SUGGESTED QUESTIONS FOR FINAL EXAMINATION REVIEW ON

TECHNOLOGY OF COFFEE, TEA, AND COCOA

DEADLINE: 03/01/2021
Question 1: The importance of cocoa fermentation (B. Phương)
Fermentation is crucial to flavor development and the final acidity of cacao beans. Without
fermentation, cacao would have no flavor at all.

1. It facilitates removal of the viscous pulp around the beans and their subsequent drying
● Yeasts quickly consume simple sugars in pulp and produce CO2, ethanol.
● LAB converts citric acid, glucose, and other carbohydrates in the pulp into lactic acid.
● AAB dominates and oxidizes ethanol and acids (citric, malic, lactic) to acetic acid. This
acetic acid is broken down into CO-2 and water.
2. It contributes to colour and flavour development of cocoa beans:

The combination of intense heat and the diffusion of ethanol and acetic acid (results of microbial
activities of yeasts, LAB, AAB) into the cacao beans breaks down the cell walls. They will no
longer germinate, and the damaged internal structure becomes a venue for chemical activities that
develop the flavor precursors associated with chocolate.

Ex: a decrease of internal pH from 6.5 - 4.8

an increased bean temperature up to 50 ◦C

a damaged internal cocoa bean structure

⇒ Development of flavour precursors and pigment degradation by endogenous enzymes, such as

invertase, glycosidases, proteases and polyphenol oxidase

3. It reduces bitterness and astringency of raw cacao beans due to the high phenolic content
of cocoa beans.

Without fermentation, the cocoa bean will create bitter taste, astringency, and will not produce
distinctive aroma of chocolate after roasting process.

Ex: Tannin content: 5 – 15% the bean weight

⇒ Tannins need to be removed in final chocolate by fermentation, especially by exchange of

compounds through diffusion between the cocoa bean cotyledons and the environment
Question 2: By your best understanding, how many types of cocoa fermentation, can you
clarify the disadvantages and advantages of each? (focus on 4 methods: heap, box, basket
and tray) (Ngân)

General characteristics: In all cases, the bottom and sides of the box or basket should be covered
with banana leaves, however banana leaves on the bottom should be not too thick and should be
also perforated by a knife to make sure that the liquid from the cocoa pulp will be well drained.
The top layer of fermenting cocoa should also be covered with banana leaves or jute bags.

Method Characteristics

Heap This method involves keeping a mass of wet beans over a layer of banana leaves.
The leaves are folded and kept over a heap of beans and a few wooden pieces kept
over it to keep the leaves in position.

Box Wooden boxes with holes at the bottom and sides of the box also are covered with
banana leaves. The beans are to be mixed on alternate days. As the quantity of beans
is high, this is best done by changing the beans from one box to another at the time
of mixing → necessitate having a minimum of three boxes.

Tray Wooden trays with battens or reapers fixed at the bottom with gaps in between, are
filled with beans. Several trays (6 -12) are stacked one over the other and an empty
tray is kept at the bottom to allow for drainage of sweating. After stacking, the
beans of the top tray are kept covered with banana leaves.

Basket The bamboo baskets had holes at the bottom from where liquids could drain. The
baskets were covered with banana leaves to reduce heat loss, and fermentation
proceeded for 6 days, with the cacao seed mass being turned at 2nd – 4th day.
Afterwards, fermented seeds were dried.
 Duration Quantity (kg) Temperature (⁰C) Rotate/
(days) Mixing (day)

Heap 5-7 25 – 2500 50⁰C. 2nd – 3rd

Box 3–5 500 – 1000 ↑ quickly 50⁰C 2nd – 4th

Tray 3-4 10 -150 50⁰C unnecessary

Basket 5-6 500 – 1000 ↑ quickly 45-50⁰C. 2nd – 4th

Table: Advantages and disadvantages of 4 methods

Method Advantages Disadvantages

Heap - Simplest method - Low quality

- Does not require fermentation - Easily to be attacked by microbes

equipment
- Requires rotating → depends entirely on
- Large quantity (25- 2500kg) the experience and technique of farmers

Box - High quality - Requires equipment - timber

fermentation boxes
- Large quantity; from 25kg up to any
amount a farmer harvest. - Requires rotating

Tray -Save time (3 days) - Small quantity

-Does not require rotating - Undesired effects on seeds’ color

-Requires equipment

Basket High quality - Small quantity: only 10-150 kg each

batch.

