UNIVERSITE DE NGAOUNDERE

ECOLE NATIONALE SUPERIEURE DES SCIENCES AGRO-

INDUSTRIELLES
Department of Process Engineering
Specialty: Génie Alimentaire et Bioprocédés

LITERATURE REVIEW PRESENTATION Master 2 G.P

The Potential of Yeast, Isolated from Raphia Palm (Raphia hookeri)

Topic: Wine, in the Alcoholic Fermentation of Guava (Psidium guajava)
Nectar
Presented by:
ASUH Rolantine
Matricule: 18S149EN
Bsc in technology in industrial food processing and biotechnology
Supervisor

Dr. ANGWANANDE Wilson Pr. NSO EMMANUEL

Academic Year 2019 - 2020

 PRESENTATION OUTLINE

INTRODUCTION

MATERIAL AND METHODS

RESULTS AND DISCUSSION

CONCLUSION AND PERSPECTIVES

2
 INTRODUCTION

Naturally present in the fermentation

of raw materials, such as grape
musts (wine fermentation) and
cereals (beer fermentation)
Photo1: Yeast strains

Mainly Saccharomyces cerevisiae

 Dominate the fermentation process,
 Due to its high fermentative capacity
 Ability to tolerate different stresses
(Warringer et al., 2011; Brice et al., 2014 and Legras et al., 2018

3
 INTRODUCTION
However, the current set of commercial S. cerevisiae strains and its
derived hybrids is insufficient to provide novel properties to beer and
wine, stressing

The need for new and improved strains for the industry
(Aquilani et al., 2015; Alperstein et al., 2020; Gibson et al., 2020)

The isolation of wild strains from different niches

(Duan et al., 2018; Pontes et al., 2020)

Wild yeast strains However, the importance of

represent alternative
native strains belonging to
genetic stocks for the
industry to overcome
domesticated lineages, such as
current challenges in palm wine, cannot be ignored.
4
 INTRODUCTION

Palm wine

Milky alcoholic beverage Contain diverse microbial

produced from palm tree population including bacteria,
yeasts or moulds

Different species of yeast Varies in palm wine depending on

exist in palm wine the source and variety of the palm
tree.

The organism has fermentative

and oxidative capabilities

(Nwachukwu et al., 2006; Amoa-awua et al., 2007; Ogunremi et al., 2017)

5
 INTRODUCTION
Table 2: Different yeast strains isolated from palm wine
Yeast Palm wine type References
Sachharomyces cerevisae Arenga palm Basir (1962); Faparusi and
Oil palm Basi (1972a);Simonart and
Raffia Laudelot (1951),Ayenor a
Schizosaccharomyces Oil palm Faparusi and Basir (1971);
pombe Raffia Faparusi and Basir (1972b);
Ahmad et al., (1954)
Saccharomyces vafer Oil palm Okafor (1972a)
Saccharomyces markii Oil palm Okafor (1972a)
Kloekera apiculata Oil palm Van pee and swings (1971);
Simonart
Saccharomyces florentius Oil palm Okafor (1972a)

Saccharomyces roseri Raphia Okafor (1972a)

6
 INTRODUCTION
Palm wine yeast

Some of these isolates show stability to high concentration of sucrose

and ethanol (Bechem et al.; 2007)

Palm wine yeast have been found to possess good sedimentation

properties for high product recovery (Ukwuru and Awah, 2013).

Efficiency of yeasts isolated from palm wine (Chilaka et al., 2010)

Applications of yeast

7
 INTRODUCTION
Alcoholic beverage produced from the fermentation of
grape juice or pulp (Combina et al.,2005)
Wine
One of the fermented functional foods having health
benefits (Singh et al.,1998)

Fermented alcoholic beverages made from the juice of ripe

fruit, fruit juices, or concentrate, without containing any
Fruit
grape
Wine
They may also have additional flavours taken from fruits,
flowers, and herb (Rivard, 2009).

Functional food is rising rapidly, and fruit wines, being considered a

functional drink, are attracting the attention of winemakers and
consumers.
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 INTRODUCTION (1/11)
Generalities on Guava Family: Myrtaceae
Genus: Psidium
Low
4-12cmevergreen
long; tree 6-25
rounded, Species: P.guajava
feet
ovoidhigh Wide spreading
or pear-shaped; sour
branches and awith
to sweet taste native
juicytoor
tropical
aromatic Americafragrance.
;130-
150genera,
Mesocarp 5000 0.1mm Species
stone
with
cells. few edible of
The colour fruits
the
(Rajan et with
flesh varies al, varieties
2012;
Nogueria
of guava et al, 2014)

9
 INTRODUCTION (1/11)

?
WHICH YEAST STRAIN
ISOLATED FROM RAPHIA
PALM WINE HAS
POTENTIAL APPLICATIONS
FOR WINE FERMENTATION

10
 INTRODUCTION (11/11)
General objective

To isolate and test yeast strains from raphia palm wine for their
potential in fruit wine production via fermentation

