LWT - Food Science and Technology 100 (2019) 322–327

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

The development of an alternative fermentation model system for vinegar T

production
Nurul Khadijah Mat Ishama, Nurdiana Mokhtara, Shazrul Fazryb, Seng Joe Lima,∗
a
Centre for Biotechnology and Functional Food, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia
b
Tasik Chini Reseach Centre, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: This study was conducted to develop and optimise an alternative fermentation model system using mushrooms
Alcohol (Pleurotus pulmonarius and Volvariella volvacea) and yeast (Saccharomyces cerevisiae) as the starter cultures, while
Fermentation standard glucose solution was used as the substrate for fermentation. The optimisation was performed on two
Mushroom factors, fermentation time and culture ratio, using D-optimal mixture design. Both fermentation time and culture
Vinegar
ratio showed significant (p < 0.05) effects on reducing sugar content and increasing the ethanol content for
Yeasts
alcoholic fermentation. The optimum fermentation conditions were 72 h of fermentation and using a S.
cerevisiae:P. pulmonarius mycelia:V. volvacea mycelia culture ratio of 1:1:1, achieving a desirability of 0.901. A
verification test was performed, and there was no significant difference (p > 0.05) in the ethanol content be-
tween the experimental value (13.45 ± 0.99%) and the predicted value (13.52%). Vinegar production was
carried out using the optimised alcoholic sample through the inoculation of mother of vinegar and Kombucha.
The concentrations of acetic acid from both samples of vinegar were between 2.05% and 2.41% with zero
residual ethanol. In conclusion, an alternative fermentation model system for vinegar production using mush-
rooms and yeast, followed by mother of vinegar and Kombucha inoculation, has been successfully developed
with the desired physiochemical properties.

1. Introduction acetic acid by acetic acid bacteria (AAB). Common AAB used in acetous
fermentation are from the Gluconabacter and Acetobacter genera
Vinegar has traditionally been used as a food preservative agent, a (Bourdichon et al., 2012; Gullo, Verzelloni, & Canonico, 2014). In ad-
food ingredient (e.g., salad dressing) in cooking or as a beverage by dition to AAB, vinegar can also be produced by the inoculation of
watering down with plain water and juices. The antimicrobial effects of mother of vinegar into the alcohol samples.
vinegar may slow the growth of microbes in food products and help to Currently, only yeasts are used in industrial-scale alcohol produc-
preserve and increase the shelf life of food products (Adams, 2014; tion. However, yeast-based alcohol production is limited by the in-
Bourdichon et al., 2012; Ho, Lazim, Fazry, Zaki, & Lim, 2017a). Acetic hibition of both substrates and products, where a high alcohol content
acid, metabolites and other bioactive compounds produced through (usually approximately 15%) after some period of fermentation results
fermentation contribute to the various therapeutic effects of vinegar on in the inhibition of yeast activity (Lin, Huan, Maua, Liou, & Fang, 2010;
cardiovascular disease, hypertension, diabetes and cancer (Ho, Lazim, Saha & Banerjee, 2013). The use of mushrooms in alcoholic fermenta-
Fazry, Zaki, & Lim, 2017b; Samad, Azlan, & Ismail, 2016). tion have been reported by Okamura et al. (2001) and Okamura-Matsui,
Vinegar is produced through a two-stage fermentation process, Tomoda, Fukuda, and Ohsugi (2003), and these studies showed that
which are alcoholic fermentation and acetous fermentation (Ho et al., mushrooms can produce the enzyme alcohol dehydrogenase (ADH),
2017b). Alcoholic fermentation usually occurs with the fermentation of which is essential in fermenting glucose into alcohol. Interestingly,
appropriate sugar or carbohydrate sources by yeasts. Any substances mushrooms, being more complex organisms than yeasts, are able to
that contain enough sugar that can be fermented (10–12% (w/v) glu- tolerate higher alcohol concentrations. Therefore, an alternative fer-
cose equivalent solution) have the potential to serve as substrates for mentation model system (mixture of yeast and mushroom mycelia) was
vinegar production (Adams, 2014). The second stage, i.e., acetous fer- developed using glucose standard solution as the substrate to optimise
mentation, is the process by which the produced ethanol is oxidised into the fermentation parameters of this model system.