- Requires rotating
Question 3: Cocoa fermentation including anaerobic phase and aerobic phase, what happens
in each phase? (Mẫn)

THE ANAEROBIC PHASE

• In cacao, the pulp surrounding packed beans creates a juicy barrier that blocks air from
entering the system.

• The pulp is composed of water, high levels of sugars (sucrose, glucose, fructose), and
various acids → ideal conditions for microorganisms.

• The main players during this phase are yeasts, LAB, and pulp enzymes.

• Yeasts: consume simple sugars → produce CO2, ethanol, and low amounts of energy.

• Lactic acid bacteria: convert citric acid, glucose, and other carbohydrates into lactic acid.

• Enzymes: break down the pulp, transforming it into a liquid that runs off and is known as
sweating.
THE AEROBIC PHASE

• As the mass is broken down, there is more space for air to enter the process.
• Citric acid is also broken down and runs off with the sweating → Increasing the overall pH
of the fermentation.
à Increasing pH and increased airflow marks the beginning of the aerobic phase of
fermentation.
• The aerobic stage is bursting with heat.
• Acetic acid bacteria dominate and oxidizes ethanol and acids (citric, malic, lactic)
→ produce acetic acid. This acetic acid is further broken down into CO2 and water by the
oxygen.
• Breaking down ethanol generates energy (expressed as heat) → increase the overall
temperature of the cacao.
• Intense heat and the diffusion of ethanol and acetic acid into the cacao beans breaks down
the cell walls → no longer germinate, the damaged internal structure becomes a venue for
chemical activities that develop the flavor precursors associated with chocolate.
 Question 4: Roles of microorganisms in cocoa fermentation. (Quỳnh chó điên)

Microorganisms Roles in cocoa fermentation

● The yeasts quickly generate an alcoholic fermentation, convert sugars in the

Yeast pulp into ethanol and CO2.
- The ethanol concentration will determine the ecology of the microbiota →
becomes a substrate for the growth of AAB.
- The CO2 is produced → contribute to the microaerobic conditions of the
bean mass, thereby encouraging the growth of LAB.
● The citric acid is used in the metabolism of the yeasts → initiates a slow rise
in the pH of the pulp material.
● The rising pH creates a self-limiting factor on further overgrowth →
pectinolytic → an important role in degradation of the pulp
● Enzymes released by yeasts attack the pectin constituents of the pulp mass
→ decreases the viscous
→ facilitates its mixing and penetration of air (O2) encouraging the growth of
AAB.
● Death and autolysis of the yeast cells → provide micronutrients, such as
vitamins and amino acids → support the growth of other bacteria.

● Ex: Lactobacillus bulgaricus, Streptococcus thermophilus

Lactic acid ● The sugary, acidic, aerobic environment of the pulp is desirable for the growth
bacteria (LAB) of LAB.
● LAB conduct 3 major reactions during bean fermentation: ferment pulp
sugars, utilize citric acid, and some may reduce fructose to mannitol →
contribute to changes in the pulp composition → impact on cocoa bean and
chocolate quality
● LAB modulates the activity of endogenous enzymes
à contribute to the generation of chocolate flavor.
● However, their production of lactic acid is not always seen as a desirable
property, as it may lead to excessive bean and chocolate acidity.
 ● Characteristic: AAB contributes to → vinegar-like aroma of cocoa beans.
Acetic acid ● Ex: A. pasteurianus
bacteria (AAB) ● These bacteria conduct two main reactions:
- Oxidation of pulp sugars and acids à give various metabolites, such as
gluconic acid, aldehydes, and ketones
- Oxidation of ethanol à acetic acid
à affect bean quality and flavor
● AAB is antimicrobial → influence the growth of yeasts, bacteria, and
filamentous fungi, which have their own specific impacts on bean and
chocolate quality.
● Indeed, residual acetic acid may limit the growth of spoilage and
mycotoxigenic fungi during the drying stage.

