. Isolation of Yeast from ACV Bioreactors
The fermented apple cider at 72 h was used for the isolation of yeast. The yeast
cultures were identified microscopically. Since commercial active dry yeast
powder containing
S. cerevisiae
had been added to hasten ethanolic fermenta-
tion, the same yeast was isolated and pure cultures were maintained on PDA
3.3. Isolation of AAB from ACV Bioreactors
The supernatant and pellicle in the ACV flasks B and D which showed maxi-
mum acetic acid production was used for isolation of AAB. Initial screening was
done on GYC and Frateur medium containing 2% ethanol. Pin point colonies
with relatively large zones of clearance as well as medium sized colonies with
CaCO3 clearance zones were preliminarily selected as AAB (Figure 3).
The selected isolates were streaked on to Carr medium and acid production
was confirmed by the appearance of yellow zones. A total of 12 isolates selected
as hyper producing AAB were obtained as pure cultures. These isolates which
appeared to be distinctly different from each other based on their colony mor-
phology were used for further characterization (Figure 4).
3.4. Cultural & Microscopic Characterization
The 12 selected isolates were all aerobic, producing pin point to small colonies
which were moist, translucent, white, beige or yellowish in color. These were
found to be gram negative or gram variable (isolates G3 and A5) small rods,
straight or slightly curved, coccoid or club shaped. All the isolates except A6 and
G7 were found to be motile.
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Figure 2. Production of ACV for isolation of AAB; Acetification with pellicle formation.
Figure 3. Screening for acetic acid bacteria on Frateur medium containing ethanol. The
colonies showing CaCO3 clearance zones were selected as AAB.
Figure 4. Pure cultures of AAB isolates.
3.5. Biochemical Characterization
Ten isolates showing significant zones of clearance on Frateur agar and which
were found to be catalase positive and oxidase negative were preliminarily iden-
tified as AAB according to the standard guidelines of Bergey’s manual of Deter-
minative Bacteriology [14].
The results of the biochemical characteristics are presented in Table 1.
Table 2 depicts the utilization of some selected carbohydrates by the ten iso-
lates putatively identified as AAB. All the isolates were able to ferment glucose
with acid production as expected whereas sucrose was not utilized except for a
variable +/− result shown by isolate G10. Mannitol was utilized by all the iso-
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Table 1. Biochemical characterization of AAB isolates.
Gram stain
Nitrate reduction
A1 ve + +
A2 ve + + + +
G3 var + + +
A4 ve + +
A5 var + + +
A6 ve +
G7 ve + + +
G8 ve + + +
A9 ve + + + +
G10 ve + + + +
ve indicates gram negative, var. indicates gram variable; +indicates positive reaction, −indicates negative
Table 2. Utilization of carbohydrates by AAB isolates.
A1 + +/− + + +
A2 + + +/− + + +
G3 + + + +
A4 + +/− +/− +/− +/− +
A5 + + + + +/− +
A6 + + + +/− +
G7 + + +/− +/−
G8 + +/− +/−
A9 + +/− +/− + + +/− +
G10 +/− +/− + + +
+indicates acid production, −indicates no acid production and +/−indicates a variable reaction.
3.6. Identification of the Genera Acetobacter Isolated from ACV
Six isolates were able to utilize glucose, arabinose, mannitol (variable) and tre-
halose but were unable to utilize sucrose (Table 2).
are distin-
guished by their capability to over oxidize alcohols to acetic acid and then to
CO2 and H2O [15]. When these six isolates were grown on MS agar, a change of
color from red (neutral pH) to yellow (acidic) was observed within 48 h which
started reverting back to red after 96 h, typical of the over oxidizing
(Figure 5).
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Figure 5.
isolates A5 and A7 showing overoxidation on MS medium con-
taining phenol red as the pH indicator.
These strains registered positive growth on lactate. Growth on DSM agar re-
sulted in a color change of the medium to purple indicative of preferential lac-
tate utilization which is characteristic of
Acetobacter spp
. (Figure 6(B)).
