chapter:4 selection of yeast for probiotic attributes and...

Chapter 4

Himanshu Kumar, PhD Thesis, 2012

70

4

CHAPTER:4 Selection of Yeast for Probiotic Attributes and Stability of Potential Probionts in Kutajarista

Chapter 4


71

4.1 Introduction

Fermented foods constitutes very important share of total diet in developing countries

and yeasts are indispensible part of these fermentation processes. The role of yeast in

Asian indigenous fermented food has been extensively explored for its potential

utility in research and product development (Aidoo et al. 2006). Moreover,

Saccharomyces cerevisiae has been predominantly associated with majority of

fermented foods and beverages. In general, S. cerevisiae is linked to three groups of

indigenous fermented products: non-alcoholic starchy foods, alcoholic beverages and

fermented milk (Jespersen 2003). All these fermentative processes are spontaneous in

nature and yeast coexists with other species. Moreover, there is increased interest in

exploitation of yeast along with other microorganisms to ensure consistency and

quality of these formulations. Microorganisms isolated from these sources are

considered to be safe for human consumption because of its long history of usage.

Several attempts have been made to isolate microorganism from traditionally

fermented food/alcoholic beverages and characterise it for probiotic attributes

(Sourabh and Sharma 2011).

Among the yeast isolates, Saccharomyces cerevisiae and Saccharomyces cerevisiae

var. boulardii have been widely commercialised and its clinical efficacy have been

well documented (Czerucka et al. 2007). S. cerevisiae has achieved its generally

regarded as safe status (GRAS) from the Food and Drug Administration (FDA, USA)

and also has QPS (Qualified Presumption of safety) status from EFSA (The European

Food Safety Authority).

Saccharomyces boulardii was isolated by a french microbiologist Henri Boulard in

1920 from litchis in Indochina. Henri Boulard observed that in cholera affected area,

consumption of a special tea could prevent cholera. The agent responsible for

preventing the development of cholera was a strain of yeast, which was named as

Saccharomyces boulardii. The patent of Saccharomyces boulardii was bought by

Laboratories Biocodex in 1947.

Apart from S. boulardii, there are very few well characterised probiotic yeast strains.

Herbal fermented products like asavas and arishtas are predominated by yeast and

could be an attractive source of isolation for potential probiotic yeast. These herbal

Chapter 4


72

preparations are produced by natural fermentation induced by autochthonous yeast

originating from dhataki flowers (Sekar and Mariappan 2008). In view of this, yeast

species isolated from herbal formulation could be characterised for probiotic

attributes. The important factors, which influence probiotic properties of yeast,

include:

Taxonomic characterisation of yeast isolates

Molecular tools are very essential in identification of Saccharomyces species and

strain differentiation. Accurate identification of yeast strains is very critical in

downstream selection or industrial application. Saccharomyces boulardii was initially

considered as separate species of Saccharomyces. Later it was found that the genetic

map of Saccharomyces cerevisae and Saccharomyces boulardii are indistinguishable

on the basis of PCR, electrophoretic karyotyping or rRNA sequencing. Hence, the

taxonomic position of boulardii strain was debatable. S. boulardii has a unique

clustering on the basis of genetic, metabolic markers which were distinct from other

strains of S. cerevisae strains (Liong 2011).

Recently, genotypic tools like PCR amplification and restriction digestion of ITS

region (Internal transcribed spacer region) has been utilized for strain differentiation

and typing of autochthonous Saccharomyces spp. in naturally fermented wine

(Agnolucci et al. 2007). Other PCR based technique like randomly amplified

polymorphic DNA (RAPD), PCR fingerprinting and enzymatic restriction of

amplified DNA could not discriminate at strain level (Hennequin et al. 2001). In lieu

of this, microsatellites have emerged as an important tool for typing closely related

yeast species like Saccharomyces and Candida species (Hennequin et al. 2001;

Malgoire et al. 2005; Sampaio et al. 2005). These are simple tandem repeats (STRs)

consist of stretches of 1 to 6 nucleotide motifs dispersed throughout the genome,

which also exhibit high level of polymorphism. Allelic length differences occur as

there is difference in number of repeated units and which can be easily assayed by

PCR amplification and sequencing.