● Ex: Bacillus subtilis

Aerobic spore- ● They give rise to:
forming bacteria - Chocolate flavor: acetic acid, lactic acids, and 2,3-butanediol
- Aroma: tetramethylpyrazine and other parazines
- Pectinolytic activity: polygalactu-ronase, pectin lyase → degrade cocoa
pulp pectin
● Under fermentative condition: the acidity and off-flavors of fermented cocoa
● Metabolic activities: Produce a range of extracellular proteolytic, lipolytic,
amylolytic, and pectolytic → impact on bean quality: off-odors and slimy
beans.

● Ex: Penicillium spp.

Mold (Filamentous ● Present in different stages of harvest, fermentation, drying and storage of
fungi) coffee beans process.
● Quite a low amount of filamentous fungi.
● Positive contribution:
- Produce pectinolytic enzymes → solubilization of the pulp at early stages
of fermentation.
● Negative contribution:
- Produce mycotoxins: ochratoxin A, aflatoxin
→ Conclusion: Not beneficial to cocoa bean and chocolate quality → should be
prevented
Question 5: Many changes during cocoa fermentation among them biochemical changes
(flavor, sugar, acids, nitrogen, alcohol,...) cocoa pulp (pH, temperature, oxygen) and enzyme
change are focused, discuss them. (Nhật Anh)

The raw cocoa beans usually have bitter and astringent flavours which are due to polyphenols such
as catechins, anthocyanin,…

- In the first 72h, anaerobic fermentation occurs.

• Sugar (glucose, fructose) in cocoa pulp → ethanol + CO2 + temp (by yeast).

_ The main carbohydrate in the ripe pulp: sucrose is hydrolyzed to glucose by pulp and yeast
invertase activity. At this fermentation point, fructose is almost not used

Under anaerobic condition:

- Yeast will convert glucose into ethanol, CO2 and generate heat simultaneously
- Ethanol production is an exothermic process

à Increase temperature in cocoa pulp

When temp gets 45-50 oC, yeast reduces due to high temperature, farmers will stir the beans and
let air travel in + aerobic condition → A desirable condition for aerobic bacteria to grow.

- In the first 36-48h of fermentation:

● Sugar + LAB → lactic acid (non-volatile) à pH drop

As the amount of yeast and LAB decreases, the volume of air increases, Acetic acid bacteria will
grow

● Ethanol + acetic acid bacteria → acetic acid (volatile)

=> Decrease pH à Create sour flavor → desirable quality in chocolate if controlled

_ The acid and heat generated kills the cocoa bean causing the cell walls within the bean to break
down, allowing enzymes to come into contact with their substrates.

_ Polyphenols will be modified by oxidation and enzymatic reaction

Anthocyanin → Anthocyanidin (by glucosidase enzyme)

Anthocyanidin → Quinone, Quinone + amino acid → melanin

=> make bitter and astringent flavours reduce.

- Biochemical and enzymatic reactions that occur within the cotyledon

=> The great majority of chocolate flavour compounds are formed

Furthermore, the volatiles such as higher alcohols, aldehydes, ketones and fatty acid esters, may
contribute to the production of cocoa flavour (precursors), once diffused into the cocoa beans or
released inside the beans by endogenous enzyme activities.

Question 6: Can you list out the enzymes related to cocoa fermentation and clarify their roles.
(Chi)
- Fresh unfermented cocoa beans contain various enzymes: α-amylase, β-fructosidase, β-
glucosidase, β-galactosidase, pectinesterase, polygalacturonase, proteinase, alkaline and
acid phosphatases, lipase, catalase, peroxidase, and polyphenol oxidase.