When grown on Carr medium, these isolates produced greenish colonies
(Figure 6(A)).
Thus, based on the morphological, microscopic and biochemical characteriza-
tion, these isolates were identified as
species and designated as A1,
A2, A4, A5, A6 and A9.
3.7. Identification of the Genera Gluconobacter Isolated from ACV
Four isolates were able to utilize glucose, mannitol (variable reaction) and ara-
binose but were unable to use trehalose and sucrose. When grown on MS agar
containing phenol red these isolates showed change of color from red to yellow
indicating acid production within 48 h but the color did not revert back to red,
typical of the under oxidizing
Gluconobacter spp.
(Figure 7).
The yellow color of the DSM medium was maintained indicating an inability
to use lactate, which is characteristic of
species. These isolates
grew as white colonies on Carr medium (Figure 6(A)).
Thus, based on the morphological, microscopic and biochemical characteriza-
tion, these isolates were identified as
species and designated as
G3, G7, G8, G10.
3.8. ACV Production Using Prepared Starter Cultures
SC-I and SC-II described above were used for the production of ACV and an
acetic acid concentration of 4%, which is prescribed for vinegar was attained
within 12 days with both the mixed starter cultures as depicted in Figure 8.
However, the acetic acid concentration in the fermentation flask with SC-I con-
tinued to increase, reaching 5% on the 14th day. On the contrary, an acetic acid
concentration of 3.8% was obtained in the control flask with no added inoculum
after four weeks. Thus, the fermentation time for ACV production using the
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Figure 6. (A) Colony morphology of isolates G7 and A1 on Carr agar supplemented with
bromothymol blue (B) Colony morphology of isolates A1, A2 and A6 on DSM agar sup-
plemented with bromocresol purple.
Figure 7.
isolates G3 and G10 showing underoxidation on MS medium
containing phenol red as the pH indicator.
Figure 8. ACV production using prepared starter cultures. SC-1: Starter Culture 1; SC-2:
Starter Culture 2; Control: No added inoculum.
starter cultures was reduced considerably as compared to the time required to
obtain an acetic acid concentration of 4% using the traditional process for ACV
production. The acetic acid concentration obtained upon using the AAB isolates
in monoculture fermentation ranged from 0.4% to 1.2%. Thus, ACV production
using a pre-formulated mixed starter culture was found to be more effective as
compared to both-monoculture fermentation as well as traditional natural fer-
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mentation with no added inoculum.
4. Discussion
Several authors have worked on the isolation and characterization of acetic acid
bacteria from sugary and starchy substrates and oriental fermented foods [16]
[17]. However, there are relatively few reports on the laboratory production of
ACV specifically for the isolation of yeasts and AAB to be used for the develop-
ment of a starter culture. The present work began with the bench scale produc-
tion of ACV using a traditional recipe with apples. The initial ethanolic fermen-
tation is carried out by several yeasts found naturally on fruits. These wild yeasts
mainly belong to the species
[18]. The yeast culture is not removed from the bioreactor after the ethanolic
fermentation and ethanol and acetic acid production may go on simultaneously
[19]. Hence samples were withdrawn throughout the course of the fermentation
in an effort to isolate high acid producing and ethanol tolerant AAB strains.
These bacteria are difficult to isolate and culture and Frateur medium, which
supports the growth of all strains of AAB. [20] was used in the initial isolation of
high acid producing strains. This medium contained ethanol as the major car-
bon source hence the clearance of CaCO3 was due to its solubilization by the
acetic acid produced by the AAB colonies. Twelve hyper acid producing isolates
were identified by taking into consideration the clearance zone: colony size ratio.