Chapter 4


73

Tolerance to physiological conditions

Yeast species, irrespective of its origin are known to tolerate various stress conditions

such as drying, heating and freezing. Stress tolerance could be an added attribute for

probiotic characterisation. For instance, yeast strains from industrial origin have

inherent property to tolerate heat stress and osmotic pressure (osmotolerance) while

processing. Due to these properties, it is now apparent that yeast possesses high

potential to tolerate simulated conditions of the digestive tract. Saccharomyces,

Debaryomyces, and Kluyveromyces species are reported to have high tolerance to bile

salt as well (Kumura et al. 2004; van der Aa Kuhle et al. 2005; Chen et al. 2010;

Kourelis et al. 2010b). The most widely studied S. boulardii has optimum growth

temperature of 37oC and it is resistant to low acidic stresses and bile, which are

important characteristics of microorganism for probiotic application (Edwards-Ingram

et al. 2007). Yeasts have been isolated from wide range of sources such as traditional

fermented foods, alcoholic beverages, cheese, sour dough and fruit juices are found to

possess excellent probiotic properties (Pennacchia et al. 2008; Roopashri and

Varadaraj 2009; Sourabh and Sharma 2011). Additionally, survival of yeast strains in

simulated gastric condition can provide accurate estimation of human gut subsistence

(Psomas et al. 2001). Probiotic S. cerevisiae and Kluyveromyces lactis can tolerate

0.15% bile salts at low pH 3.0 (Kourelis et al. 2010b).

Strains which can tolerate these in vitro conditions should be able to withstand in vivo

physiological conditions. In vivo colonisation studies with S. boulardii and S.

cerevisiae proved their safety, adherence and persistence at high population in

digestive tract of the host. After regular feeding of S. cerevisiae for 3 days, the yeast

population reached a steady state in gut but it was eliminated within 2-5 days if the

dose is discontinued (Blehaut et al. 1989).

Property of adherence to intestinal cells

Adherence to intestinal cells is one of the important probiotic attribute, as it ensures

the maintenance of potential probiont at intestinal tract for longer duration. Yeast

strains of Saccharomyces, Debaryomyces, Candida, Isaatchenkia and Kluyveromyces

species possess very high adhesive capacity to different cells (Kumura et al. 2004;

Chapter 4


74

Kourelis et al. 2010b). As well established in prokaryotic probiotic microorganisms,

adhesive property in eukaryotic probionts is also strain specific. S. cerevisae strains

from blue veined cheese possess high adhesive capacity (van der Aa Kuhle et al.

2005). However, some strains of S. cerevisiae have very less adherence to some of the

cell lines (Tasteyre et al. 2002; Kumura et al. 2004). In an in vitro study, K. lactis

showed better adherence to Caco2 cell line than other yeast strains of Kluveromyces

or Saccharomyces.

Aggregation and co-aggregation property of probionts

Aggregation is one of the desirable attribute of potential probiotic microbes, which

helps them to achieve adequate mass to exert its beneficial effect on host (Collado et

al. 2008). Auto-aggregation also helps in adhesion of probiotic microbes to intestinal

epithelial cells, which in turn prevents pathogen colonisation. Moreover, the ability of

bacteria to co-aggregate with other bacteria such as pathogen helps in removing the

pathogen from intestinal environment. Castagliuolo et al. 2005, reported the

aggregation (auto-aggregation and co-aggregation) of Lactobacillus crispatus M247

which exhibited preventive effect in mouse model of colitis. According to one report,

Lactobacillus species might act as barrier which in turn prevents colonization with

pathogenic bacteria (Schachtsiek et al. 2004). Auto-aggregating lactobacilli have

reported to form bio film on vaginal epithelia which prevents the manifestation of

infection (Juarez Tomas et al. 2005).

