Lactic Acid Bacteria Involved In Cocoa Beans

Fermentations From Ivory Coast: Species

Diversity And Citrate Lyase Production
Presented by:
Puri Nurwidayanti (10414001)│Ahdina Karima (10414015)

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Content

Introduction

Material and methods

Result

Discussion

Conclusion

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INTRODUCTION

3
Introduction

Involved yeast Lactic Acid first stage in the chocolate

Bacteria (LAB), Acetic Acid production process
Bacteria (AAB), and Bacillus,

Microbial activity generates Characterized by aprogressive

metabolite and creat conditions Fermentation of in the pH : breakdown citric
that inactivates the embryo of cocoa beans acid
bean

An array of essential biochemical Citric acid impart an initial pH of

reactions are triggered for the around 3.5 and favorableto fungal
complex flavor of chocolate growth and yeast

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 Introduction
 Citrate catabolism involved a particular enzyme, Citrate lyase

Citrate lyase: catalyzed

breakdown of citrate into
oxaloacetate and acetate

Subunit γ: catalyzed the Subunit α: replace the acyl Subunit β: split citryl-S-ACP
activation of citrate lyase by group with a citryl group to into oxaloacetate and
introducing an acetyl group form the citryl-S-ACP regenerated acyl-S-ACP
at the thioester residue of
the prosthetic group linked
to its acyl carrier protein
(ACP) to form acetyl-S-ACP

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I
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Introduction

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Introduction

The aim of this study: to evaluate by molecular typing,

the diversity of LAB isolated from fermenting cocoa and
to asses the impact of environmental conditions
encountered during cocoa fermentation on the activity
of citrate lyase in these strain.

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MATERIALS
&
METHODS

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Materials and methods

Fermentation, 16S rRNA Citrate lyase

PCR Screening of
isolation and gene gene
amplification LAB for citrate
growth restriction and detection
of 16S rRNA lyase
condition of sequence using the PCR
gene production
LAB analysis reaction

Effect of
Assay for citrate Quantification of
environmental
lyase activity and lactic, acetic and
conditions on
protein citric acids using
citrate lyase
concentration HPLC
production in LAB

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Fermentation, isolation and growth condition of LAB

 Cocoa pods were harvested on farms from six cocoa producing regions of Ivory Coast
 Spontaneous fermentations were conducted for six days, using banana leaves
 Numeration and isolation of LAB were carried out using the decimal dilution method (0.1%
pepton water (pH 7.2), diluted in tryptone salt solution and plated on to MRS)
 Characteristics of LAB: Gram negative, oxidase and catalase negative, and unable sporulation.

PCR amplification of 16S rRNA gene

 Bacteria grown for 24 h on agar plate were suspended in 100 uL of sterile distilled water
 PCR component: bacterial suspension, Taq DNA polymerase, 10X standard buffer, deoxynucleoside
triphosphate, primer, and water
 The presence and yield of specific PCR products were monitored using agarose 0.8% (w/v) gel
electrophoresis

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16S rRNA gene restriction and sequence analysis

 PCR product were directly digest with two restriction enzyme, HaeIII
(370C) and TaqI (650C) in separate reactions
 Component: PCR product, , incubation buffer, water, and restriction
enzyme
 Digestion product were run on a 2% agarose gel in Tris-Borate EDTA
buffer at 35 V overnight and gel were stained with ethidium bromide,
visualized by transillumination, and digitalized with a gel print system
 Strain were grouped according to the restriction profiles.
 Further identification: four representative strains were randomly
chosen for amplification of hyper-variable region of the 16S rRNA, and
then sequenced using the primer F27.
 The phylogenetic tree was constructed from the partial sequence of
RNA 16S gene alignment using maximum likelihood method.

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Screening of LAB for citrate lyase production
 Screening was did on Kempler and McKay medium and supplemented with potassium ferricyanide solution
(10% w/v) and solution containing both iron citrate (2.5% w/v) and sodium citrate (2.5% w/v).
 The breakdown and consumption of citrate by bacteria leads to the complexion of free iron ion with
ferricyanide to form a blue complex.

Citrate lyase gene detection using the PCR reaction

 Citrate lyase gene was analyzed by PCR using

degenerated oligonucleotide primer.
 These primers were designed from the multiple
alignment of citE genes of various LAB species.
 Reaction mixture: bacterial suspension, Taq DNA
polymerase, 10X standard buffer, dNTP, primer,
and water

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Effect of environmental conditions on citrate lyase production in LAB
 Strain were grown in a standard liquid medium (0.8% citric acid; 0.2% ammonium citrate; 1% casein
peptone; 0.4% yeast extract; 1% glucose; 0.2% K2HPO4; 0.02% MgSO4 and 0.004% MnSO4) pada pH 3.8.
 Asses the individual impact of various environmental condition (temperature 30-450C; pH variation 3.5-6;
and influenced of each compound (ethanol, citric acid, lactid acid, and acetic acid)).