Microscopic and biochemical characterization indicated the isolates to be
AAB. These were differentiated into
by tradi-
tional methods, mainly based on their ability/inability to over-oxidize ethanol to
carbon dioxide and water, their ability/inability to oxidize lactate and their
comparative ability to utilize selected sugars. Differential media such as DSM
agar, MS agar and Carr medium were used to differentiate
DSM agar contains lactate as the major carbon source and bro-
mocresol purple as the pH indicator. Isolates belonging to the genus
were able to utilize lactate resulting in an increase of pH causing a color change
of the medium from yellow to purple whereas isolates belonging to the genus
were unable to oxidize lactate and the yellow color of the me-
dium was maintained as there was no increase of the pH [21].
MS agar contains mannitol as the major carbon source and phenol red as the
pH indicator. All the isolates grew well, produced acid and turned the medium
yellow. However,
species possess a functional tricarboxylic acid
cycle and can further oxidize the acetic acid to CO2 and H2O when there is a
high level of dissolved oxygen and no ethanol in the medium. This acetate over-
oxidation resulted in a reversal of the color back to red.
on the other hands, are underoxidizers and hence no color reversal is observed
[22]. The growth pattern on Carr’s medium further confirmed the differentia-
tion of
The main species responsible for the production of vinegar belong to the ge-
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10.4236/aim.2019.96034 566 Advances in Microbiology
cause of their superior capacity to oxidize ethanol and resistance to acetic acid
released into the fermentative medium. AAB are fastidious organisms and
strains are known to lose several features, including their ability to produce
higher concentrations of acetic acid upon subculturing [23]. Thus maintenance
of AAB isolated from bioreactors too is not easy since these are known to quick-
ly pass into the viable but not culturable state (VNBC). This is mainly due to the
lower oxygen availability and the considerable drop in pH because of the conti-
nuous acetic acid production which is an energy related or primary metabolite
for AAB [24]. In the present work, the isolates were maintained on GYP agar
slants containing CaCO3. The inclusion of CaCO3 ameliorates this problem to a
great extent by neutralizing the acetic acid produced and relieving the physio-
logical stress which drives the cells into the VBNC state [25].
In the present study, the selected AAB isolates were used in mono culture as
well as mixed culture fermentations to assess their hyper acid production capac-
ity in the shortest time. The yield of acetic acid in the single culture fermenta-
tions was very poor even after four weeks. Sossoou
et al.
[26] have also reported
a similar fermentation time of 23 - 25 days for production of vinegar with
strains isolated from pineapple juice. But Nanda
et al.
[27] isolated
hyper producing
from various fruits and have reported the
production of around 3% - 4% acetic acid within 4 days in GYP medium con-
taining 4% ethanol using their isolates in monoculture.
In the mixed culture fermentations carried out in this study using SC-I and
SC-II, an acetic acid concentration of 4% which is prescribed by FAO for vinegar
was attained within 12 days. The starter cultures formulated in this study possi-
bly exhibit some synergistic enhancement of acetic acid production leading to a
50% reduction in the fermentation period as compared to the natural vinegar
fermentation period of over four weeks. In the natural fermentation of sugary or
alcoholic substrates, there is a succession of microbes beginning with high sugar
tolerant yeast strains and moving on to AAB [28]. During acetification, in the
early stage, low acid tolerant AAB such as
Acetobacter pasteuranus
and in the later stages, high acid tolerant AAB such as
Gluconacetobacter intermedius
predominantly present. Al-
though a few reviews and studies [29] [30] using known AAB strains for acetic
acid production exist, no preformulated mixed culture studies with yeast and
AAB isolates are documented to the best of our knowledge. Moreover, our start-
er cultures are composed of locally isolated strains which may be better adapted
to the prevailing conditions. The importance of locally isolated AAB has been
highlighted by other researchers too
[31]. Thus the consortia developed in this
study may be successfully used for the commercial production of ACV. The
main factors deterring the availability of starter cultures are difficulties in cul-
turing the AAB and preserving their acid forming potential in the laboratory.