∗
Corresponding author.
E-mail address: joe@ukm.edu.my (S.J. Lim).

https://doi.org/10.1016/j.lwt.2018.10.065
Received 14 September 2018; Received in revised form 15 October 2018; Accepted 21 October 2018
Available online 22 October 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
N.K. Mat Isham et al. LWT - Food Science and Technology 100 (2019) 322–327

Response surface methodology (RSM) is the analysis of a response inoculated into a 250 mL conical flask containing 25 mL PDB and in-
that is influenced by several factors to obtain the optimal conditions of cubated in an incubator shaker for 14 days at 28 °C and 120 rpm. Broth
the response (Francis et al., 2003). A specific form of RSM, the mixture with a 0.4 optical density (OD) at 600 nm on a spectrophotometer
design, which is widely used for optimising culture media and enzyme (Biotek Epoch, US) were used as the starter cultures for alcoholic fer-
ratios, is usually applied for optimising complex processes (Tang, mentation.
Abdul, Engliman, & Hassan, 2018; Yin, Chen, Gu, & Han, 2009). In the
study of Yin et al. (2009), the D-optimal mixture design was utilised to 2.2.2. Preparation of the medium
obtain the optimum levels of different variables for the culture medium Glucose standard solution at a 10% (w/v) concentration was used as
for selenium-enriched yeast fermentation to produce biomass and se- the alcoholic fermentation medium. The glucose solution was auto-
lenium. claved for 20 min at 121 °C prior to inoculation. Conical flasks (250 mL)
The objective of this study was to optimise the alcoholic and acetous were used as fermentation flasks, and the flasks were covered using
fermentation parameters of the alternative model system using the D- cotton and parafilm to ensure anaerobic respiration. Fermentation was
optimal mixture design. In addition, this study also aimed to identify performed in an incubator shaker (ET 25 Electron, INFORS HT) at 28 °C
the metabolic changes that occur during fermentation with yeast and with a speed of 120 rpm. The fermentation process was carried out
mushrooms. according to the D-optimal mixture design, as shown in Table 1, where
the culture ratio and fermentation time were manipulated.
2. Materials and methods
2.2.3. Verification of the optimum point
2.1. Materials A verification test was carried out at the predicted optimum point of
alcoholic fermentation, which was a mixture of S. cerevisiae:P.
Three types of starter cultures were used in alcoholic fermentation: pulmonarius:V. volvacea at ratio of 1:1:1 and at 72 h of fermentation.
yeast (Saccharomyces cerevisiae) and mushroom (Pleurotus pulmonarius The actual values from the analyses were compared to the predicted
and Volvariella volvacea) mycelia. The yeast culture was purchased from values.
Giant Supermarket, Bangi, Malaysia. The P. pulmonarius and V. volvacea
mycelia were obtained from the Malaysian Nuclear Agency. 2.3. Chemical analysis of the alcoholic fermentation samples
The two types of AAB cultures that were used for the production of
vinegar were Acetobacter acetii from mother of vinegar (apple cider 2.3.1. pH value
vinegar) and Kombucha. The apple cider vinegar (Bragg's) was pur- The pH values of the samples during alcoholic and acetous fer-
chased from Section 15, Bandar Baru Bangi, Selangor, Malaysia, with an mentation were determined using a pH metre (PHM 210, France).
expiration date of 11 May 2020. The Kombucha was obtained from the
Molecular Biology Laboratory, Biological Science Building, Universiti 2.3.2. Determination of the total sugar content
Kebangsaan Malaysia. The total sugar content was determined using the phenol-sulphuric