Assay for citrate lyase activity and protein concentration

 Bacterial cells were collected by centrifugation, washed twice with distilled water, and then suspended in
buffer A and were broken by sonication. The crude cell extract was clarified by centrifugation and the
supernatant was used as final crude enzyme extract (40C).
 Crude enzyme was incubated for 1 min in the presence of anhydride with a couple spectrophotometry assay
with malate and oxaloacetate.
 The formation of NAD+ as a result of citrate lyase activity was measured in a spectrophotometer at 340 nm.
 The protein concentration was determinated using the method of Bradford.

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 Quantification of lactic, acetic acids using
HPLC
Peak areas were then compared with standard curves, realized for
each acid in order to measure the amount in each sample in mg/ml.

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 RESULT
&
DISCUSSION

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16S rRNA gene PCR-RFLP and identification
of isolates

■ 782 LAB isolates could be classified into eight distinct

groups based on Restriction Fragment Length
Polymorphism analysis
■ Group 1  corresponding to the Lactobacillus
plantarum species, accounted for 527 isolate
■ Group 2  corresponding to Leuconostoc
mesenteroides species, accounted for 190 isolates
■ Group 3  Lactobacillus curieae species, accounted for
45 isolates
■ Lactobacillus plantarum, Leuconostoc mesenteroides and
Lactobacillus curieae, represented more than 96% of
total isolates and constituted the major LAB encountered
in the six regions studied.

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Distribution of LAB species in the
different cocoa producing regions
 Lactobacillus plantarum and Leuconostoc
mesenteroides are dominant, representing more
than 90% of the total LAB and consistently
present in all the selected regions
 Leuconostoc mesenteroides and four of the minor
species (Lactobacillus casei, Weissella
paramesenteroides, Weissella cibaria,
Lactobacillus curieae) may constitute a specific
microbiota to Ivory Coast cocoa fermentation
 Diversity of bacterial composition could be the
main determining factor in the variability of cocoa
bean quality
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Screening of LAB capable to consume citrate

293
isolate

489
isolate

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 Screening of LAB capable to consume citrate

Non-citrate consumers First Group Second Group

metabolize citrate with acidification able to consume citrate with

possess the citE gene encoding the
of the medium but without gas acidification and gas production in
subunit β (citryl-S-ACP lyase)
production the medium

35 isolates were able to produce

metabolic pathway leading to
acetoin from glucose which is used
succinic acid
as a food flavoring

• CL activity in LAB is a key factor for adaptation to the fermenting cocoa ecosystem

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 Citrate consumption during bacterial
growth

 During the first 12 hours of growth, a sharp decrease in citrate

concentration in the medium  indicated a rapid consumption of
citric acid
 After bacterial growth, HPLC analysis showed the appearance of
acetic acid and lactic acid, in both medida
 Acetic acid and lactic acid  diffuse deep into beans or to
volatilize under heat and mixing of the fermenting mass  NOT
provoke a decrease of the cocoa pulp pH and activate the
formation of aroma precursors
 Leuconostoc mesenteroides strain T8AB6 and Lactobacillus casei
strain T10G5 are the best CL producers, they could serve as
valuable microbial starters for an effective fermentation of cocoa.

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Influence of temperature and pH on the
production of citrate lyase (CL) in LAB
 CL is optimally produced when bacteria are grown below pH 4
and the CL level decreases as soon as pH increases
 The best CL producers : Leuconostoc mesenteroides (T8AB6)
Lactobacillus plantarum (T10AG26) and Lactobacillus casei
(T10G5)
 Maximum enzyme production in these strains occurred at 35°C,
except for the Lactobacillus plantarum strains T10AG26 for
which a maximum occurred at 40°C
 5 strains under investigation retained significant activity within
the pH range of 3.5 to 6.0 and at temperatures between 30°C
and 45°C.

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 Influence of temperature and pH on the production of citrate lyase (CL)
in LAB
 CL induction is linked to the uptake of citrate through a specific transporter (citrate permease) which functions
best at a low pH.

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 Influence of acids and ethanol on CL
production in bacteria

 In the presence of 2.5% citric acid, increase in enzymatic activity

 Lactic acid and acetic acid inducing an increase in CL production
by the bacterial strains but with variable patterns
 Among the different acids, lactic acid appeared to be more
favorable for CL production since the highest level of enzyme
activity (maxima from 0.2 to 0.394 U/mg of protein
 Ethanol in a concentration range from 2 % to 8 %, induced a
gradual increase in CL activity in the 5 LAB strains tested  CL
production is tolerated at a relatively high concentration of
ethanol (up to 12%),

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 ■ Eight distinct species were identified from these six
location in Ivory Coast

CONCLUSION ■ Lactobacillus plantarum and Leuconostoc

mesenteroides were the dominant species recovered
from all the locations
■ For the first time, the production of citrate degrading
enzyme (CL) by LAB involved in cocoa
fermentation.
■ One of the main roles of LAB could be related to
the breakdown of citric acid at an early stage of the
cocoa fermentation process

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TERIMA KASIH

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