Also, the costs for production of the starter cultures may be higher compared
with using seed cultures from previous production batches. The advantage of the
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10.4236/aim.2019.96034 567 Advances in Microbiology
starter culture is that it facilitates a more controlled process that is easier to re-
produce and control and gives a standardized product [32]. A comprehensive
optimization programme of the various process parameters affecting acetic acid
production, studying the acid tolerance profile of the isolates and a systematic
trial of permutations and combinations of our isolates in the formulation of the
starter cultures will lead to further increase in acetic acid concentration within a
shorter fermentation period.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this
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... The yeast was capable of fermenting sugars in Garcina kola and Acer pseudoplatanus into alcohol. The yeast culture was removed after the alcohol fermentation and acetic acid production follows this was in correspondence to [21]. Several authors have worked on isolation and characterization of acetic acid bacteria from sugary and starchy substrates and oriental fermented foods [22]. ...
... The acetic acid concentration obtained upon using the AAB isolates in monoculture fermentation ranged from 0.4% to 1.2%. Thus, ACV production using a pre-formulated mixed starter culture was found to be more effective as compared to both-monoculture fermentation as well as traditional natural fermentation with no added inoculum [21]. ...
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Vinegar is the product made from the conversion of ethyl alcohol to acetic acid by a genus of bacteria Acetobacter. This work is based on the ability of vinegar to be produced from Garcina kola and Acer pseudoplatanus. The production of vinegar from bitter kola and sycamore to avoid waste or spoilage of the fruits which can serve for preservation and food preparation was the essence of the study carried out. It is a useful means to help ensure that losses incurred with fruits are reduced and the vinegar produced can help to properly preserve some foods against spoilage. Bitter kola and sycamore were processed, cut and eventually blended to evaluate the production and quality of the vinegar being produced. The bitter kola and sycamore were fermentation was carried out with added inoculant and naturally by indigenous inoculant for 7d at 30 o C. Results showed that pH, alcohol content and specific gravity were 4.0, 0.5 and 1.001g/cm 3 respectively. The acetic acid yields of the vinegars produced were within the range of 0.43%-1.84% due to the use of monoculture which was indigenous in the fruit and Braggs vinegar with mother. Microbiological and biochemical analysis was carried out during alcoholic and vinegar fermentation. The antimicrobial potential of the vinegars was also tested and found effective on clinical pathogens. The test proved that the G. kola had the most antimicrobial properties against the bacterial isolates than the A. Pseudoplatanus which had the lowest.
... The ratio or difference in the speed of light is called refractive index & refractometers are called instruments that calculate this parameter. A liquid's refractive index is related to its concentration and so the concentration can be displayed in relevant units such as brix by a refractometer ( percent sugar) [11]. the experiment was conducted according to the method described in. ...
... Pure bacterial colonies underwent macro and microscopic observations by studying the shape, size, arrangement, Gram staining, pigmentation and motility test, by inoculating the colonies in YPG medium at an incubation temperature of 30 °C for 72 h. Classical biochemical tests, such as catalase activity, cytochrome oxidase, growth in peptone, presence of pigmentation in YPG medium and growth on YPG medium containing D-glucose > 30% were used according to the protocol described by [5,39]. Acetate oxidation and ethanol overoxidation to C2O and H2O were performed to distinguish between the genera Acetobacter and Gluconobacter. ...
