INTRODUCTION

Figure 1

Fermentation is the slow decomposition of complex organic

compound into simpler compounds by the action of enzymes.
Enzymes are complex organic compounds, generally proteins.
Examples of fermentation are: souring of milk or curd, bread
making, wine making and brewing.
The word Fermentation has been derived from Latin (Fervor
which means to ‘boil’).As during fermentation there is lot of
frothing of the liquid due to the evolution of carbon dioxide, it
gives the appearance as if it is boiling. Sugars like glucose and
sucrose when fermented in the presence of yeast cells are
converted to ethyl alcohol. During fermentation of starch,
starch is first hydrolyzed to maltose by the action of enzyme
diastase. The enzyme diastase is obtained from germinated
barley seeds.
Fermentation is carried out at a temperature of 4–16 °C
(40–60 °F). This is low for most kinds of fermentation, but is
beneficial for cider as it leads to slower fermentation with less
loss of delicate aromas.
1
THEORY

Louis Pasteur in 1860 demonstrated that fermentation is a

purely physiological process carried out by living
micro-organism like yeast. This view was abandoned in 1897
when Buchner demonstrated that yeast extract could bring
about alcoholic fermentation in the absence of any yeast cells.
He proposed that fermenting activity of yeast is due to active
catalysts of biochemical origin. These biochemical catalysts
are called enzymes. Enzymes are highly specific. A given
enzyme acts on a specific compound or a closely related
group of compounds. Fermentation has been utilized for many
years in the preparation of beverages. Materials from Egyptian
tombs demonstrate the procedures used in making beer and
leavened bread. The history of fermentation, whereby sugar is
converted to ethanol by action of yeast, is also a history of
chemistry. Van Helmont coined the word iogaslt in 1610 to
describe the bubbles produced in fermentation. Leeuwenhoek
observed and described the cells of yeast with his newly
invented microscope in 1680. The fruit and vegetable juices
contain sugar such as sucrose, glucose and fructose. These
sugars on fermentation in the presence of the enzymes
invertase and zymase give with the evolution of carbon
dioxide. Maltose is converted to glucose by enzyme maltose.
Glucose is converted to ethanol by another enzyme zymase
Invertase

C​12​H​22​O​11​ + H​2​O C​6​H​12​O​6​ + C​6​H​12​O​6

2
Sucrose Glucose Fructose

Zymase

C​6​H​12​O​6​ + C​6​H​12​O​6​ 2C​2​H​5​OH + 2CO​2

Glucose Fructose Ethanol

Diastase

2(C​6​H​10​O​5​)​n​ + nH​2​O nC​12​H​22​O​11

Starch Maltose

Maltose

C​12​H​22​O​11​ + H​2​O 2C​6​H​12​O​6

Maltose Glucose

Zymase

C​6​H​12​O​6​ 2C​2​H​5​OH + 2CO​2

Glucose Ethyl alcohol

Glucose is a reducing sugar and gives red coloured

precipitates with Fehling’s solution, when warmed.
When the fermentation is complete, the reaction mixture stops
giving any red colour or precipitate with Fehling solution.

3
EXPERIMENT

Requirements:

Conical flasks (250 ml), test tubes and water bath, Carrot juice
and Fehling’s solution.

Procedure:

1.Take 5.0 ml of carrot juice in a clean 250 ml conical flask

and dilute it with 50 ml of distilled water.
2. Add 2.0 gram of Baker’s yeast and 5.0 ml of solution of
Pasteur’s salts to the above conical flask.
3. Shake well the contents of the flask and maintain the
temperature of the reaction mixture between 35-40°C.
4. After 10minutes take 5 drops of the reaction mixture from
the flask and add to a test tube containing 2 ml of Fehling
reagent. Place the test tube in the boiling water bath for about
2 minutes and note the colour of the solution or precipitate.
5. Repeat the step 4 after every 10 minutes when the reaction
mixture stops giving any red colour or precipitate.
6. Note the time taken for completion of fermentation.
Pasteur’s Salt Solution – Pasteur salt solution is prepared by
dissolving ammonium tartrate 10.0g; potassium phosphate 2.0
g; calcium phosphate 0.2g, and magnesium sulphate 0.2 g
dissolved in 860ml of water.