The symbiotic association of yeast and Lactobacillus species are known to occur in

traditional foods such as sake, sause, wine, kefir etc. S. cerevisiae and L. plantarum

isolated from sake fermentation, showed co-aggregation property, which was due to

electrostatic interaction between the positively charged Lactobacilli sp. and negatively

charged S. cerevisiae (Furukawa et al. 2011). Another report by (Katakura et al.

2010) revealed that co-aggregation between Lactococcus lactis and S. cerevisiae is

mediated by cell surface DnaK protein and mannan respectively. Lactobacillus

paracasei H9 and Saccharomyces cerevisiae, isolated from kefir showed significant

co-aggregation property which in turn helped in enhancing the tolerance of

Lactobacillus towards gastric stress condition (Xie et al. 2012). Apart from co-

aggregating property of Lactobacillus sp. and yeast, these species also form mixed

species biofilm. Yeast and Lactobacillus sp. are known for their potential application

Chapter 4


75

in fermentation of food and have proven safe application; therefore co-culture could

help in developing functional food containing probiotic microorganisms.

Stability of potential probiotic microbes in a herbal formulation

Probiotics are found in many forms and formulations; it includes dairy, vegetable,

fruit, cereal and meat based foods or products. Majority of food includes dairy based

products such as milk (Reilly and Gilliland 1999), ice cream (Hekmat and McMahon

1992), frozen fermented dairy desserts (Laroia 1991), yogurt (Shah et al. 1995) and

cheese (Dinakar and Mistry 1994). Number of different factors need to be considered

before incorporation of microorganisms in food matrix. These factors include type of

culture, dose, adequate physiological condition, stability, survival, processing, storage

and other parameters like sensory properties. For incorporation of Bifidobacteria in

fruit juices, the strains were selected on its capacity to resist acidic pH. Bifidobacteria

animalis was found to be more acid resistant than the other strains of Bifidobacterium

species (Saarela et al. 2011). It has also been reported that viability of

Bifidobacterium and Lactobacillus acidophilus dropped during incorporation and

storage in dairy products as these probiotic microorganism were found to be sensitive

to heat treatment and exposure to oxygen or acidic environments (Dave and Shah

1997; Kailasapathy and Rybka 1997). Encapsulation or immobilization can help in the

survival of probiotic bacteria in these food matrix (Adhikari et al. 2000). For example,

some of the authors have tried encapsulation or other protective treatment to protect

Bifidobacteria against acids and low pH in fruit juices (Ding and Shah 2008).

Maintaining the stability of any commercial probiotic microorganism in food from

manufacturing process till consumption is quite demanding process and needs lot of

improvement in method development. Incorporation of autochthonous probiotic

microorganism in herbal formulation will have multiple advantages and additionally

quality of the finished product can also be regulated. Moreover, autochthonous strains

have higher viability in comparison to other industrially or commercially available

strains. Thus, present study was envisaged to isolate yeast species from herbal

formulations and characterise them for probiotic attributes. The stability of these yeast

isolates along with bacterial probiotics were characterised in Kutajarista.

Chapter 4


76

4.2 Materials and Methods

4.2.1 Sampling and physicochemical analysis

Samples were collected and physicochemical analysis was conducted as described

previously 2.3.1. Type strain of S. boulardii was isolated from commercially available

Ultra-Levure batch No. 111 from Laboratories Biocodex, Montrouge, France and

Econorm (B.No. D80061) Dr Reddy‘s Laboratory, India. For stability assessment of

probiotic microorganisms in Kutajarista, product was taken from company. The

details of all the samples have been described in Table 4.1.

Table 4.1 Physicochemical parameters of Prasham Asawarishta, Panchakolasava and

Kutajarista

S.No.