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The production of vinegar on an industrial scale from different raw materials is subject to constraints, notably the low tolerance of acetic acid bacteria (AAB) to high temperatures and high ethanol concentrations. In this study, we used 25 samples of different fruits from seven Moroccan biotopes with arid and semi-arid environmental conditions as a basic substrate to isolate thermo- and ethanol-tolerant AAB strains. The isolation and morphological, biochemical and metabolic characterization of these bacteria allowed us to isolate a total number of 400 strains with characters similar to AAB, of which six strains (FAGD1, FAGD10, FAGD18 and GCM2, GCM4, GCM15) were found to be mobile and immobile Gram-negative bacteria with ellipsoidal rod-shaped colonies that clustered in pairs and in isolated chains. These strains are capable of producing acetic acid from ethanol, growing on peptone and oxidizing acetate to CO2 and H2O. Strains FAGD1, FAGD10 and FAGD18 show negative growth on YPG medium containing D-glucose > 30%, while strains GCM2, GCM4 and GCM15 show positive growth. These six strains stand out on CARR indicator medium as isolates of the genus Acetobacter ssp. Analysis of 16S rDNA gene sequencing allowed us to differentiate these strains as Acetobacter fabarum and Acetobacter pasteurianus. The study of the tolerance of these six isolates towards pH showed that most of the six strains are unable to grow at pH 3 and pH 9, with an ideal pH of 5. The behavior of the six strains at different concentrations of ethanol shows an optimal production of acetic acid after incubation at concentrations between 6% and 8% (v/v) of ethanol. All six strains tolerated an ethanol concentration of 16% (v/v). The resistance of the strains to acetic acid differs between the species of AAB. The optimum acetic acid production is obtained at a concentration of 1% (v/v) for the strains of FAGD1, FAGD10 and FAGD18, and 3% (v/v) for GCM2, GCM4 and GCM15. These strains are able to tolerate an acetic acid concentration of up to 6% (v/v). The production kinetics of the six strains show the highest levels of growth and acetic acid production at 30 °C. This rate of growth and acetic acid production is high at 35 °C, 37 °C and 40 °C. Above 40 °C, the production of acid is reduced. All six strains continue to produce acetic acid, even at high temperatures up to 48 °C. These strains can be used in the vinegar production industry to minimize the load on cooling systems, especially in countries with high summer temperatures.
... Comparing the vinegars produced with yeast and that of natural it was observed that the vinegar produced with yeast yielded a little bit higher acetic acid value when compared to the vinegar produced without yeast.The acetic acid concentration obtained upon using the AAB isolates in monoculture fermentation ranged from 0.4% to 1.2%. Thus, ACV production using a pre-formulated mixed starter culture was found to be more effective as compared to bothmonoculture fermentation as well as traditional natural fermentation with no added inoculum [25]. The use of starter cultures for vinegar fermentation gives a more controlled process that is easily to reproduce and gives a standardized product. ...
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Vinegar can be defined as an acetic acid liquid produced through fermentation from a suitable raw material of agricultural origin which contains starch or sugars as a carbon source also fit for human consumption.This work is aimed at determining the vinegar production capability of Garcina kola(Bitter Kola)and Artocarpusheterophyllus(Jack fruit). The bitter kola and jack fruit fermentation was carried out with added inoculant and naturally by indigenous inoculant for seven days at 30 o Cfor alcoholic fermentation and 28 day acetic fermentation where some of the samples were boiled and one of the groups was subjected addition of inoculant while the other contains no added inoculant. Proximate analysis of the produced vinegar was also carried out to have an insight on its nutritional quality. Results showed that the range of the pH of the vinegar was between pH 2.6-2.9 for the bitter kola, 3.20-3.73 for jackfruit with peel and pH 3.20-3.40 for jackfruit without peel. The range of acetic acid yield of the vinegar produced is between 0.80%-2.30% for bitter kola, 0.80-1.92% for jackfruit with peel and 0.98-1.92% for jackfruit without peel. The range of alcohol content was between 0-0.5. The specific gravity ranges from 1.001-1.083. The suspected organism present in the samples is Acetobacter sp. Sample 4A Natural showed to poses the highest colony count of Acetic acid bacteria with 3.49logcfu/ml. The jackfruit with peel vinegar had the highest protein, ash and total solid content with 2.45%, 1.5% and 17.3%. The jackfruit without peel vinegar had the highest moisture content with 88.36%. Generally, the substrates used which includes bitterkola and jackfruit showed to be able to be used in vinegar production and contain some nutritional properties although optimization process needs to be carried out to increase production of vinegar with better qualities.
