THE UNIVERSITY OF BAMENDA

DEPARTMENT OF FOOD
COLLEGE OF
1 TECHNOLOGY
AND BIORESOURCE
TECHNOLOGY

TWO STAGE OXYGEN ASSISTED FERMENTATION FOR THE

PRODUCTION OF ACETIC ACID (VINEGAR) FROM AGRO-
FOODWASTE (PINEAPPLE PEELS)

Presented by
MELDRADE NYUYKONGI
Matricule: UBa20PP040

SUPERVISOR
Dr. NGWASIRI PRIDE NDASI

ACADEMIC YEAR 2022/2023

 PLAN
2  Introduction
 Problem Statement
 Significance of the study
 Objectives
 Material and Methods
 Results and discussions
 Conclusions
 Perspectives
3
INTRODUCTION
 INTRODUCTION

4  Food’ is defined as any substance, whether processed, semi processed or

raw, that is intended for human consumption as well as the ‘inedible parts’
associated with food that are not intended to be consumed by humans .
 ‘Food waste’ means any food and inedible parts of food, removed from the
food supply chain that can be recovered or disposed
 Agro food waste can either be avoided, reused, recycled or disposed safely
 The most preferred is to avoid (like food rotting/spoilage) it but most of the
waste cannot be avoided.
(Anwar et al., 2014).
 These agro waste contain lignocellulose biomass.
(Bhatia et al., 2020)
 INTRODUCTION
5
It can be used to produce biofuels and other
bioproducts.

The main compounds found in lignocellulose

biomass are cellulose (35–50%),
hemicelluloses (25–30%), and lignin (25–30%)

Bioconversion of lignocellulosic biomass to

biofuel and value-added products is globally
gaining significant importance.
 INTRODUCTION
6
 Due to increase in population, food shortages arise and researchers are looking for
different ways of producing important bio products needed by the population
from agro food waste and thus allowing the available food for consumption.
 Consumption of fruits generates a significant amount of fruit waste, especially the
fruit peels.
 This generates a problem of waste disposal
Environment
waste
management Health

Ecosystem.
(Cruess 1958., Suman et al., 2014) (De Ory et al., 2002).(Suman et al., 2014).
 INTRODUCTION
7
 Pineapple and other fruit peels have been identified as one of the common fruit
wastes in Cameroon.
 These peels contain lignocellulose which can be converted into fermentable forms for
the production of ethanol and other bio-products
 Vinegar is one of the bio products in high demand, not only in Cameroon but in the
world at large.

Drink (boost strength and promote wellness

vinegar
Medicine, corrosive agent,

Beverage, aid in dieting, etc.

 Till now, research focused on the valorization of food waste by production of vinegar from
pineapple peel biomass and other fruit peels is still in its infancy stage.
Su et al., (2016), Roda et al., (2017)
 PROBLEM STATEMENT 1/2
8  Acetic acid is highly corrosive, and the production processes needs to be
more sustainable and environmentally friendly by reducing the amount of
energy required in production.
 The demand for acetic acid will always keep growing which necessitates the
development of an eco-friendly process
 In order to minimize the losses of raw materials and the consumption of
auxiliary media, it is necessary to optimize the technologies used
 Most of the fermentation techniques used to produce vinegar from pineapple
peels have proven to have setbacks

Gunjan and Haresh 2020) (Piotrowski and Kubica 2021).

 PROBLEM STATEMENT 2/2
 Research has shown that the use of pure oxygen, injected into a fermentation vessel
9 by a proper injection technique, significantly maintaining a high level of safety.
 The use of oxygen assisted fermentation has been shown to reduce energy
consumption and cycle of time used for the process.

s e b iom a s s u s ing
r f ro m li gnocellu lo
to pr o duce v ine ga
Is it pos s ib le
e on ly availa b le gas?
oxygen as th
We therefore found it necessary to use the two stage oxygen assisted
fermentation method for the production of acetic acid from pineapple peel
biomass and to check the effect of the process parameters on some properties
of acetic acid.
 SIGNIFICANCE OF THE STUDY

10 Valorization of pineapple peel to vinegar will have;

Environmental benefits: having pineapple peels converted to food grade vinegar will
reduce its litter around from pineapple fruits and will significantly reduce
environmental pollution.

Socioeconomic benefits: valorizing pineapple peels will reduce or compensate for the
losses incurred by companies during processing as well as with farmers and consumers
providing another source of income in the food chain.