- During fermentation, aminopeptidase, cotyledon invertase, pulp invertase and

polyphenol oxidase are significantly inactivated, carboxypeptidase is partly inactivated,
whereas endoprotease and glycosidases remain active.
- During the anaerobic phase, the complex pigment components are attacked by
glycosidases and are converted by hydrolysis to sugars and cyanidins. Sucrose is converted
to glucose and fructose by invertase, proteins are converted to peptides and amino acids by
proteinase, and polyphenols are converted to quinones by polyphenols oxidase.
- During these processes, the color of the cotyledons changes slowly, the deep purple tissue
is converted to a red-brown color. Fermentation protease and peptidase activities seem
critical for good flavor quality.
- In the aerobic phase of fermentation, Polyphenol oxidase (PPO) is the major oxidase
responsible for some flavor modifications.

+ Oxidation of epicatechin during the aerobic phase of fermentation and drying is largely
responsible for the characteristic brown color of fermented cocoa beans.

+ Polyphenols oxidase: polyphenols are converted to quinones, which in turn can polymerize with
other polyphenols or complex with amino acids and proteins to yield characteristic color
compounds and high-molecular-weight insoluble material that result in the reduction in
astringency and bitterness.
Question 7: In your opinion, list out factors that may affect the final quality of cocoa. (Thanh
Phương)
- Cocoa genotype: influences the type and quantity of bean storage proteins, carbohydrates
and polyphenols which are degraded during fermentation and drying to form flavor
precursors.
- Depulping: removes portion of the pulp prior to fermentation hence, reduces fermentable
sugars during fermentation leading to the production of less acids
- Pod storage: reduces pulp volume peer seed due to water evaporation and inversion of
sucrose; reduces total sugar content and increases micro-aeration within the pulp and
eventually reduces alcohol fermentation and acetic acid formation.
- Fermentation: generates flavor precursors, namely free amino acids, peptides and reducing
sugars from which flavor volatiles are formed. Polyphenols are oxidized and polymerized
to insoluble high molecular-weights (tannins) leading to a significant reduction of its
concentration and, thus reducing the bitterness and astringency of flavor.
- Drying: Physic loss of acidity through outward migration of volatile acids as well as
biochemical oxidation of acetic acid from the beans leading to less acid in the beans.
Oxidation and polymerization of polyphenols resulting in reduction of its concentration.
Non-enzymatic reactions, that is Maillard reactions to form volatile fractions such as
pyrazines.
- Roasting: evaporation of volatile acids from the beans causing a reduction in acidity, hence,
reducing sourness. Flavor precursors namely free amino acids, short chain peptides and
reducing sugars undergo the Maillard reaction and Strecker degradation to produce the
desirable flavor compounds.
Question 8: What do you know about chemical compositions in cocoa (fats, proteins and
amino acids, carbohydrates, organic acids, polyphenols). (Thông)
1. Fats
• Cocoa beans contain 54% fat.
• Three primary fatty acids that make up cocoa butter are Oleic acid, Stearic acid and
Palmitic acid.
• In general, about 37.5% of the fat within cocoa butter is unsaturated fat and 61.4% is
saturated fat.
• Cocoa butter is solid at room temperature and melts at temperature between 30-40oC,
depending on the polymorphic form.
2. Protein and Amino Acids
• Cocoa beans contain 10-15% protein.
• Four main fractions of protein in cocoa beans: Albumin, Globulins, Prolamins and
Glutelins.
• During fermentation, proteins are degraded by enzymes to polypeptides and amino acids
which are the chocolate flavor precursors.
• The free amino acids content in fermented beans are responsible for the formation of
important compounds for cocoa aroma.
3. Carbohydrates:
• Carbohydrates contribute about 31% of dry weight of cocoa beans.
• Most carbohydrates in cocoa beans are starch, soluble and insoluble dietary fibers, simple
sugars.
• The various sugars present are Glucose, Fructose, Sucrose, …
• The concentration of glucose and fructose are decreased after roasting but the levels of
non-reducing sugar and sucrose are not affected.
4. Organic Acids: 1.2-1.6% in cocoa beans, mostly formed during the fermentation step and
the most represented are acetic acid, citric acid and oxalic acid. Acetic acid mostly lost during
cocoa processing.
5. Polyphenols:
• The unfermented cocoa beans contain about 180g/kg of polyphenolic compounds.
• Three major groups of polyphenols: Catechins, Proanthocyanidins and Anthocyanin.
• The polyphenols in cocoa beans are stored in pigment cells of the cotyledons.
• Depending on the amounts of anthocyanins, pigment cells are white to deep purple.
• During fermentation step, polyphenols diffuse with cell liquid from their storage cells and
undergo oxidation to condensed high-molecular mostly insoluble tannins.
 Question 9: What do you know about drying and grinding of cocoa? (Quang)