... The ratio or difference in the speed of light is called refractive index & refractometers are called instruments that calculate this parameter. A liquid's refractive index is related to its concentration and so the concentration can be displayed in relevant units such as brix by a refractometer ( percent sugar) [11]. the experiment was conducted according to the method described in. ...
Full-text available
The goal of the present study was to ascertain the effect of the addition of certain natural materials such as sugar or Jaggery Syrup, Black Gram, or extracts there of as fortified materials on the consistency of apple vinegar and the types of microorganisms in it. The findings revealed that the addition of whole legume or their extracts during processing enhanced the properties of the resulting vinegar . The findings showed that the better pH was for apple vinegar produced with the inclusion of sugar and protein extract, which had a pH of 3.1. This was accompanied by vinegar resulting from the addition of jaggery with whole legume, where the pH was 3.8, followed by vinegar resulting from the addition of whole legume plus sugar, where the pH was 3.9. Though apple vinegar obtained by adding protein extract with jaggery had a pH of 4.As it was influenced by the acidity of the resultant vinegar due to the content of legumes and sugary compounds, the maximum acidity was 6.5 in the vinegar to which entire legume jaggy was applied. Then vinegar, to which whole legume sugar was added, and vinegar, to which jaggy with protein extract was added, the acidity reached 3. Finally, vinegar made by combining sugar with protein extract, where the acidity reached 2. The best types of vinegar were selected from among the samples (vinegar developed by the addition of protein extract jaggary) and the contents of its microbiology were analysed. The results showed that the vinegar was free of bacteria and yeasts, while its content of moulds was . The four samples of vinegar also showed good antioxidant activity. It was 86.77%, 85.38%, 79.0% and 19.11% in vinegar containing jaggery with extract protein, sugar with extract protein, jaggery with whole legume and sugar with whole legume respectively. The total soled of vinegar were different, 4.8%, 4.5%, 4.0% and 3.0% in vinegar containing jaggery with whole legume, jaggery with extract protein, sugar with whole legume and sugar with extract protein. The colour analysis of the vinegar sample containing the jaggery with extract protein was 75.91, 4.87, 51.4 for the lighter side, red colour and greener side, respectively.
Vinegar is a common food additive produced by acetic acid bacteria (AAB) during fermentation process. Low yield and long incubation time in conventional vinegar fermentation processes has inspired research in developing efficient fermentation techniques by the activation of AAB for acetic acid production. The present study intends to enhance vinegar production using acetic acid bacteria and light emitting diode (LED). A total of eight acetic acid bacteria were isolated from Korean traditional vinegar and assessed for vinegar production. Isolate AP01 exhibited maximum vinegar production and was identified as Acetobacter pasteurianus based on the 16S rRNA sequences. The optimum fermentation conditions for the isolate AP01 was incubation under static condition at 30 °C for 10 days with 6% initial ethanol concentration. Fermentation under red LED light exhibited maximum vinegar production (3.6%) compared to green (3.5%), blue (3.2%), white (2.2%), and non-LED lights (3.0%). Vinegar produced using red LED showed less toxicity to mouse macrophage cell line (RAW 264.7) and high inhibitory effects on nitric oxide and IL-6 production. The results confirmed that red LED light could be used to increase the yield and decrease incubation time in vinegar fermentation process.
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The flavored vinegars made from wines and fruit are y highlighted in the food gastronomy market. However it is not easy to have a good starter. It is easy to found acetic acid bacteria (AAB) in the natural fermentation of fruits where they are mixed with yeasts. A medium was adapted have only AAB. For test this medium overripe fruits were fermentedby 3 days at room temperature and sampled as inoculum. Bacteria presenting AAB characteristics were identified in microscope. Samples with0.5mlwere placed into Petri dishes containing a modified Frateurmediumcomposed of agar, yeast extract, alcohol, and calcium carbonate. As fungistaticswere tested the gentian violet (1% methylrosanilinium chloride) and nystatin water solution (105 IU) both used at 0.5/1.0/1.5mlon 20mlof the mediumdirectly placed into sterile plates. Petri dishes were incubated at 25°C for five days and AAB colonies recognized by forming a halo. The data showed that only nystatin at dose 1.0 mlcontrolled the wild yeasts growth. Biochemical assays (Gramstaining, oxidase, catalase, indol and H2S formation)confirmed the genus Acetobacter. The data proving that the combination of Frateur medium with 1.0 mlof water solution of nystatin 105 IU)may be a good option for isolating AAB from fermenting fruit.