FERMENTATION OF ORANGE JUICE

4
Oranges are appreciated as fruit throughout the world. The
high productivity of oranges, approximately 17,618,450 tons
annually, especially in southeast Brazil, generates post-harvest
losses. An alternative to disposing of the fruit to reduce waste
and increase income to farmers is the sale of processed fruit to
generate industrial products such as jams, juices, wines and
spirits. The use of the fruit as a substrate for producing high
added value products has been accomplished; an example is
spirits obtained by the fermentation and distillation of fruit.
Fruit spirits are produced all over the world using various
fruits, according to the availability in different countries and
seasons. In this way, the current commercialization of known
alcoholic beverages obtained from fruit could facilitate the
market penetration of such spirits. Some fruits that have been
used to produce distillates are melons, mulberries, plums and
cherries jabuticaba, black mulberries and blackcurrants and
pears.

Figure 2

According to Brazilian law, a fruit spirit is a beverage with an

alcohol content between 36 and 54% v/v at 20°C that is
obtained from simple fruit alcoholic distillates or by the
distillation of fermented fruit. The volatile compound content

5
should be ≥2000 mg/L of anhydrous alcohol, but never >6500
mg/L.
The process needed to produce fruit spirit is complex and
involves various factors that influence the quality of the final
product. However, the main physico-chemical and sensorial
differences among spirits are due to the particular composition
of their corresponding raw materials (fruit, cereals,
vegetables, etc.) and the fermentation process.
Market-orientated yeast strains are currently being developed
for the competitive production of alcoholic beverages with
minimized resource inputs, improved quality and low
environmental impacts. Thus, Saccharomyces
cerevisiae strains are being developed to improve
fermentation and biopreservation abilities, as well as to
improve the sensory qualities of the beverages.

Materials and methods

Fruit

One-hundred kilograms of oranges (variety Pêra Rio) were

harvested in November 2011 in Lavras, Minas Gerais, Brazil.
The fruit was washed with bleach (10 g/L) and cut
longitudinally, and the juice was obtained using an electric
juicer (Cemaf, São Paulo, Brazil). The seeds and pulp were
separated from the juice manually using a metal mesh strainer.
The fruit was analysed in relation to total weight and the
seed–juice weight ratio. Samples of orange juice were
characterized in relation to total soluble solids (ºBrix),
titratable acidity and pH, reducing sugars and pectin.

6
Orange must

The orange must was prepared according to the methods of

Dias et al. and Duarte ​et al​. with minor modifications. In the
orange juice, the initial sugar concentration was
approximately 11​o​Brix, and the pH was 3.7. The orange juice
was mixed with a sucrose solution (1:1 v/v) to adjust the sugar
concentration to 16°Brix. The orange must was thermally
treated at 100°C for 10 min by autoclaving. After cooling to
approximately 35°C, the juice was transferred to three
stainless steel fermentation vats.

Figure 3

Calcium carbonate (CaCO​3​) was added to increase the pH to

4.5. Sulphur dioxide, in the form of potassium metabisulphite
(K​2​S​2​O​5​), was added at a concentration of 100 mg/L to inhibit
bacterial growth.

Yeast strain

7
Saccharomyces cerevisiae UFLA CA1174, a strain isolated
from sugar cane distilleries in the South of Minas Gerais,
Brazil, was previously identified as showing good
fermentative characteristics in acid fruit juices. It was first
grown in YPD (10 g/L of yeast extract, 20 g/L of peptone and
20 g/L of glucose) to reach a concentration of
2 × 10​8​ cells/mL after 48 h. To adapt the microorganism to
the orange juice and to achieve the necessary volume of
biomass for fermentation, the strain was grown in sterile
(121°C for 15 min) orange juice (11°Brix and pH 3.7) and
inoculated at 8% (maximum) of the total volume of
fermentation must. The initial population in the vats was
approximately 3 × 10​7​ cell/mL.

Fermentation assays

Three batch fermentations of 20 L each were conducted at

approximately 25°C. At the end of fermentation, the vats were
allowed to rest for 4 h to aid sedimentation of the solid
material from the orange juice. The fermentation was
considered complete when the Brix level was stable. Samples
were collected at 0, 4, 8, 12, 18 and 24 h to determine the
concentrations of sugars, organic acids, glycerol, ethanol,
methanol, volatile compounds, Brix value, pH, temperature
and viable yeast cells.
Evaluation of Fermentation Performance

8
 Figure 4

The conversion factors of the substrates into ethanol (​Y​p/s​),

biomass (​Y​x/s​) and glycerol (​Y​g/s​); the volumetric productivity
of ethanol (​Q​p​); the biomass productivity (​P​x​); and the
conversion efficiency (​E​f​) were calculated.