Samples of

Fermentative

process

Specific

gravity

Sugar

Content

(%)

pH Alcohol

(%) Acidity

1 Panchakolasava

1.07 24 3.84 7 0.453

2

Kutajarista

(Marketed

product)

1.07 27 3.5-4.0 10 0.368

Composition of the formulation was as follows:

a) Panchakolasava: Each 10 ml Asava contains approx. 0.1 g of Pimpali (Piper

longum), Chavak (Piper chaba), Chitrak (Plumbago zeylancia), Shunthi (Zingiber

officinale); Manuka (Vitis vinifera) 0.4 gm, Dhayati (Woodfordia fruticosa) 0.1gm,

Madhur dravya Q.S. Composition of Kutajarista and Varunadi kwath and their

physicochemical parameters have already been described in previous section 2.3.1.

4.2.2 Isolation of Saccharomyces strains

Samples were taken at different time points during fermentation and the dilutions

were plated on two different media: SDA (Sabourad‘s dextrose agar) and YPD (yeast

peptone dextrose agar). Plates were incubated at 28oC for two days. After incubation

morphologically distinct colonies were recorded. For isolation, single colonies were

re-streaked and purified on its corresponding media. S. boulardii was isolated from a

Chapter 4


77

commercial probiotic product. The purified isolates were stored at -80oC in YPD

broth containing 20% (v/v) glycerol.

4.2.3 Molecular characterisation of yeast strains

i) For DNA isolation, overnight grown culture was washed with PBS and

suspended in lysis buffer [composition: 2% (v/v) triton X 100, 1% (v/v)

SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 2), 1 mM EDTA]. Alternate

temperature treatment of -80oC and 80

oC for 5 minutes each was given.

ProteinaseK treatment (20 mg ml-1

) along with 10% SDS treatment was

given for overnight at 55oC. After incubation, Lysis buffer was added and

content was incubated at 70oC for 30 min. Content was mixed with 500 µl

of chloroform/isoamyl alcohol (24:1) and centrifuged the tubes at 12,000

rpm for 10 min. After centrifugation supernatant was transferred to new

tubes with isopropanol and stored at -20oC for 30 min. Tubes were

centrifuged at 12000 rpm for 15 min at 4oC. Pellet was washed with 70%

ethanol and resuspended in 60 µl of TE buffer. The DNA was quantified

using Nanodrop.

ii) Yeast were identified using internal transcribed spacer ITS1 (5‘-

TCCGTAGGTGAACCTGCGG-3‘) and ITS4 (5‘-

TCCTCCGCTTATTGATATGC-3‘) primer set reported previously

(Esteve-Zarzoso et al. 1999). The amplification reaction for ITS region

were performed using following conditions: each 50 µl reaction mixture

contains 20-30 ng template DNA; 1xPCR buffer (Banglore genei), 0.2 mM

each of dNTP‘s, 0.5 µM of each primer and 1 U of Taq DNA polymerase

(Banglore genei, India). Amplification was performed in a Eppendorf

thermocycler with the following programme: initial denaturation at 95oC

for 5 min; 1 min 94oC for denaturing, annealing at 55

oC for 1 min,

extension for 1 min at 72oC, repeated for 35 cycles; and a final extension

for 1 min at 72oC. All the positive PCR products were sequenced as

described previously. The corresponding sequences were finally compared

with sequences present in public database (NCBI) using BLAST search

programme to identify and determine the closest known relatives.