Health benefits: The method used will improve the quality of the vinegar, giving
more bioactive compounds in the product which will improve the health of the
consumers.
 OBJECTIVES

Specific objectives
11
To evaluate the effect of two stage oxygen
General objective
assisted fermentation process on the
To contribute in the
physicochemical and antioxidant properties of
valorization of
vinegar produced from pineapple peel biomass
pineapple peels by
maximizing its use to
To evaluate the effect of process parameters
produce vinegar u
on the physicochemical and antioxidant
properties of the vinegar produced by two
stage oxygen assisted fermentation process.
 MATERIAL AND METHODS
Pineapple peels
12
Oven dry at 600C
Crush and sieve

Weigh sample
Take brix before Hydrolyse in 2.2% H2SO4
and after pre-
Neutralise with sodium hydroxide
treatment
Why acid hydrolysis? -mild reaction
Sieve to remove solid particles and liquid conditions, -acids can penetrate lignin
without any preliminary pretreatment
used for alcoholic fermentation.
of biomass, (Binod et al, 2011:
Lenithan et al., 2010).
Par eira et al., (2022), Casabar et al., (2019) Sample preparation and hydrolysis
 MATERIAL AND METHODS
Pre-treated pineapple peel
13 Acetic acid
bacteria culture
Alcoholic fermentation at 300C for
Inoculation with yeast
48hours

Pasteurise to stop Bacteria identification

fermentation and viability

Acetic acid fermentation in pure Inoculate with viable acetic

oxygen, at 330C acid bacteria

Measure pH, acetic acid

Pasteurisation and Analysis
content, Brix
Schematic diagram for two stage fermentation
 MATERIAL AND METHODS
Sampl Oxygen Bacteria inoculum
14 e saturation (ml) (ml)
1 20 15
2 40 15
3 60 15
4 80 15
5 100 15
6 0 15
7 0 0

Variation of oxygen volume

 MATERIAL AND METHODS
Experimental design
15 Three vital factors i.e fermentation time (3-5 days), A. aceti volume(10-15ml) and
oxygen volume (60-120ml), were varied using Central Composite Design (CCD) and
Response Surface Methodology (RSM) with coded values A, B, and C, respectively,
in STATGRAPHICS.
 The acetic acid concentration in the fermentation broth (gL−1) was analyzed as the
sole response.
 Other responses included ferric reducing antioxidant power (FRAP), Ph, TSS,
temperature, specific gravity, and density
 MATERIAL AND METHODS

16 Experimental design sample A B C

1 120 3 10
2 90 5 15
Coded Factors
3 120 7 20
values 4 39.5462 5 15
A/% C/ hrs B/ml 5 60 7 10
6 120 3 20
-1 60 3 10
7 60 3 10
+1 120 7 20 8 90 8.36359 15
The coded value and their corresponding real 9 90 5 23.409
values for all three factors 10 90 5 6.59104
Factor A is the volume of oxygen, B is the 11 60 7 20
fermentation time and C. is substrate (acetic acid 12 90 1.63641 15
bacteria inoculum) 13 90 5 15
14 140.454 5 15
15 60 3 20
16 120 7 10
The experimental matrix in real form
 ANALYSIS OF VINEGAR

17
Physicochemical analysis of samples
 Measuring TSS and specific gravity (Joshi and Devrajan, 2008)
 Determination of acidity (San 2005).
 Determination of PH (Piyush and Nardeshwar 2022)
 Determination of alcohol content
 Determination of Density (Ademiluyi and Mepha, 2013)
Determination of antioxidant activity of vinegar.
 The antioxidant activity of the vinegars was evaluated using FRAP (Ferric
reducing/antioxidant power) assay (Benzie and Strain, 1996).
 Modelling and model validation
18
Modelling:

Y = β0 + β1A + β2B + β3C + β12AB + β13AC + β23BC + β11A2 + β22B2 + β33C2 +E

Criteria for model validation:

- Coefficient of determination (R2 > 70%)

Statistical analysis
The data collected was analyzed using Microsoft excel and
STATGRAPHICS.
 RESULTS
Results of bacteria identification
19 Character evaluated Results • The results were similar to that
Inoculation on GYC medium Growth observed obtained by Mathew et al.,
Shape Rod 2019, who Isolated Acetic Acid
Color Whitish
Elevation Flat Bacteria and Prepared a
Surface Smooth and Starter Culture for Apple
shiny Cider Vinegar Fermentation
Gram stain Gram negative • Results obtained were also
Catalase Positive
Oxidase Negative
similar to that obtained by
Odor Like that of Zahoor et al., 2006.
vinegar
Identity Acetobacter
 RESULTS
Effect of two stage fermentation on the physicochemical and anti-oxidant
20 properties of vinegar
effect on pH • The pH ranged from 5.38 on day one to 4.06 on
6
day six. Samples 1 to 5 showed a reduction in
5 pH, with sample 5 recording the lowest pH.
• Decrease in pH was in line with the findings of
4
Ezenekwe et al (2021).
3
• The reduction in pH could be attributed to the
fact that the sample was becoming acidic as the
2
fermentation time increased because the
1
alcohol in the solution was being converted to
the weak acid, acetic acid by the bacteria
0
S1 S2 S3 S4 S5 S6 S7
present.
• Raji et al, 2012.
0th hour 24th hour 48th hour 72nd hour • However, the pH values obtained at the end of
96th hour 120th hour 144th hour
this experiment were higher than that of
Ezenekwe et al., (2021) and Raji et al., 2012.
 RESULTS
Effect of oxygen assisted fermentation on specific gravity