Specific objectives
01. Characterize the microbiological profile of raffia palm wine
harvested in Ngaoundere
H1. The Ngaoundere raphia palm wine harbours microorganisms of
industrial interest, especially yeasts
02. Carryout a test fermentation of guava nectar in the production of
guava wine; with the hypothesis that

H2. The isolated yeasts could ferment guava nectar and produce wine
with acceptable aroma profile. 11
MATERIALS AND METHODS

12
 INTRODUCTION
Palm
Guava wine
Physicochemical analysis of
raffia palm wine
Production of guava must Microbial load analysis of
raphia palm wine
Characterization of guava
Isolation and Purification of
must
of the isolated yeast
Prelimenary identification of
Pre-fermentation treatments yeast
Multiplication and Preparation
of innoculum
Fermentation of guava
must

Characterization of guava
Figure 1: Synoptic wine
diagram of work Sensory analysis 13
 MATERIAL AND METHODS
MATERIALS

Viniferm (J) NEVADA (C)

pink guava cultivar Oenofrqnce (A)

Reagents Equipements Tools

 Autoclave,  Stat graphics
 Petri dishes
 incubators,  ANOVA
 glasswares
 centrifuge,  Duncan’s multiple
 Reagents
 Refrigerator range test
 Culture
 Weighing  Excel
mediums
balance 14
 MATERIAL AND METHODS
METHODS
Physicochemical analysis of raphia palm wine

 Total soluble solids: Hand Refractometer

 Alcoholic content: Alcoholic meter

 pH: HI 223 pH meter

 Total acidity: Titration with NaOH with phenolphthalein used

as colour indicator (Rangana, 1986)

 Reducing sugar: By spectrophotometry

 Moisture content: By AFNOR 1998

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 MATERIAL AND METHODS
Methods
Determination of microbial load in raphia palm wine
Table 1: Methods used in the enumeration of microorganism
Microorganisms Culture Method of Incubator
medium enumeration temperature/ time
Aerobic mesophilic PCA Depth plate count 37°C, 48hrs
bacteria
Lactic acid bacteria MRS Surface plate count 37°C, 48hrs

Yeast SDA Surface plate count 25°C 72hrs

Mould SDA Surface plate count 25°C 120hrs

Coliform EMB Surface plate count 37°C, 48hrs

E: coli EMB Surface plate count 44°C, 48hrs

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 MATERIAL AND METHODS
Isolation and Purification of yeast from raphia palm wine
Distilled sterile
water
10ml of
raffia Centrifugation
palm 3000rmp/4mins sediment Resuspension 1
wine
Supernatant

SDA SDA

Centrifugation sediment Innoculation Pure

1 Restreaking
3000rmp/4mins 25°C, 2days cultures
25°C, 2days

Supernatant

Figure 2: Isolation of yeast (Okafor, 1972)

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 MATERIAL AND METHODS
METHODS
Preliminary identifications of pure isolates

• Macroscopic test (Frazier et al., 1995)

1. Morphological test
• Microscopic test (Frazier et al., 1995)

2. Physiological and biochemical test

• Temperature tolerance test (Maravilla, 1974)

• Determination of ethanol tolerance (Martini A, 1992)

• Flocculation test (Nwokeke , 2001)

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 MATERIAL AND METHODS
METHODS
Production and fermentation of Guava Must
water water

Guava Sorting and Washed

Crushing Dilution 1
fruits washing fruits
Powder Waste water+
Diluted sucrose bad fruits
Pasteurisation
pulp
Chaptalization (65OC ; Cooling
1 2
30mins) (allow standing)
CO2
Guava
wine Fermentation Rackling Filtration 3
2
Yeast

Pasteurisation
3 Guava wine
(65OC ; 30mins)

Figure 3: Production diagram of guava wine 19

 MATERIAL AND METHODS
METHODS
Characterization of Guava must
 Total soluble solids: Hand Refractometer

 Phenolic compounds

 pH: HI 223 pH meter

 Total acidity: Titration with NaOH with phenolphthalein used

as colour indicator (Rangana, 1986)

 Reducing sugar: By spectrophotometry

 Moisture content: By AFNOR 1998

20
 MATERIAL AND METHODS
METHODS
Sensory analysis of guava wine by davis 20 score card
Name

Appearance 2
Colour 2
Aroma and boquet 4

Acescent 2
Total acid 2
Sugar 1
Body 1

Flavour 2
Astringency 2
General quality 2
21
Total
RESULTS AND DISCUSSIONS

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 RESULTS AND DISCUSSIONS
Table 1: Physicochemical analysis of raphia palm wine
Time (hour)
Parameter 0 24 48
pH 6.37 4.25 3.5
TSS (°B) 13.34 11.57 10.11
Alcohol content (%)
7.9 15.8 18.4

Titratable acidity (%)

0.03 0.05 0.08

Reducing sugar (%)

3.09 2.73 0.65

Moisture content (%)