acid method, as described by Lutpi, Jahim, Mumtaz, Harun, and Abdul
2.2. Alcoholic fermentation (2016). A sample (1 mL) was mixed with 1 mL of phenol solution (1%
w/v) in a test tube. Next, 5 mL of concentrated sulphuric acid was
The optimisation of alcoholic fermentation was carried out using D- added to the test tube and left for 15 min. The absorbance of the sample
optimal mixture design with Design Expert software 10.7.1 (Stat Ease). was measured at 490 nm using a microplate spectrophotometer, Biotek
Two factors were optimised in the alcoholic fermentation: the fer- Epoch (US). A standard curve was prepared using standard glucose
mentation time (hour) and ratio of starter culture. The D-optimal solution at 200 ppm–1000 ppm. The total sugar content was calculated
mixture design contained 21 experiments for each fermentation time using the following formula:
(24 h, 72 h and 120 h) that included 3 independent replications for each 100
Total sugar content (%) = R × DF ×
ratio of starter culture to evaluate the effect of the combination of the 3 1000000
independent variables, S. cerevisiae (A), P. pulmonarius mycelia (B) and
where R = reading from the standard curve and DF = dilution factor.
V. volvacea mycelia (C), as shown in Table 1. The significance of the
values for all parameters were statistically determined at the prob-
2.3.3. Determination of the total reducing sugar content
ability p = 0.05 (Yin et al., 2009). The responses that needed to be
The concentration of reducing sugars was determined using the
optimised in this study were the total sugar, reducing sugar and ethanol
dinitrosalicylic acid method (DNS) as described by Loh, Hassan, and
contents, where the goal was to achieve the maximum ethanol content
Yusop (2014). A sample (1 mL) was mixed with 3 mL of DNS solution
with the minimum amount of total sugars and reducing sugars. Desir-
(1% w/v) in a test tube. The mixture was left in boiling water (100 °C)
ability was predicted using contour plots and calculated based on the
for 5 min and cooled to room temperature. The absorbance of the
following equations:
sample was measured at 540 nm using a microplate spectrophotometer
D = (d × d × …. × dn)1/n = {∩di}1/n, where D = the overall de- Biotek Epoch. A standard curve was prepared using standard glucose
sirability, d = desirability, and n = the number of responses. solution at 200 ppm–1000 ppm. The total reducing sugar content was
calculated using the following formula:
100
2.2.1. Preparation of the starter culture Total reducing sugar content (%) = R × DF ×
1000000
The mushrooms (P. pulmonarius and V. volvacea) mycelia were
subcultured on potato dextrose agar (PDA) and incubated at 25 °C for where R = reading from the standard curve and DF = dilution factor.
14 days in the dark until new mycelia were observed. After 14 days, the
mycelia were subcultured again, and their reproduction was main- 2.3.4. Determination of the total ethanol content
tained on PDA at 25 °C for one week in dark conditions; they were The concentration of ethanol was determined using a spectro-
subsequently kept at 4 °C as stock culture. The mushrooms mycelia photometric method, as described by Betiku and Taiwo (2015). A
were prepared in potato dextrose broth (PDB) for inoculation in glucose sample (1 mL) was mixed with 5 mL of sodium dichromate solution
medium. The PDB was autoclaved for 20 min at 121 °C. A 9 mm dia- (2.5% w/v), 5 mL of acetate buffer solution (0.1 mol/L, pH 4.3) and
meter circle of mycelia was removed from the agar (14 days old), 25 mL of sulphuric acid (1 mol/L). The mixture was thoroughly mixed