In cocoa processing, besides roasting, drying and grinding are the two most distinguished
important steps that contribute to the success of final flavor for the cocoa product.

Drying

· Introduction

• After conducting fermentation, drying cocoa is the next essential step to that manufacturer
aim, so as to successfully finish all the necessary chemical reactions. The main reason is to
help produce a pleasant bitterness and other expected flavors.
• Cocoa beans during drying must be controlled under 65oC (higher temp will restart the
chemical reactions) over 5-7 days (more than 7d motivates the growth of MOs) with 7-10%
moisture content and dark brown color.
• Natural/sun drying and Artificial drying are 2 typical types of cocoa drying

Method

• Natural or Sun drying uses solar energy to evaporate the water content of cocoa beans that are
being spreaded out on the mats, trays or on concrete floors. In fact, beans are normally turned
to ensure they are completely exposed to the sun.
• Artificial drying uses heat exchangers or conduction to allow hot air ventilate through the
products which use other fuels as wood, gas,… as the main energy source.

· Pros and cons

Advantages Disadvantages

- Natural drying
- Inexpensive and best way to obtain optimal - Depend on weather
quality. - Harmed by insects and hard to be controlled with
- Slow migration of moisture è transport flavor MOs
precursors during fermentatio

- Artificial drying
- Variety in using areas where being affected by - Poor quality due to contamination from the
weather is beaten. smoke
- Well controlling in insects and MOs affection.
- Creating great economic profit in saving space - Expensive and highly required technology.
and time.
Grinding

Introduction

• The grinding step is to turn the cocoa beans into nib particles. The nib consists of 55% cocoa
butter and 45% solid material coming from the plant cells. The latter must be ground very
finely. When eating chocolate or drinking chocolate milk, no grittiness should be felt in the
mouth. For both cocoa powder and chocolate it is important that the final particle size
distribution is narrow. This facilitates the pressing operation and improves the rheological
properties of chocolate.

• Grinding produces a smooth texture by reducing the size of the particles of cocoa mass from
about 80 – 90 microns to about 30 – 40 microns (Payne et al., 2010). At this point, the cocoa,
which has the required ingredients mixed, still has rough texture, because it is a form of
chocolate paste or chocolate liquor. Therefore, refining is necessary to turn the roughness into
smoothness. In addition, the cocoa liquor is mixed with cocoa butter and sugar and this is
further refined by reducing the particle size of the added milk powder solids and sugar down
to the desired fineness (Pugh, 2014
- The smoother the chocolate desired, the more rolling milling required. Because of that, in
order to ensure that its products' qualities meet their expectations, it refines its chocolate
paste to 19 micrometers while other major markets refine their chocolate to only 40
micrometers.
- The grinding process has certain parameters that can be changed that might alter the
flavour. Whether a product is fine, medium or coarse ground will determine the pallete’s
flavour perception. The particles will be coated with fat, which is the flavour carrier. When
these particles enter the mouth, the melt, sweetness and mouthfeel, and all will influence
how the product tastes (Stauffer, 2000).
Question 10: You have read many papers related to coffee, tea, and cocoa, choose one and
show your knowledge about recent research. (Doan)
● Name: Cocoa shell: A by-product with great potential for wide application
This paper gives an overview of possible applications of the cocoa shell in different areas for
solving economic problems by polluting the environment because there are huge quantities of by-
products that are discarded.
1. Use in Feedstuff:
- Cocoa shells that were boiled for 15 min (reduce theobromine) could be used in laying hen feed
up to 20% without an influence on egg production and feed conversion.
- In pig nutrition, a positive effect on the balance of the intestinal microbial ecosystem. 20% was
the optimal biological level of cocoa shells as an energy substitute for corn in a pig diet.