87.48 89.70 91.30

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 RESULTS AND DISCUSSION
Table 2: microbial profile of microorganism found in raphia palm
wine
Microorganism Microbial load/cmf
Aerobic mesophilic 6 X
bacteria
yeast 15 X
Lactic acid
Aerobic
Lactic mesophilic
bacteria bacteria 18 X
bacteria
Coliform Presence
E. coli Absent
Mould Present Yeast

cfu/ml = Colony-forming unit per millilitres

Mould
Coliform 24
 RESULTS AND DISCUSSION
Table 3: Morphological characteristics of the isolated yeast

Isolate code Morphology

SFCT Flat transparent(colourless) colony
SYCC Small round creamy colony
SWEC Smooth Small elongated white colony
YCMC Round creamy colony
LTYC Large, thick, round and creamy colony
SLWC Round and white colony with white at the centre
YWCC Round creamy colony
SCTC Small round transparent colony

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Table 4: Microscopic characteristic of the isolates
ISOLATES FORM Gram test Form Buddin Viability
g
SFCT Oval positive In clusters and in chains +++ viable
SWEC Oval positive In clusters ++ viable

SCTC Oval positive In chains, clusters and ++ viable

single
SLWC Oval positive In clusters +++ viable

YWCC Oval positive Single cells chain cells + viable

Cells in clusters
YCMC Oval positive Single cells chain cells ++ viable
Cells in clusters

SYCC Spherical positive Single cells ++ viable

LTYC Oval positive Single cells chain cells ++ viable

Cells in clusters

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 RESULTS AND DISCUSSION
Table 5: Representation of stress exclusive test results
Temperature
Isolates Ethanol tolerance Tolerance Flocculation
0% 5% 10% 15% 20% 25°C 37°C Tolerance

LTYC + ++ + - - ++ ++ -
YWCC ++ ++ ++ - - + + -
YCMC ++ + ++ - - ++ ++ +
SLWC +++ +++ +++ - - + - +
SWEC +++ ++ +++ - - ++ + +
SCTC +++ ++ +++ - - -
SFCT ++++ ++ ++++ - - +++ + +
Key: + = less ++= moderate; +++= intensive; ++++ = more intensive - =
negative ++++ +++
SYCC ++++ - - +++ + ++ 27
 RESULTS AND DISCUSSION

Table 6: Characterization of guava must

Parameters Content
Total soluble solids (°Brix) 10.20 ± 0.30
Moisture content (%) 86.03 ± 0.58
pH 4.30 ± 0.00
Titratable acidity(g citric acid/100g 0.75 ± 0.11
fresh mass)
Reducing sugar (mg/ml) 1.18 ± 0.01
Total phenolic compounds(mg/100g) 4 .38 ± 0.008

28
 RESULTS AND DISCUSSION
4.00
3.50
3.00
2.50
2.00
pH

1.50
1.00
0.50
0.00
C C C T A C J
M Y EC FC
YC
T LW
S S SW S

Wine samples from different yeast

Figure 4: Variation in pH among different wine samples

29
 RESULTS AND DISCUSSION
0.45
0.40
Total polyphenol (mg/100g)

0.35
0.30
18.00
0.25
16.00
0.20
0.15 14.00

Alcohol content (% v/v)

0.10 12.00

0.05 10.00

0.00 8.00
C C C T A C J
M TY EC FC
6.00
YC LW
S S SW S
4.00
Wine samples 2.00
0.00
T J
Figure 5: Total Polyphenol M
C
T Y C
LW
C EC FC A C
YC S S SW S
content of the different wine
Wine samples from different yeast
samples
Figure 6: Alcohol content of
the different wine samples 30
 RESULTS AND DISCUSSION
Appearance

General Acceptance 10 Colour

5
Astringency Aroma

Flavour Acescent

Body Acidity

YCMC STYC Sweetiness

SLWC SWEC SFCT

Figure 7: Level of appreciation of sensory attributes

31
 Conclusion and perspectives
The principal goal of this study was to isolate potential indigenous
starter yeast from locally harvested raphia palm wine in Ngaoundere.

Microbial analyses of freshly harvested raphia palm wine revealed the

presence of lactic acid bacteria and different yeast strains.

Eight yeast strains were isolated and purified, then screened based on
their ethanol, temperature and flocculation tolerances. Five of the
eight strains were selected for further testing as a ferment. (not q
cncusion: on whqt bases were the 5 selected)

32
 Conclusion and perspectives

The yeast strain SLWC produced more alcohol than all the other yeasts
and wines from strains SWEC and STYC were most appreciated
.

PERSPECTIVES

 Carryout complete identification of the isolated yeast strains as well

as the lactic acid bacteria strains. These microorganisms are of
technological importance.
 Optimise fermentation conditions with the different yeast strains so
as to have appropriate working conditions for better quality product.
 Multiply and lyophilise the best yeast strains for future availability
and proper conservation.
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THANK YOU FOR
YOUR KEEN
ATTENTION

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