323
N.K. Mat Isham et al. LWT - Food Science and Technology 100 (2019) 322–327

Table 1
Pseudo levels of independent variables with observable values for response variables, total sugar, reducing sugar and ethanol content in alcohol samples 24 h, 72 h
and 120 h.
A B C Total Sugar Content (%) Reducing Sugar Content (%) Ethanol Content (%)

24 h 72 h 120 h 24 h 72 h 120 h 24 h 72 h 120 h

0 0 1 7.32 5.94 2.38 2.92 1.04 1.14 −7.68* −5.06* 1.68

0 0 1 7.32 6.64 2.48 2.98 1.38 1.06 −7.43* −5.31* 1.68
0 1 1 16.58 14.90 4.45 0.50 0.54 0.34 −6.56* −6.06* 7.29
1 1 1 0.26 −0.53* −0.93* 1.30 0.28 −0.14* −2.56* 15.65 1.18
0 1 0 7.65 4.82 0.98 1.80 1.08 0.46 −8.05* −5.43* 3.18
1 0 1 1.64 −0.89* −0.80* 1.48 −0.16* −0.14* 0.93 6.17 13.53
1 0 1 1.34 −0.82* −0.86* 1.54 −0.04* −0.02* 0.56 6.30 13.41
0 1 0 8.27 5.39 1.04 1.86 0.88 0.34 −7.80* −5.06* 2.55
1 0 0 1.38 1.31 0.17 −0.22* −0.18* −0.14* 1.30 5.80 11.42
1 0 0 1.36 1.74 0.26 −0.22* −0.16* −0.18* 2.05 5.80 11.16
0 0 1 6.55 6.62 2.40 3.14 1.38 1.14 −7.18* −4.68* 1.30
1 1 1 0.39 −0.74* −0.84* 1.38 0.10 −0.24* −2.06* 15.78 0.93
1 1 1 0.39 −0.57* −0.67* 1.66 0.10 −0.22* −2.06* 15.40 0.56
1 1 0 9.37 2.06 −0.84* 2.14 −0.22* −0.18 3.05 5.67 15.40
0 1 0 8.02 5.98 0.94 1.60 1.24 0.19 −8.18* −5.06* 2.55
0 1 1 16.36 14.2 5.05 0.36 0.36 0.30 −6.68* −5.93* 6.92
1 0 1 1.64 −0.06* −0.48* 1.56 −0.18* −0.10* 0.43 6.67 13.28
1 1 0 9.56 2.95 −0.84* 1.86 −0.14* −0.22* 3.30 6.05 15.53
0 1 1 16.32 14.6 4.67 0.36 0.44 0.34 −6.18* −6.06* 7.42
1 1 0 9.56 2.45 −0.80* 2.34 −0.26* −0.10* 3.05 5.67 15.90
1 0 0 1.42 0.98 −0.08* −0.22* −0.36* −0.16* 1.43 5.67 11.16

A = S. cerevisiae culture B = P. pulmonarius culture C = V. volvacea culture.

*The value is considered 0%.

Fig. 1. Surface response plot of ethanol content for alcoholic fermentation at 24, 72 and 120 h of fermentation using S. cerevisiae (A), P. pulmonarius (B) and V.
Volvacea (C).

Table 2
Comparison between the experimental value and the predicted value in the optimum alcohol sample.
Response Experimental Value Predicted Value/Numerical Optimisation Percentage Difference (%)

Total Sugar (%) 0.49 ± 0.30 a

−2.27 b#
-ˆ
Reducing Sugar (%) 0.16 ± 0.10a −0.18b# -ˆ
Ethanol (%) 13.45 ± 0.99a 13.52a 0.48

a-b
Means with different alphabet within the same row are significantly different at p < 0.05.
#
Value is considered 0%.
ˆ
Percentage difference was not calculated because value is considered 0%.

for 1 min and left for 2 h at 27 °C. The absorbance of the sample was where R = reading from the standard curve and DF = dilution factor.
measured at 578 nm using a microplate spectrophotometer Biotek
Epoch (US). A standard curve was prepared using 5–40% ethanol. The
ethanol content was calculated using the following formula: 2.4. Acetous fermentation

Ethanol content (%) = R × DF The acetous fermentation was performed using a 2 × 2 factorial
design. The two factors that were selected in this process were the type

324
N.K. Mat Isham et al. LWT - Food Science and Technology 100 (2019) 322–327

Table 3
Acetic acid content, pH and ethanol concentration for K and M vinegar samples.
Sample Acetic acid content (%) pH Ethanol concentration (%)

5% inoculation 10% inoculation 5% inoculation 10% inoculation 5% inoculation 10% inoculation

K 2.37 ± 0.34 a
2.28 ± 0.27a
2.86 ± 0.14 2.93 ± 0.07 −1.31 ± 0.91 a#
−1.88 ± 1.10a#
M 2.05 ± 0.13a 2.41 ± 0.24b 3.02 ± 0.06 3.04 ± 0.18 −0.35 ± 3.58a# −2.08 ± 1.27a#

K = vinegar sample with Kombucha inoculation.