2. Use in Biofuels: good potential for biogas production, with cumulative methane yields.
3. Use as an Adsorbent: used as a low-cost adsorbent to entrap pollutants such as heavy
metal ions, gases, or industrial dye.
4. Use as a Dye: cocoa shell pigment has potential applications as natural dye for fabric
dyeing and in the production of UV protective cotton fabric
5. Use in Food Products:
- Produce corn snack products enriched with cocoa shells -> nutritional fortification agent.
- Cocoa shell flour for production of muffins and biscuits
- Safety warning: it is treated with different pesticides, and may contain heavy metals and toxins.

Conclusion: Cocoa shells can be used for further applications as biofuel, an ingredient in food
processing - or in other industries such as pharmaceutical, cosmetic, or agricultural industries.

Question 11: What are similarities or differences between cocoa fermentation and coffee
fermentation? (Nhi Đinh)
● Similarities
- Cocoa and coffee fermentation are a combination of microbial reactions that occur
in the pulp and the biochemical reactions they subsequently initiate within the bean
itself.
- Remove mucilage pulp and enhance flavor
- Involve similar microorganisms (bacterials, yeast, fungi)
- Happen spontaneously after being removed from the pulp
à The purpose of fermentation of cocoa and coffe bean are both to break down complex structure
to simple structure inside the bean à develop and contribute the unique characteristic of cocoa
and coffee
 ● Differences

Coffee fermentation Cocoa fermentation

Purpose Coffee mucilage contains polysaccharides

- can prolong the time needed to dry the
coffee bean
- also lead to mould development
à Breakdown the coffee mucilage layer to
enhance the final quality of the coffee
Raw cocoa beans are unpleasant taste because
of their bitter and astringent flavour
à Destroy the seed coat, and kill germ of the
seed to form a distinctive flavor of chocolate,
reduce bitter taste
à Development of desirable characteristics of
cocoa beans

Method
2 methods (Wet, Dry) 4 methods (Heap, Tray, Box,Basket)

Process
Mucilage is hydrolyzed by microbial 2 phases: aerobic and anaerobic
pectinase or pectate which is contained in
coffee cherries.

1. Wet: Soak coffee cherries under water and 1. Microbial action on mucilage pulp, produce
let them ferment. Stop when all the alcohol, acid and heat --> 20% pulp is
mucilage is removed and washing removed
completely the parchment 2. The combined effects of increasing
2. Dry: Put coffee cherries in a tank, temperature and penetration of the alcohol
fermented by the effect of temperature and and acids into the beans --> biochemical
oxygen level. and enzymatic reaction

Fermentat Around 5-6 days based on the method

ion time 24-48 hour depend on enzyme concentration,
temperature, and thickness of the mucilage
layer.

Main
enzyme pectin lyase Protease (Aspartic endoprotease,

polygalacturonase Carboxypeptidase, aminopeptidase)

pectin methyl esterase. Hydrolase (Glycosidase)

Oxidase (Polyphenoloxidases,
glutamate decarboxylase)
Question 12: What do you know about tea processing? What is tea fermentation? Discuss it.
(Nhi Nguyễn)
Tea processing is the method in which the leaves from the tea plant Camellia sinensis are
transformed into the dried leaves for brewing tea. Tea processing involves different manners and
degree of oxidation of the leaves, ending the oxidation, forming the tea and drying it.
There are various tea leaf processing, the four most common types of tea:

❖ The main difference between un-oxidized and unoxidized tea processing.