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The group of Gram-negative bacteria capable of oxidising ethanol to acetic acid is called acetic acid bacteria (AAB). They are widespread in nature and play an important role in the production of food and beverages, such as vinegar and kombucha. The ability to oxidise ethanol to acetic acid also allows the unwanted growth of AAB in other fermented beverages, such as wine, cider, beer and functional and soft beverages, causing an undesirable sour taste. These bacteria are also used in the production of other metabolic products, for example, gluconic acid, L-sorbose and bacterial cellulose, with potential applications in the food and biomedical industries. The classification of AAB into distinct genera has undergone several modifications over the last years, based on morphological, physiological and genetic characteristics. Therefore, this review focuses on the history of taxonomy, biochemical aspects and methods of isolation, identification and quantification of AAB, mainly related to those with important biotechnological applications.
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Thermotolerant microorganisms were collected, identified and characterized under different physiological conditions from various rotten fruits in Bangladesh for vinegar production. Among the 15-isolates characterized previously, the strains F-1, F-3 and F-10 represented Staphylococcus, Bacillus and Acetobacter spp. respectively. After checking various parameters for growth, acetic acid production rate was optimized further. Among the 3-starins analyzed here, the strain F-10 gave maximum acetic acid (7.0 g/100 ml) at 37˚C in 2% ethanol concentration. The strain F-10 is capable of producing high yield of acetic acid at relatively high temperature, which is an ideal condition for vinegar production, which may reduce the water cooing expenses as well as the risk of contamination.
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Organic apple cider vinegar is produced from apples that go through very restricted treatment in orchard. During the first stage of the process, the sugars from apples are fermented by yeasts to cider. The produced ethanol is used as a substrate by acetic acid bacteria in a second separated bioprocess. In both, the organic and conventional apple cider vinegars the ethanol oxidation to acetic acid is initiated by native microbiota that survived alcohol fermentation. We compared the cultivable acetic acid bacterial microbiota in the production of organic and conventional apple cider vinegars from a smoothly running oxidation cycle of a submerged industrial process. In this way we isolated and characterized 96 bacteria from organic and 72 bacteria from conventional apple cider vinegar. Using the restriction analysis of the PCR-amplified 16S-23S rRNA gene ITS regions, we identified four different HaeIII and five different HpaII restriction profiles for bacterial isolates from organic apple cider vinegar. Each type of restriction profile was further analyzed by sequence analysis of the 16S-23S rRNA gene ITS regions, resulting in identification of the following species: Acetobacter pasteurianus (71.90%), Acetobacter ghanensis (12.50%), Komagataeibacter oboediens (9.35%) and Komagataeibacter saccharivorans (6.25%). Using the same analytical approach in conventional apple cider vinegar, we identified only two different HaeIII and two different HpaII restriction profiles of the 16S-23S rRNA gene ITS regions, which belong to the species Acetobacter pasteurianus (66.70%) and Komagataeibacter oboediens (33.30%). Yeasts that are able to resist 30 g/L of acetic acid were isolated from the acetic acid production phase and further identified by sequence analysis of the ITS1-5.8S rDNA- ITS2 region as Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. This study has shown for the first time that the bacterial microbiota for the industrial production of organic apple cider vinegar is clearly more heterogeneous than the bacterial microbiota for the industrial production of conventional apple cider vinegar. Further chemical analysis should reveal if a difference in microbiota composition influences the quality of different types of apple cider vinegar.