M = vinegar sample with mother of vinegar inoculation.
a-b
means with different alphabets are significantly different at (p < 0.05).
#
Value is considered 0%.

of starter culture (mother of vinegar (M) and Kombucha (K)) and the observable values as the response variables, which included the total
respective concentration for inoculation (5% and 10%). The response sugar, reducing sugar and ethanol contents in the alcohol samples with
factors were the acetic acid content, ethanol content and pH. 24 h, 72 h and 120 h of fermentation. The minimum total sugar content
was from the mixture of S. cerevisiae:P. pulmonarius:V. volvacea at a ratio
2.4.1. Preparation of the starter culture of 1:1:1. The mixture of S. cerevisiae:P. pulmonarius:V. volvacea at a ratio
The two types of AAB cultures that were used for the production of of 1:1:1 took a shorter time (24 h) than the other mixtures to use the
vinegar were AAB contained in mother of vinegar (apple cider vinegar) sugar as a substrate to produce alcohol. The maximum total sugar
and Kombucha. content (16.58%) was found in the mixture of P. pulmonarius and V.
volvacea (1:1) at 24 h of fermentation. The mixed mushroom culture, P.
2.4.2. Preparation of the medium pulmonarius:V. volvacea at a ratio of 1:0 and 1:1, required a longer
The medium for acetous fermentation was the alcohol solution re- fermentation time, which was 120 h, to show a decline in the sugar
sulting from the previous optimised fermentation of the alcohol sample. content.
Acetous fermentation was performed via the inoculation of mother of Based on Table 1, the yeast culture (ratio 1:0:0) did not have any
vinegar (M) and Kombucha (K) into the optimised alcohol medium at a reducing sugars at 24 h of fermentation due to the glucophilic proper-
5% (v/v) and 10% (v/v) concentration to produce vinegar. To prepare ties of the yeast, which means that yeast can use glucose within a short
the 5% (v/v) concentration, 1 mL of Kombucha and mother of vinegar period of time (Lin et al., 2010). The mixtures of S. cerevisiae and P.
was added to different conical flasks (250 mL) containing 19 mL of the pulmonarius or V. volvacea (ratio 1:1) did not show the presence of
alcohol sample. The medium was incubated in an incubator shaker (ET glucose after 72 h of fermentation, while the mushroom cultures (P.
25 Electron, INFORS HT) at 30 °C and a speed of 150 rpm. For the 10% pulmonarius:V. volvacea at ratio 1:0 or 0:1 and P. pulmonarius:V. volvacea
(v/v) concentration, the above steps were repeated by adding 2 mL of at ratio 1:1) required 120 h of fermentation to substantially use the
Kombucha and mother of vinegar into different conical flasks con- glucose.
taining 18 mL of the alcohol sample. Only cotton was used to cover the Microorganisms require different amounts of time to adapt to the
conical flasks to allow airflow to provide aerobic conditions. Acetous new media, which is known as the lag phase. The differences in lag time
fermentation was conducted for one week, and the fermentation of each showed that the mushroom cultures took longer to adapt to a new
sample of vinegar was performed in triplicate. media compared to yeast, as shown by the use of sugar. However, the
mushrooms culture has amylase, which can break down starch to
2.4.3. Determination of the acetic acid content simple sugars, such as glucose, thus maximising the production of
The concentration of acetic acid was determined using the titratable ethanol (Hur, Ka, Joo, & Terahita, 2001; Hur, Park, Chung, & Joo, 1998;
acidity method as described by AOAC (1990). A sample (1 mL) was Terashita & Kono, 1989).
pipetted into a 250 mL Erlenmeyer flask, and 20 mL of distilled water At 24 h, the maximum amount of ethanol produced (3.3%) was from
and 5 drops of phenolphthalein were added to the flask. The sample the mixture of S. cerevisiae:P. pulmonarius (1:1). At 72 h of fermentation,
solution was titrated with 0.1 N sodium hydroxide until a pink colour the maximum ethanol content (15.78%) was seen in the mixture of S.
was present. The acetic acid content was calculated using the following cerevisiae:P. pulmonarius:V. volvacea at a ratio of 1:1:1. In addition, the
formula: minimum ethanol content was observed in the mixed mushroom cul-
ture of P. pulmonarius and V. volvacea (at ratios of 1:0 or 0:1 and 1:1),
V (mL) × N × 60.05
Acetic acid content (%) = which did not contain ethanol at 24 and 72 h of fermentation. However,
10 × W (g ) at 120 h of fermentation, the mixture of S. cerevisiae: P. pulmonarius: V.
where V = volume of the final 0.1 N NaOH solution, N = normality of volvacea at a ratio of 1:1:1 produced the least amount of ethanol
the NaOH solution, 60.05 = molar mass of acetic acid (g/mol), and (0.56%), while the mixture of S. cerevisiae: P. pulmonarius (1:1) pro-
W = weight of the vinegar sample. duced the largest amount of ethanol (15.9%).