21
effect on specific gravity
 The specific gravity decreased as the
1.009

1.008
number of hours increased for the first six
1.007 samples. Values ranged from 1.008 to
1.006 1.004.
1.005  There was no effect on specific gravity for
1.004
sample seven.
1.003
 These results differ from the results of
1.002
S1 S2 S3 S4 S5 S6 S7
Ezenekwe et al., (2021), who recorded an
0th hour 24th hour 48th hour 72nd hour 96th hour 120th hour 144th hour
increase in specific gravity as the
fermentation period increased.
 RESULTS
• Acetification is an aerobic process, and oxygen is critical
22
to the growth of the bacteria (Mas et al., 2014).

5
Acetic Acid Content • This therefore suggests that the higher the oxygen

4.5 saturation, the, the more the bacteria will produce

4
vinegar. This was in line with the results of Song et al.,
3.5
3 (2022), who studied the effect of dissolved oxygen on the
2.5
acetic acid produced.
2
1.5 • There was an increase in acetic acid formation rate as
1
0.5 oxygen concentration increased. Also, the result is in
0 keeping with the work of Sossou et al., (2009) This result
S1 S2 S3 S4 S5 S6 S7

is also in keeping with the work or Ezemba et al., (2021)

0th hour 24th hour 48th hour 72nd hour
96th hour 120th hour 144th hour and Raji et al., (2012), who noted an increase in the
acetic acid with decrease in pH as the fermentation time
increased.
 RESULTS
 All samples had higher antioxidant activity as
23
compared to the commercial vinegar.
antioxidant activity  Samples without oxygen had the highest
30
25.1 24.97 antioxidant activity compared with the treated
25
20.13 20.3 20.76 samples.
20
16.7 17.1
15  These results indicate that processing alcohol to
10
5.133
vinegar reduces the antioxidant activity of the
5
product.
0
 Megan 2018 stated that processing certain foods
can either increase or decrease its antioxidant
activity.
 RESULTS
Effect of process parameters on the physicochemical and antioxidant properties of vinegar produced.
Responses
Runs pH TSS Temperature acetic acid concentration specific gravity FRAP Density
(mgFeSO4/g)
24 (%) (degree C) (g/100ml)
1 4.66 5.5 31.2 1.5 1.007 24.7 1.2056
2 4.55 5.7 30.5 3.34 1.006 17.03 1.2059
3 4.98 5.8 31.2 3.4 1.007 23.9 1.2073
4 4.48 6 30.1 1.99 1.008 14.43 1.2045
5 4.83 6 30.4 2.2 1.007 19.7 1.2085
6 4.54 5 31 2.99 1.006 20 1.2037
7 4.47 5.8 30.3 1.56 1.008 28.9 1.2066
8 4.83 6 31.3 3.24 1.007 14.9 1.209
9 4.48 5.2 30 3.97 1.007 14.9 1.2031
10 4.55 6 29.7 1.56 1.008 15.6 1.2072
11 4.76 6 31 3.12 1.006 18.5 1.2053
12 5.89 5 31.5 0.66 1.008 14.33 1.0057
13 4.48 5.4 31.9 4.26 1.006 14.9 1.204
14 4.69 5.8 31.7 4.52 1.007 16.6 1.2038
15 4.72 5.4 29.9 2.5 1.007 21.6 1.2049
16 4.76 6 31 2.64 1.008 18.1 1.2104
 RESULTS
Effects of process parameters on acetic acid content of vinegar produced from pineapple peel by two stage
25 fermentation process.
Analysis of Variance for Acetic acid content
R-squared = 86.2238 percent  Production of acetic acid from ethanol
Source Sum of Squares Df Mean Square F-Ratio P-Value depends on the presence of acetic acid
A:A 2.13911 1 2.13911 5.23 0.0622
bacteria (Song et al., 2022)
B:B 3.74234 1 3.74234 9.15 0.0232  Time has both a positive and negative
C:C 4.87937 1 4.87937 11.94 0.0136
effect on acetic acid content of vinegars.
Aye and Ko (2016) found out that the
AA 0.430807 1 0.430807 1.05 0.3442
acidity of the vinegar produced
AB 0.0105125 1 0.0105125 0.03 0.8779 decreased when the fermentation
AC 0.0190125 1 0.0190125 0.05 0.8364 period was longer.
BB 4.24642 1 4.24642 10.39 0.0181  They further explained that the
negative effect observed could be
A is the volume of oxygen, B is the fermentation time and C. is acetic acid
bacteria inoculum volume caused by the further oxidation of acetic
acid
 RESULTS
P-VALUES FOR ANALYSIS OF VARIANCE
26 Source TSS/Brix
TSS
A:A 0.0366 • Bacteria inoculum had a negative significant effect
B:B 0.0002 on the TSS, together with oxygen concentration,
C:C 0.0018 while time and doubling oxygen concentration had
AA 0.0213
significant positive effects.
• The brix values however were higher than that
AB 0.2066
obtained by Akarca et al., (2020), who had mean brix
AC 0.4282 value of 3.63±0.07 °Brix
BB 0.7363