The equations used in this work are presented below:

where ​Pi​​ is the initial concentration of ethanol, ​P​f​ is the

ethanol concentration at the end of fermentation, ​S​i​ is the
initial substrate concentration (sum of sucrose, fructose and
glucose), ​Sf​​ is the substrate concentration at the end of
fermentation, ​X​i​ is the initial biomass concentration, ​X​f​ is the
biomass concentration at the end of fermentation, ​gi​​ is initial
glycerol concentration, ​gf​​ is glycerol concentration at the end
of fermentation and ​tf​​ is the total time of fermentation.

9
Enumeration of microorganisms and viability

For the determination of cellular populations, 100 μL of an

appropriate serial dilution of the sample was plated (in
triplicate) onto YPD plates. The plates were incubated at 30°C
until colonies appeared (1–3 days), and the number of
colony-forming units per millilitre of cell culture was
determined. Microscopic cell viability was measured using
methylene blue in a Neubauer chamber.

Distillation

After fermentation, the distillation process was performed in a

copper alembic equipped with a condenser and gas heater and
with a working capacity of 50 L. The temperature of the
fermented orange must was maintained between 91 and 97°C.
The distillate was separated into three fractions. The first
fraction (head fraction) was collected separately and
standardized to a volume corresponding to approximately
10% of the total volume of spirit. The intermediate fraction
(heart fraction) was then collected until an ethanol
concentration of approximately 400 g/L was reached. The last
fraction (tail fraction), corresponding to 10% of the volume of
spirit produced, was also collected. The final beverage was
stored in glass bottles and maintained at 20°C for
physico-chemical and sensory analysis.

HPLC analysis

10
Ethanol, glycerol, organic acids (acetic, malic, succinic and
propionic acids) and carbohydrates (glucose, sucrose and
fructose) were quantified by high-performance liquid
chromatography (HPLC), using a Shimadzu chromatograph,
model LC-10A​i​ (Shimadzu Corp., Japan), equipped with a
dual detection system consisting of a UV detector (SPD-10A​i​)
and a refractive index detector (RID-10A​i​). A Shimadzu ion
exclusion column (Shim-pack SCR-101H, 7.9 mm × 30 cm)
operated at a temperature of 50°C was used to achieve
chromatographic separation. The quantification of alcohols,
sugars and acids was performed using calibration curves
obtained from standard compounds. All samples were
examined in duplicate. Malic, succinic, citric and propionic
acids, glucose, sucrose and fructose were purchased from
Sigma-Aldrich (St Louis, MO, USA). Acetic acid, glycerol
and ethanol were purchased from Merck (Darmstadt,
Germany).

Sensory evaluation

The sensory analysis was performed with non-trained tasters.

The orange spirit was evaluated by 50 panellists of both sexes
ranging from 18 to 60 years of age. Ten-millilitre samples
were served in clear glasses with a capacity of 25 mL.
The evaluation sessions took place between 9:00 and 10:00
a.m. at room temperature (20–22°C) under white light.
The samples were evaluated for taste, aroma, appearance and
overall impression according to the hedonic scale of nine
categories:

11
extremely dislike = 1;
very much dislike = 2;
moderately dislike = 3;
slightly dislike = 4;
neither like nor dislike = 5;
slightly like = 6;
moderately like = 7;
very much like = 8 and
extremely like = 9.

ALCOHOLIC FERMENTATION

The acidic pH and the use of K​2​S​2​O​5​ in the must (100 mg/L)
contributed to the reduction of the bacterial population and
consequently allowed yeast growth.
Viable cell counts during the fermentation period failed to
detect bacteria, indicating that the metabisulphite was efficient
at controlling bacterial growth during the fermentative
process. Dias ​et al​. found that the use of SO​2​ in the
elaboration of fruit wine was effective for the inhibition of
undesirable bacteria during fermentation.
Throughout the fermentation process,
when ​S​. ​cerevisiae​ UFLA CA1174 was inoculated, there were
stable viable populations of approximately 7 log CFU/mL.