2.5. Statistical analysis 3.2. Optimum point of alcoholic fermentation and verification

Analysis of variance (ANOVA) and Tukey's test were conducted on The experimental data on ethanol production were analysed and fit
the results from the verification test as well as the acetous fermentation, a cubic model with high R2 values (24 h: 0.9974; 72 h: 0.9995; and
using Minitab 16. The means were considered significantly different 120 h: 0.9986). The following polynomial models were derived from
when p < 0.05. the cubic models:

3. Results and discussion Ethanol 24 h = 1.59*A - 8.01*B - 7.43*C + 25.37*AB + 14.23*AC +

4.99* BC -69.26*ABC
3.1. Alcoholic fermentation
Ethanol 72 h = 5.76*A - 5.18*B - 5.02*AC + 22.04*AB + 24.04*AC -
3.67*BC + 334.22*ABC
Table 1 shows pseudo levels of the independent variables with the

325
N.K. Mat Isham et al. LWT - Food Science and Technology 100 (2019) 322–327

Ethanol 120 h = 11.25*A + 2.76*B + 1.55*C + 34.43*AB + produced, it was found that the optimum parameters for the production
28.03*AC + 20.21*BC - 364.01*ABC of the alcohol samples were 72 h of fermentation and a mixture of S.
cerevisiae:P. pulmonarius:V. volvacea at a ratio of 1:1:1. The verification
where,
tests of the optimum point for alcohol sample production showed that
the experimental value (13.45 ± 0.99%) of the amount of ethanol
A = S. cerevisiae,
produced was not significantly different (p > 0.05) from the predicted
B = P. pulmonarius, and
value (13.52%). The vinegar produced from the optimum alcoholic
C = V. volvacea
fermentation sample had an acetic acid content ranging between 2.05%
and 2.41%, and the ethanol concentration was less than 1%. Therefore,
Fig. 1 shows the surface response plots, derived from the polynomial
the alternative fermentation models using a mixture of mushroom and
models above, of the 3 fermentation times (24, 72 and 120 h) based on
yeast in this study were found to be potential methods for alcohol and
the ethanol content. It was evident that the optimum point was only
vinegar production. Such applications could contribute economically
achieved at 72 h of fermentation time and with a culture ratio of S.
towards increasing the yield and production in the brewery industry.
cerevisiae:P. pulmonarius:V. volvacea of 1:1:1 and with desirability of
0.901. The optimum point was determined by minimising the total
Declarations of interest
sugar and reducing sugar contents and maximising the ethanol content.
Verification tests were performed at the optimum point of alcohol
None.
production, and the results are shown in Table 2. The response of total
sugar (0.49 ± 0.30%) and reducing sugar (0.16 ± 0.10%) were sig-
Acknowledgements
nificantly different (p < 0.05) from the predicted values (−2.27% and
−0.18%, respectively) mainly due to the fact that the predicted value
This study was supported by the Dana Top Down-Geran
was negative. Negative values for the total sugar and reducing sugar
Penyelidikan Universiti-Industri, Universiti Kebangsaan Malaysia
contents are not possible and thus are considered 0. Therefore, although
(INDUSTRI-2014-005) and Geran Ganjaran Penerbitan (GP-K020181).
the total sugar and reducing sugar contents were significantly different
The authors would like to acknowledge the Faculty of Science and
(p < 0.05), their effect is very minimal, as the experimental values are
Technology, Universiti Kebangsaan Malaysia, which provided the fa-
very low (approaching zero) and the predicted values are considered to
cilities necessary for this research.
be 0. This result shows that almost all the sugars in the alcohol sample
were used by the yeast culture and mushrooms to produce ethanol. The
References
produced ethanol (13.45 ± 0.99%) did not show a significant differ-
ence (p > 0.05) compared to the predicted value (13.52%), which is
Adams, M. R. (2014). Vinegar. In C. A. Batt, & M. L. Tortorello (Eds.). Encyclopedia of food
desirable. This result shows that the production of ethanol using S. microbiology (pp. 717–722). (2nd ed.). Amsterdam: Academic Press.
cerevisiae:P. pulmonarius:V. volvacea at 1:1:1 has been successfully op- AOAC (1990). Official methods of analysis (16th ed.). Arlington VA, USA: Association of
timised and verified. Official Analytical Chemists.
Betiku, E., & Taiwo, A. E. (2015). Modeling and optimization of bioethanol production
from breadfruit starch hydrolysate vis-a-vis response surface methodology and arti-
3.3. Acetous fermentation ficial neural network. Renewable Energy, 74, 87–94.
Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J. C., Gerds, M. L., Hammes, W. P.,
et al. (2012). Food fermentations: Microorganisms with technological beneficial use.