- Unoxidized tea such as white tea and green tea (it is not totally unoxdized because there is a
little bit oxidation after the leaves are plucked and let them until undergo processing), fresh
harvest tea leaves and unfermented leaves are immediately steamed to prevent fermentation
and inactivate enzymatic activity
à to keep green color and remain the natural polyphenol (catechin)

- On the other hand, fresh tea will not be steamed, and it undergoes fermentation steps to
contribute to the unique characteristics of oolong tea and black tea.
Plucking
- Using young tender leaves, include: 1 terminal bud with 2 leaves immediatly below the
bud
- Plucked by hand
Withering:
● Withering allows: water to evaporate from the leaves and a very slight amount of oxidation.
● There are 2 functions in this stage:
1. Withering is the preparation for the next step (rolling). If there is too much water
presence in tea leaves, the tea leaves will not shape easily in the rolling stage → interfere
shaping of tea.
2. Withering helps reduce the volume of tea leaves, enzyme and substrate have a chance
to react → enhance the fermentation process to contribute the taste and color of tea.
Bruising and rolling
● The tea leaves are bruised in order to break down the structure of leaf cells in order to damage
leaf edges to promote and quicken oxidation
● Cell damage → Enzymes are released on the whole tea leaves → enhances oxygen exposure
→ oxidoreductase (in favorable condition) → the reaction occurs faster.
Fermentation
● After rolling, the rolled tea usually goes through the oxidation process depending on the
desired qualities in the final tea.
1. Oolong tea: partial fermentation (20-60% oxidation)
2. Black tea: full fermentation (100%)
Fired/ Dried
● Stop the tea leaf oxidation at a desired level
● The moisture content level: less than 7.5% done to finish the tea for sale.
Grading and Packaging
● Remove physical impurities
● The dried leaves are size graded and separated by sorter machine
Then, the sorted teas are packaged for storage and trading.

Tea fermentation: Fermentation of tea is caused by enzymatic reactions of an oxidase (oxidizing

enzyme) derived from the tea-leaf enzymes themselves. The longer is the fermentation, the darker
is the color.

● During fermentation, the enzymes polyphenol oxidase (PPO) and peroxidase (PO) act on
catechins in the presence of oxygen.

● Catechins start to form larger molecules through condensation, and non-volatile

compounds such as theaflavins (TF) and thearubigins (TR) are formed, belonging to a
group called polyphenols → responsible for the color and taste

● TFs are responsible for the brightness, briskness, and quality of tea liquor

● TRs are responsible for the color pigments, taste, and body
Results
Table 1: Changes in total count of AAB, yeast, LAB and pH values in Kombucha beverages
during 10 days of fermentation at 20°C, 25°C and 30°C shows that:
- The fastest increase of yeasts was found in the beverages fermented for 10 days at 25°C
- The lowest yeast count was found in the beverages fermented at 30°C
- In the cases of fermentation in 25°C and 20C, the logarithmic phase of yeast growth
lasted until the seventh day of the fermentation and it remained stable (7 log CFU ml−1
on average) until the end of the process
=> It can be concluded that the fermentation in case of this product could last longer than the
assumed 10 days

Table 2: Changes in the organic acid content during 10 days of Kombucha tea fermentation at
20°C, 25°C and 30°C shows that:
- The main organic acid in the beverage was acetic acid but the acetic acid content was
relatively low
=> Because the part of glucose was probably consumed by the synthesis of cellulose during the
fermentation causing the insufficient amount of glucose for the acetic acid synthesis
- The highest amount of D-glucuronic content was found 0.063g on the 10th day at 25oC
- The lactic acid was not found of Kombucha tea fermentation

Table 3. Changes in sugar and ethanol concentrations during 10 days of Kombucha tea
fermentation at 20°C, 25°C and 30°C shows that:
- A decrease in the amount of sucrose
- The content of reducing sugars increased during fermentation (greatest amounts: glucose
and fructose on the 10th day at 25oC) due to hydrolysis of sucrose
- Content of ethanol increased with the fermentation time

Figure 1: . Sensory profiles of the Kombucha beverages after 10 days of fermentation process at
20°C, 25°C and 30°C à Kombucha fermented at 25°C was assessed as the highest

Conclusion
The results of the study revealed that optimal conditions for the fermentation of Kombucha
beverages were a temperature of 25°C and a period of 10 days.