12
 Graph 1

The maximum population (8.33 log CFU/mL) was reached

after 8 h of fermentation. The cell viability was high during
fermentation.
The differences in the growth of the yeast can be associated
with the yeast's ability to adapt to the substrate. According to
Ivorra ​et al,​ several factors, including heat-shock, oxidative
and osmotic stress, available nitrogen, sugar and ethanol
concentrations, affect yeast growth during fermentation.
Yeasts with a lower resistance to these factors have
difficulties in the fermentation process, which ultimately leads
to a reduction in their growth and survival rates, and may
result in lower fermentation efficiencies.

Kinetic parameters

​ value was 0.50 g/g. This value was similar to values

The ​Yp/s​
reported in the literature related to sugar cane juice
fermentation for ​cachaça​ production, ethanol production and
for the fermentation of raspberry juice. The UFLA CA1174

13
strain achieved a very low ​Yx/s​​ value of <0.030 g/g. The value
for the volumetric productivity of ethanol (​Qp​​ ) found in this
study was 1.78 g/L/h. This value indicates that the yeast used
was able to grow in orange must and efficiently convert the
substrate into ethanol.
The conversion factor of the substrate into glycerol (​Y​g/s​) was
0.04 g/g, corresponding to moderate ​Y​g/s​values. Glycerol is
quantitatively the most important fermentation product after
ethanol and carbon dioxide. During alcoholic fermentation,
the main roles of glycerol are to equilibrate the yeast
endocellular oxidation–reduction potential (or NAD​+​/NADH
balance) and to act as an osmoregulatory metabolite in
response to the high osmotic pressure of the sugar solution in
the fermenter. The ​Y​g/s​ value found in this work may be a
result of the addition of SO​2​ to the orange juice. The added
SO​2​ may combine with the ethanol formed at the beginning of
the fermentation and increase the glyceropyruvic fermentation
rate and the overall amount of glycerol. Table ​2​ shows the
results of ethanol, glycerol, sugars, organic acids and volatile
compounds identified during the fermentation process of
orange must.
The ​Px​​ and ​Ef​​ parameters were 58.47 g/g and 97.83%,
respectively. The yeast used showed a very high ​E​f​level and
achieved better results than when the strain was employed in
the fermentation of raspberry juice. Different levels
of ​E​f​ might be justified by the Gay–Lussac equation for
alcoholic fermentation, which states that, under anaerobic
conditions, each kilogram of glucose consumed could produce
0.51 kg of ethanol. The remaining carbon source was used to
generate biomass, glycerol and the volatile compounds.

14
Chemical analyses during fermentation

Figure 5

The chemical compounds produced by microbial activity were

analysed throughout the fermentation process. The rapid
decrease in sugar content and the increase in the concentration
of ethanol during the inoculated fermentation confirmed that
the use of the selected yeast promoted a rapid increase in the
concentration of ethanol, as the strain dominated the
fermentation process.

Graph 2

15
Based on the initial sugar levels (16°Brix), a theoretical
alcohol content of approximately 8% (v/v) was expected after
fermentation. As the determination of °Brix by refractometry
indicated the total soluble solids, which are not necessarily
composed of only sugars, the final alcohol yield might appear
to be low if based on the Brix value. The highest ethanol
concentration (58.13 g/L) was achieved after 24 h of
fermentation.
Sucrose, glucose and fructose were determined to be the sugar
components in the orange juice and wine. The total amounts
of sugar were 116.74 and 9.86 g/L in juice and wine,
respectively. Glucose and fructose were found in high levels
at the beginning of fermentation, but were consumed during
the process. At the end of fermentation, the glucose, fructose
and sucrose contents were 8.07, 1.48 and 0.31 g/L,
respectively. The sugar content in Pêra oranges differed from
the value found by Kelebek ​et al.​ in Kozan orange juice,
which were as follows: 32.30 g/L of glucose, 25.55 g/L of
fructose and 59.34 g/L of sucrose. This difference may be due
to the use of different varieties of oranges or heat treatment
imposed on the juice before inoculation; the sucrose was
probablyhydrolysed to glucose and fructose.
The production of glycerol by ​S.​ ​cerevisiae​ was evaluated.
Glycerol is a non-volatile compound with no aromatic
properties, but it significantly contributes to wine quality by
providing sweetness and fullness. Glycerol was approximately
9% of the total amount of ethanol produced by strain CA
1174. Its concentration ranged from 0.86 to 5.45 g/L, which
was similar to the values of 6 and 10 g/L suggested by Vogt ​et
al.​ as a characteristic capable of conferring body and texture
to a beverage.