BC 0.0948

CC 0.6289
R-Squared (%) 95.3166 A= oxygen
B=time
C=acetic acid inoculum
 RESULTS
P-VALUES FOR ANALYSIS OF VARIANCE
27
Temperatur Specific Gravity
e Temperature
A: These results show that increasing the oxygen
0.0173 A: 0.4418
A
A
concentration increases the temperature, while
increasing bacteria inoculum decreases the
B: 0.6268 B:B 0.4418
temperature of the vinegar produced, compared
B
to the temperature of the environment.
C: 0.6906 C:C 0.0319
C
Specific Gravity
AA 0.5641 AA 0.0738 Bacteria inoculum had a significant negative effect
AB 0.3883 AB 0.0430 on the specific gravity of the vinegar produced
AC 0.8819 AC 1.0000 with p-value of 0.0319.
BB 0.5899 BB 0.0738 A= oxygen
B=time
BC 0.3196 BC 1.0000 C=acetic acid inoculum
 RESULTS
FRAP Density
28
A:A 0.9363 A:A+AA 0.9655

B:B 0.5047 B:B 0.1125

C:C 0.6799 C:C+AC 0.7432

AA 0.4456 AB 0.9632

AB 0.5497 BB 0.0603

AC 0.5497 BC 0.9837

BB 0.5486 CC 0.7159

BC 0.3152

CC 0.4744
37.6234 56.4918
A= oxygen
B=time
C=acetic acid inoculum
 RESULTS
 Results of multiple response optimization analysis revealed optimum conditions
29
of 8 days, 18.81 bacteria inoculum volume and 140ml oxygen saturation and
predicted optimum pH, TSS, temperature, acetic acid content, specific gravity and
density to be 5.21, 6.0, 31.6, 3.1, 1.007 and 1.19882 respectively.

 The predicted optimum ferric reducing antioxidant activity was 28.97. The
predicted acetic acid concentration which was the main response fell below the
standard. This could be because of other factors. However, the predicted acetic
acid content was closer to that of Jang et al., 2009.
30

CONCLUSION, RECOMMENDATIONS AND

PERSPECTIVES
 CONCLUSION
 Two stage oxygen assisted fermentation had effects on the pH, specific gravity and
31 acetic acid content of the vinegar produced. However, there was no significant
effect on the ferric reducing antioxidant activity of the vinegar produced.

 The process parameters time, bacteria inoculum and oxygen volume had effects
on some of the physicochemical properties studied.

 The use of two stage oxygen assisted fermentation technique can be a highly
suitable process for industrial production of vinegar from pineapple peel and
other agro food waste
 The high antioxidant capacity of pineapple peel vinegar content suggest that it can
be used in the treatment of many diseases.
 RECOMMENDATIONS

32
This research recommends that;

 Vinegar consumption from organic sources should be encouraged because of its

high antioxidant capacity.
 In order to reduce food shortages and environmental pollution, food wastes
should be converted to value added products such as vinegar.
 PERSPECTIVES
For the continuation of the work, we propose that;
33
 The effects of other factors and interaction should be studied such as temperature
and initial ethanol concentration.
 Proximate analysis which gives the nutritional information should be analyzed, in
order to ascertain the nutritional composition, quality of vinegars and to know if
it is safe for human consumption.
 The vinegar should be subjected to sensory analysis, descriptive and consumer
acceptance, for further evaluation and to investigate the amount of toxic
compounds in pineapple peel vinegar.
34

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