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For the isolation of acetic acid bacteria from the »seed vinegar« culture, laboratory vinegar production was initiated with a culture for spirit vinegar production. The second and the third cycles of the process were followed. Samples for the isolation of acetic acid bacteria from the bioprocess liquid were taken four times during each cycle. A total of forty-seven strains were isolated. They were phenotyped as Acetobacter sp. Forty-one of them were identified as A. europaeus. Differentiation among 13 strains of A. europaeus and four other Acetobacter strains not identified as A. europaeus was done by random amplification of polymorphic DNA (RAPD) analysis. Seven different RAPD profiles were identified. From this it was assumed that in spirit vinegar genotypically different strains of acetic acid bacteria were present.
A rapid historical survey from the XVIth to the XXth centuries serves to order and situate the principal landmarks which have contributed to the present level of scientific knowledge, and of the technological advances in vinegar production. The first proposition of an elaboration of a chemical equation, the description of the mother of vinegar, the identification of the importance of wood shavings and of air led to the development of a rapid and more stable method. As soon as scientific knowledge established rules, the resultant profit contributed to the rapid development of corresponding technologies. This progress prepared the way for the valuable contribution of Pasteur: the description of "Mycoderma aceti", the role of oxygen in the atmosphere for the oxidation of ethanol to acetic acid, the possibility of continuous production, and eventually the acetification of high strength vinegar. The submerged fermentation system and computerization have both contributed to these last aspects of technological advance. From the end of the 19th century and throughout the 20th century, the development of industrial equipment and process significantly increased the production and the quality of vinegar. Probably the most significant step forward was the development of the submerged system in 1949. This communication attempts a rapid historical review, with corresponding references, of the fundamental advancements which have contributed to the present understanding and knowledge of the elaboration of vinegar.
Thermotolerant acetic acid bacteria were isolated from 15 kinds of flower namely Allamanda cathartica L., Antigonon leptopus Hook.t Arn., Bidens bipinnaata L., Brunfelsia hopeana Benth., Cassia surattensis Burm.f., Ervatamia divaricata, Euphorbia milii Desmoul., Ixora lobbii Loud., Jasminum multiflorum, Lantana trifolia L., Lonicera japonica Thun., Nerin indicum Mill., Petrea volubilis L., Plumeria acutifolia and Zinnia angustifolia Kunth. using potato medium supplemented with 4% ethanol (v/v) as an enrichment medium. Three different dyes namely 0.0016% bromocresol green, 0.0016% bromocresol purple and 0.0016% bromophenol blue were also incorporated in the enrichment broth for comparison. The numbers of successful isolation obtained from each dye were nearly the same and there were no significant differences between the dyes tested (α>0.05). However, the use of 0.0016% bromocresol purple as indicator for the isolation of thermotolerant acetic acid bacteria from flowers was recommended due to its most easily observed colour change. Morphological and biochemical determinations indicated that most of thermotolerant isolates obtained were members of the genus Gluconobacter.
Sixty thermotolerant acetic acid bacteria were isolated from 13 kinds of fruit using sterile distilled water supplemented with 4% ethanol (v/v) as an enrichment medium. Successful isolations were obtained from apple, Jamaican cherry, longan, mango, pineapple and rambutan. Morphological and biochemical examinations revealed that 43 isolates were members of the genus Acetobacter whereas the remaining 13 isolates were members of the genus Gluconobacter. Preliminary screening showed that isolates No. 13, 34, 36 and 37 gave the widest zone of acidity on overoxidation medium. These isolates were identified as A. aceti and selected for acetic acid production at 30 and 37°C by shaking culture for 14 days in ethanol-yeast extract medium. It was found that A. aceti isolate No. 37 from rambutan gave the highest acetic acid yield of 13.53 and 8.97 g L-1 at 30 and 37°C, respectively after 7 days of fermentation.