16
Citric, malic, succinic and propionic acids were separated and
identified in the orange juice and wine. Citric acid was found
to be the major organic acid in orange juice (9.72 g/L) and
wine (8.55 g/L). Reported results for the citric acid level in
Kozan orange juice and wine were 12.66 and 6.03 g/L,
respectively . Malic acid was the second most abundant
organic acid in orange juice (4.43 g/L) and wine (0.92 g/L).
The lower concentration of organic acids in the wine,
compared with the juice, can be explained by losses during
fermentation. Acetic acid was not detected during
fermentation, which is beneficial because wine containing
acetic acid in high concentrations has a pronounced
vinegar-like taste. Methanol was not detected in any samples
of the fermented must.
Several volatile compounds, which consisted of higher
alcohols, ethyl esters and acetate of higher​ ​alcohols, were
identified and quantified by gas chromatography during the
fermentation of orange juice. In general, the higher alcohols
quantified in this study (1-propanol, 1-butanol,
2-methyl-1-propanol, 2-phenylethanol, 2,3-butanediol and
1,2-propanediol) were not detected or were found in low
concentrations during the fermentation of orange juice. The
exception was 2,3-butanediol, which was found to be present
at a concentration of 382.52 mg/L. The total amount of
isoamyl alcohols [2-methyl-1-butanol (2M1B) and
3-methyl-1-butanol (3M1B)] for the orange wine was
approximately 31.39 mg/L. Alcohols similar to
2-phenylethanol have aromatic descriptions of ‘rose-like’,
‘sweet’ and ‘perfume-like’ and can positively influence the
beverage aroma. In this study, 2-phenylethanol was present
after 8 h of the fermentative process, reaching 62.30 g/L after
24 h of fermentation. Selli ​et al.​ also found that, among higher
17
alcohols, isoamyl alcohol showed the highest concentration
(79.04 mg/L) in orange wine; another alcohol present at a very
high concentration was 2-phenylethanol (27.26 mg/L). At
concentrations below 300 mg/L, higher alcohols can
contribute to the desirable complexity of wine, but when their
concentrations exceed 400 mg/L, higher alcohols are regarded
as having a negative effect on quality. These higher alcohols
are an important group of volatile molecules produced by the
yeast during alcoholic fermentation.
Ethyl acetate was the main ester detected in the orange wine
with a concentration of 134.35 mg/L. This concentration is
dependent upon several factors, particularly juice
composition, fermentation temperature, yeast strain and
aeration degree. The values of ethyl acetate found in this work
were higher than those described by Duarte ​et alf​ or the
production of the jabuticaba spirit and were similar to values
found by Hernández-Gómez ​et al​. in fermented beverages
from sugar cane, orange and grape. The production of esters
by yeasts during fermentation significantly affects the ‘fruity’
flavour of the final wine.
Similar concentrations of acetaldehyde were found in
fermented wine and in the heart fraction (approximately 45
mg/L). The amount of these alcohols in wine is influenced by
the presence of SO​2​. Despite the high concentration of SO​2​ at
the beginning of the fermentation of the orange must (100
mg/L), it was eliminated by decanting and during the
fermentative process.