Malaysian Food Regulations (1985) state that the minimum acetic International Journal of Food Microbiology, 154(3), 87–97.
acid content in vinegar should be 4%. Based on Table 3, the acetic acid FAO (2000). Food standards programme. Rome: Codex Alimentarius Commission.
Francis, F., Sabu, A., Nampoothiri, K. M., Ramachandran, S., Ghosh, S., Szakacs, G., et al.
contents in all samples were less than the prescribed standard; both (2003). Use of response surface methodology for optimizing process parameters for
vinegar samples were in the range of 2.05%–2.41% acetic acid. Ac- the production of α-amylase by Aspergillus oryzae. Biochemical Engineering Journal,
cording to Ubeda et al. (2011), an inadequate oxygen supply during 15(2), 107–115.
Gullo, M., Verzelloni, E., & Canonico, M. (2014). Aerobic submerged fermentation by
acetous fermentation leads to the accumulation of acetyldehyde and a
acetic acid bacteria for vinegar production: Process and biotechnological aspects.
low production of acetic acid. Additionally, according to Saha and Process Biochemistry, 14(2), 1–38 (This reference is one of the key references because
Banerjee (2013), a reduction in acetic acid in vinegar occurs due to the it reviews the acetous fermentation, which is the 2nd part of the fermentation in the
current study. It highlights the type of acetous fermentation parameters, which is
evaporation of acetic acid during acetous fermentation.
essential for the current study).
The total ethanol content of the optimum alcohol sample was Ho, C. W., Lazim, A. M., Fazry, S., Zaki, U. K. H., & Lim, S. J. (2017a). Effects of fer-
13.45 ± 0.99% and was reduced to zero (negative values are con- mentation time and pH on soursop (Annona muricata) vinegar production towards its
sidered zero), which shows that all of the ethanol was completely used chemical compositions. Sains Malaysiana, 46(9), 1505–1512.
Ho, C. W., Lazim, A. M., Fazry, S., Zaki, U. K. H., & Lim, S. J. (2017b). Varieties, pro-
during acetous fermentation. According to the Food and Agricultural duction, composition and health benefits of vinegars: A review. Food Chemistry, 221,
Organization (FAO, 2000), the maximum residue of alcohol in the vi- 1621–1630 (This reference is one of the key references because this review gives an
negar should be between 0.5% for wine vinegar and 1.0% for vinegar overview on the production of vinegar, which is very useful for the current study.
They types, legislation, processing, and bioactivities of vinegars were described in
other than wine vinegar, and the vinegar samples in this study met this this reference).
standard established by the FAO. All the samples did not contain vi- Hur, T. C., Ka, K. H., Joo, S. H., & Terahita, T. (2001). Characteristics of the amylase and
negar ethanol during acetous fermentation. To improve acetic acid its related enzymes produced by Ectomycorrhizal fungus Tricholoma matsuke.
Mycobiology, 29(4) 183 -18.
production, a closed system with a continuous oxygen supply is es- Hur, T. C., Park, H., Chung, J. H., & Joo, S. H. (1998). Changes in soil physicochemical
sential. properties and dehydrogenase activity by formation of fairy ring of Tricholoma mat-
sutake. Journal of Korean Forestry Society, 87(2), 270–275.
Loh, P. L., Hassan, N. S., & Yusop, M. R. (2014). Investigation of the effects of ionic liquids
4. Conclusions pretreatments and enzymatic hydrolysis of kempas, Koompassia malaccensis. Oriental
Journal of Chemistry, 30(4), 1535–1543.
In conclusion, the alternative fermentation system using mixtures of Lin, P. H., Huan, S. Y., Maua, J. L., Liou, B. K., & Fang, T. J. (2010). A novel alcoholic
beverage developed from shiitake stipe extract and cane sugar with various
S. cerevisiae starter culture and mushroom mycelia (P. pulmonarius and Saccharomyces strains. LWT – Food Science and Technology, 43, 971–976.
V. volvacea), followed by AAB in Kombucha and mother of vinegar have Lutpi, N. A., Jahim, J. M., Mumtaz, T., Harun, S., & Abdul, P. M. (2016). Batch and
successfully increased the ethanol yield in alcoholic fermentation and continuous thermophilic hydrogen fermentation of sucrose using anaerobic sludge
from palm oil mill effluent via immobilisation technique. Process Biochemistry, 51,
subsequently converted into acetic acid in acetous fermentation. 297–307.
Generally, fermentation time and cultures had a significant impact on Malaysian Food Regulations (1985). Standards and particular labelling requirements for
the production of the alcohol and vinegar samples. From the models food. Vinegar sauce, chutney and pickle. Putrajaya: Malaysia Ministry of Health.