Analysis of the distillate

18
The three fractions (head, heart and tail) of the distillate were
subjected to HPLC and GC analysis. The main objective for
the analysis of the individual fractions was to ensure that the
heart fraction had a low concentration of toxic and detrimental
sensory compounds, acceptable concentrations of ethanol and
compounds that are favourable to the aroma and flavour of the
spirit.
Ethanol and all by-products produced potentially influence the
quality of the final product. The composition and
concentration of the by-products may significantly vary in the
final product from a few nanograms to hundreds of milligrams
per litre. Table ​3​ shows the composition of the head, heart
and tail fractions found in orange spirit.
As expected, a reduction in the concentration of some
compounds was observed when the head and tail fractions
were compared. Acetaldehyde, ethyl acetate, phenyl acetate
and 2,3-butanediol were found at higher concentrations in the
head fraction than in the tail fraction. On the other hand, the
amounts of isoamyl alcohols, 1-hexanol and 2-phenylethanol
in the tail fraction were higher than the amounts measured in
the head fraction. The compound 1-butanol was not detected,
a highly positive finding, as this substance adversely affects
the final aroma of the distillate. Acetaldehyde is one of the
components responsible for pungency in distillates. A critical
concentration of this compound (1281.83 mg/L) was present
in the head fraction, and the heart fraction contained 44.06
mg/L of acetaldehyde, thus showing that the separation was
efficient. The increase in the concentration of 2-phenylethanol
in the tail fraction could be explained by its high boiling point
of approximately 220°C. The compound 1-propanol was
mainly collected in the heart fraction. The compounds
2-methyl-1-butanol, 3-methyl-1-butanol and
19
2-methyl-1-propanol have boiling points lower than 200°C,
are alcohol-soluble and are completely or partially
water-soluble; thus, they distil mainly in the heart fraction.
The concentrations of these compounds were the highest
values found and were observed only in the heart fraction of
the distillate.
The esters eluted principally in the head fraction of the
distillates. Esters have been associated with pleasant aroma
descriptors. Small amounts (44.06 mg/L) of ethyl acetate
contribute to aroma complexity and have a positive effect on
product quality, but high quantities of this compound are
responsible for strong and pungent smells. Ethyl octanoate
was found in low concentration (6.47 mg/L) only in the heart
fraction. A major contribution to orange flavour is due to the
minor oxygenated constituents, especially the aldehydes,
esters and alcohols.
Volatile fatty acids were found in lower concentrations in the
orange spirit, and they included octanoic (6.08 mg/L) and
decanoic (2.12 mg/L) acids. The presence of high
concentrations of acids may negatively influence the qualities
of the wines and spirits, as octanoic acid has aroma
descriptors that include ‘rancid’ and ‘harsh’ and decanoic acid
has aroma descriptors that include ‘fatty’.
According to Brazilian law, spirits must have a standard
quality measured by parameters set by the Ministry of
Agriculture. It shows several results for parameters evaluated
in a routine analysis of spirits and the limits set by Brazil for
each parameter.​ The relative density value of 0.95 is
considered a normal value for a distillate beverage. The
volatile acidity (such as acetic acid) was found in the orange
distillate at a concentration of 37.99 mg/100 mL of anhydrous
alcohol. This value of volatile acidity is lower than the value
20
found by Duarte ​et al​. in a spirit produced from jabuticaba
wine. Madrera ​et al.​ investigated the influence of cider
maturation on the chemical and sensory characteristics of
fresh cider spirits and found volatile acidities between 10 and
15 mg/100 mL of acetic acid. Acidity has a negative influence
on the sensory quality of a beverage. The main source of these
compounds is the metabolism of yeast during fermentation.
However, some compounds in spirits
are from the fruit that is used as a raw material.

​BY-PRODUCTS OF FERMENTATION

Ethyl carbamate

Ethyl carbamate is a naturally occurring by-product in

fermented foods and beverages. It is formed by the chemical
reaction between urea and ethanol. Urea is a product of the
microbial degradation of L-arginine and citrulline.
Differences in the L-arginine concentrations of the fruit juices
determine the urea and thus the ethyl carbamate levels of the
final fermented product to some extent. Orange juice contains
about 44 mg/1 00 mL L-argininc.

21
 Figure 6

However, for the present case more important than differences

of L-argininc concentrations in the fruit juice is thefact that
Schizosaccharomycespombe contains urease, whichprevents
an accumulationof urea in fermented musts. Indeed, the urea
contentof white wines fermented with
Schizosaccharomycespombe alone was lower than in
wines made with mixed fermentations and with
Saccharomyces cerevisiae alone. Accordingly, the levels of
urea in the desugared fermented orange juice and
consequently the formation of ethyl carbamate is also
expected to be low.

Biogenic amines

The public health risks of biogenic amines, such as histamine

and tyramine, have beenevaluated by different authoritative
bodies. Inwine, tyramine and histamine typically occur in
concentrations that do not present a riskto human health.
However, histamine concentrations of more than 10 mg/L
have alsobeen reported.

22
The tyrosine content of orange juice was reported to increase
from 284 ± 46 mg/L to 425± 18 mg/L during fermentation for
9 daysusing Pichiakluyveri forstarting the fermentation.
Whether the fermentation oforange juice with
chizosaccharomyces porn be leads to a similar increase of
tyrosine isnot known.However, no indication could be found
in the scientific literature of the presence oftyrosine
decarboxylase or histidine decarboxylase in
Schizosaccharomycespombe.Therefore, there is no indication
that tyramine and histamine is formed during thefermentation
of orange juice with hizosaccharomycespombe. Direct
evidence for thisis provided by a comparative study on the use
of different yeasts in red wine productionin which the
biogenic amines after fermentation were measured. For none
of theanalyzed amines there was a difference between
fermentations withSchizosaccharomycespombe,
Saccharomyces cerevisiae, and
Kluyveromycesthermotolerans.

Figure 7

Recently, the formation of melatonin in orange juice during

alcoholic fermentation with Pichiakluyveri was reported. The
observed concentrations increased from 3.15 )lg/L on day 0
(melatonin naturally present in range)to 21.8 )lg/L on day 15

23
of fermentation. These concentrations are well within the
range of melatonin concentrations that have been found, using
the essentially same analytical method, in Schizo
saccharomycescerevisiae, fermented red wines Whether
Schizosaccharomycespombe has the same capability of
forming melatonin like Saccharomyces cerevisiae is not
known.

Methanol

Methanol is yet another potential undesirable by-product of

alcoholic fermentations including the fermentation of orange
juice. In a comparative study on the volatile compounds
formed in the production of white wine with either
Saccharomyces cerevisiae or Schizo saccharomycespombe
insignificantly lower concentrations were found when Schizo
saccharomycespombe was applied. However, methanol due to
its lower boiling point will be removed during the ethanol
evaporation step even more completely than ethanol.
Therefore, at the most, trace amounts of methanol might occur
in desugared fermented orange juice.

NUTRITIONAL AND HEALTH RELATED

CONSEQUENCES

The consumption of fruit is generally associated with better

health. However, the consumption of fruit falls short of
national recommendations. In general, whole fruit contributes
about 65% of all fruit servings while fruit juice contributes
about 35% depending upon age.

24
 Figure 8

Thus, the consumption of 100% fruit juice provides a

significant amount of vitamins and minerals as well as other
bioactive compounds with the potential to positively affect
human health. Fruit juice is under consumed rather than over
consumed and adverse effects to humanhealth, directly or
indirectly, are not known.
A comparative analysis of orange juice and desguared
fermented orange juice has shownthat ascorbic acid,
carotenoids, flavonoids and folates are not affected by the
fermentationof the orange juice
withSchizosaccharomycespombe. In contrast, the content
of total sugars and thus energy is decreased by about 98 and
87%, respectively. The introduction in the food supply of
desugared fermented orange juice under the
Intended conditions of use and at the estimated highest
achievable levels is not associatedwith any risk to human
health directly or indirectly but may indeed help to increase
fruitconsumption because it offers an acceptable alternative to
consumers who are concernedabout the sugars consumption
that is inherentlyassociated with the consumption of
100%fruit juice or even whole fruits.

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Among children in which the intake of energy with fruit juice
or fruit drinks is notproblematic from an energy intake point
of view, a reduction inthe frequency of sugar intake,
particularly between meals, may still have a beneficialeffect
on dental health.

HISTORICAL AND PRESENT USE OF FERMENTED

ORANGE JUICE

The term "orange wine" encompasses quite different

beverages such as white wine (madefrom grapes) flavoured
with macerated orange peel or wine with an orange color that
itgot from contact with grape skins. However, this term also is
applied to ·'fruit wine"made from orange juice rather than
grape juice. This type of orange wine has a longhistory
ofhuman consumption, for example, in Southern Europe
andon the French island of Martinique, where it was already
known more than two hundredyears ago.However, the
fermentation of orange juice was known at about that time
notonly on the Cara'ibes but also in Portugal. Typically,
sucrose was added to the orange juice and baker's yeast was
used for starting thefermentation. From Portugal, the product
reached France and other regions.In recent times, the
production of orange wine has attracted renewed interest in
differentcountries including Spain, Brazil, Colombia, Mexico,
Honduras, and Argentina. In all those studies,Saccharomyces
cerevisiae was used for the fermentation.The fermentation of
orange juice with Pichiakluyveri was studied more recently.
Thisyeast was isolated from the natural microbiota present in
the orange fruit but it fermentsonly reducing sugars and
therefore results in a fermented product which still

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containssignificant concentrations of sugar and a low ethanol
content (only 0.87%).