326
N.K. Mat Isham et al. LWT - Food Science and Technology 100 (2019) 322–327

Okamura-Matsui, T., Tomoda, T., Fukuda, S., & Ohsugi, M. (2003). Discovery of alcohol 2(9), 501–514.
dehydrogenase from mushrooms and application to alcoholic beverage. Journal of Samad, A., Azlan, A., & Ismail, A. (2016). Therapeutic effects of vinegar: A review. Current
Molecular Catalysis B: Enzymatic, 23, 133–144 (This reference is one of the key re- Opinion in Food Science, 8, 56–61.
ferences because it reviewed the use of mushrooms for production of alcoholic bev- Tang, P. L., Abdul, P. M., Engliman, N. S., & Hassan, O. (2018). Effects of pretreatment
erages. Aside from that, it also reviewed the biochemical pathway of the conversion and enzyme cocktail composition on the sugars production from oil palm empty fruit
from carbon source (sugar/carbohydrate) to ethanol, as well as the synergistic effects bunch fiber (OPEFBF). Cellulose, 25(8), 4677–4694.
of yeasts and mushrooms for alcoholic fermentations, which is the key factor in the Terashita, T., & Kono, M. (1989). Purification and some properties of carboxyl proteinase
current study). from Tricholoma matsusake. Transactions of the Mycological Society of Japan, 28(3),
Okamura, T., Ogata, T., Minamimoto, N., Takeno, T., Noda, H., Fukuda, S., et al. (2001). 245–256.
Characteristics of wine produced by mushroom fermentation. Bioscience Biotechnology Ubeda, C., Callejȏ, R. M., Hidalgo, C., Torija, M. J., Mas, A., Troncoso, A. M., et al. (2011).
& Biochemistry, 65(7), 1596–1600 (This reference is one of the key references because Determination of major volatile compounds during the production of fruit vinegar by
it showed that certain strains of mushrooms could produce the alcohol dehy- static headspace gas chromatography-mass spectrometry method. Food Research
drogenase enzyme for alcoholic fermentation of suitable carbon source, which is a International, 44, 259–268.
key factor in the current study. It uses 3 mushrooms species, i.e. Agaricus blazei Yin, H., Chen, Z., Gu, Z., & Han, Y. (2009). Optimization of natural fermentative medium
MWU-C20, Flammulina velutupes MWU-C3 and Pleurotus ostreatus MWU-C1 for the for selenium-enriched yeast by D-optimal mixture design. LWT – Food Science and
alcoholic fermentation). Technology, 42, 327–331 (This reference is one of the key references because it out-
Saha, P., & Banerjee, S. (2013). Optimization of process parameters for vinegar produc- lines the experimental design for the alcoholic fermentation, which is the D-optimal
tion using banana fermentation. International Journal of Renewable Energy Technology, mixture design. The same experimental design was used in the current study).

327