Figure 9

In a study conducted in Turkey, orange juice with added sugar

was subjected tospontaneous fermentation. The alcohol and
total sugar concentration in the final analysesreached 12.2
and 6%, respectively. While the flavor compounds were
analysed in detail,no attempt was made to characterize the
microbiology of this product.Schizosaccharomycespombe
which has been found naturally occurring in fermentersused
for the fermentation of sugar cane was examined for usc in
maloalcoholicfermentation about 30 years ago but was
proposed for theproduction of fruit wine only more recently.
Energy and nutrient content of orange juice before
and after fermentation
Desugared
Content Orange Juice Fermented Difference
Orange Juice

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Energy (kcal 40.2 5.3 -87%
/100 mL)

Total Sugars 8.53 0.13 -98%

(g/100 mL)

Total vitamin C 48.43 44.88 -7%

(mg/100 mL)

Total 3.28 2.88 -12%

Carotenoids
(pg/100 mL)

Total Flavonids 284 315 +11%

(mg/100 mL)
Folates 20.3 19.8 -2%
(pg/100 mL)

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PHOTO GALLERY:

29
CONCLUSION

​Based on the characteristics of the juice and the acceptance of

the orange spirit in the sensory analysis, orange fruit showed
good potential for use in the production of a distilled
beverage. It was observed that, from the acceptability of the
beverage, this technology could be an alternative use for citric
fruit juice and could provide a new industrial outlet for
oranges.

Figure 10

​A more systematic production process, as well as a better

standardization process of the product, would offer an

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opportunity to improve the distillation process and to obtain
an orange spirit of high quality.
FUTURE WORKS:

Lactic acid (LA) fermentation is considered a simple and

useful form of biotechnology to keep and/or enhance the
safety, nutritional, sensory and shelf life properties of
vegetables and fruits (Demir et al. 2006). As shown in the data
from literature of the last decade, the combination of this
ancient method of bio-preservation with the current
biotechnology tools should allow controlled fermentation
processes and the selection of starter cultures to increase the
consumption of fresh-like vegetables and fruits. Lactic acid
bacteria (LAB) convert the carbohydrate contents of the
vegetables and fruits into LA, which decreases the pH of the
fermented products to around 4.0 ensuring stability. They are
often considered as probiotic, benecial for human health and
active in lowering the serum cholesterol level. Fruits are
commonly processed for alcoholic fermentation of wine and
beer as they are rich in sugars, vitamins, minerals. As juices
are slightly acidic, they are therefore a suitable medium for
the growth of yeasts, and fruit sugars are rapidly converted
into ethanol. Fermentation of fruits and vegetables can occur
‘spontaneously’ by the natural lactic acid bacterial surface
microora, i.e., Lactobacillus,Leuconostoc, Pediococcus, etc.;
however, the use of starter culture such as
Lactobacterplantarum, Lb. rhamnosus, Lb.gasseriand Lb.
acidophilus (all probiotic strains) provides consistency and
reliability of performance. Pasteurizing or adding
preservatives after fermentation, which are commonly done
during the industrial production of lactic acid fermented

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vegetables (e.g., sauerkraut), destroy most of the LAB present,
thus cancelling any possible probiotic effects .

PHOTO CREDITS:
TITLE OF IMAGE Pg #

Figure 1: orange fermentation 01

Figure 2: orange fermentation 05

Figure 3: orange fermentation 07

Figure 4: orange fermentation 08

Figure 5: orange fermentation 14

Figure 6: orange fermentation 20

Figure 7: orange fermentation 22

Figure 8: orange fermentation 23

Figure 9: orange fermentation 25

Figure 10: orange fermentation 28

Graph 1: fermentation 12

Graph 2:fermentation 14

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BIBLIOGRAPHY
cssf.usc.edu/History/2010/Projects/J0426.pdf

https://books.google.co.in/books?isbn=8184091192

www.jbc.org/content/66/1/49.full.pdf

inside.mines.edu/~jobush/gk12/lessons/Fermentation.pdf

https://www.fermentedfoodlab.com
https://en.wikipedia.org/wiki/Fermentation_in_winemaking

jfoodprotection.org/doi/pdf/10.4315/0362-028X-45.9.874

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