Chapter 4


78

4.2.4 Microsatellite typing of yeast strains

For microsatellite typing, seven microsatellites were selected from previous report

(Hennequin et al. 2001). The details of the conditions and modifications are as

follows:

i) Amplification was performed using previously described reaction

conditions (Hennequin et al. 2001). The positive PCR products were

purified using Qiagen PCR purification kit (Qiagen). For sequencing, the

PCR amplified products were cloned into pGEMT easy vector system

(Promega), as per the manufacturer‘s protocol. The sequencing protocol

has been described in previous chapter. The sequences obtained were

manually checked for any mis-incorporation and edited using Applied

Biosystem Sequence scanner software v 1.0 and chromasPro (version

1.34). The number of repeats were determined manually and compared to

standard strain.

ii) Native PAGE: As an alternative method to sequencing, all the PCR

products were also loaded into Native PAGE (non denaturing

polyacrylamide gel electrophoresis). Since, it is simple, low cost, high

throughput system for detection of microsatellite markers (Wang et al.

2003). The final concentration of resolving gel was 18% (w/v) after

diluting 30% acrylamide with 0.5 M Tris-HCl (pH 8.9), polymerized with

0.1% v/v TEMED and 0.1% APS (w/v). Concentration of stacking gel was

4% (w/v). PCR products were loaded along with 10 bp ladder (Invitrogen)

and gels were run with running buffer (0.5X TBE). After complete run,

gels were stained with 10 mg mL-1

of ethidium bromide. Gels are then

visualized and documented using UV gel documentation system (Gene

Genius Bioimage System).

4.2.5 Screening of isolates for physiological tolerance

The methodologies for tolerance to different conditions have been adopted from

previously published reports with little modifications (Graff et al. 2008; Pennacchia et

al. 2008; Rajkowska and Kunicka-Styczynska 2010). All the yeast strains were

inoculated in SDB broth and incubated at 37oC for 24 h in order to get the optimum

Chapter 4


79

growth. To simulate acidic conditions and assess tolerance at different pH, pH of SDB

broth was adjusted 2, 4 and 6. For bile tolerance test, SDB broth was supplemented

with 0.3% bile salts (Oxgall, Himedia, India). Gastric juice was prepared as described

in previous chapter. For all tolerance tests, culture of yeast cells (Optical density ≈ 1)

were harvested by centrifugation at 2000 g for 10 min, washed twice with PBS. It was

added to SDB broth with above mentioned modifications for acid, bile and gastric

juice. Then the number of viable S. cerevisiae strains was determined by serial

dilution and plate count method.

4.2.6 Analysis of adherence of yeast isolates to HT-29 cell line by Flow

cytometry

The protocol has been adopted from previously published report with little

modifications (Bouzaine et al. 2005). Overnight grown culture of S. cerevisiae were

harvested by centrifugation at 3000 rpm for 10 min and washed twice with PBS. Cells

were resuspended in PBS to give final concentration of 109 CFU ml

-1. S. cerevisiae

was stained by using Vybrant cFDA-SE tracer kit (Molecular Probes, USA) as per the

manufacturer‘s protocol and methodology was adopted from previous report (Bunthof

and Abee 2002). cFDA-SE is a colourless and non-fluorescent but when incorporated

in cells it reacts with intracellular amines to form fluorescent conjugates which are

intracellularly retained. S. cerevisiae were labelled with 40 µmol l-1

of cFDA-SE at

37oC for 30 min. Labelled cells were harvested by centrifugation washed twice with

PBS and resuspended in 500 µl of DMEM. To observe adherence, these labelled cells

were added to the wells of 6 well culture plates with confluent HT-29 cells.

Qualitative determination of adherence: Monolayer of HT-29 cells was washed twice

with 1 ml antibiotic free DMEM and suspended in 1 ml of antibiotic free DMEM. The

adherence of each strain was observed by inoculating 1 ml of labelled bacterial

suspension to the wells (Bianchi et al. 2004). After 1 h incubation at 37oC and 5%

CO2, the medium containing non adherent bacteria were harvested from the wells and

monolayer were washed twice with 1 ml PBS. Cover slips were placed on

microscopic slides and were analysed using confocal microscope with setting of 12-V,

50W halogen lamp for transmitted light illumination, a fluorescein isothiocyanate

filter set (excitation wavelength, 450 to 490 nm; emission wavelength, > 520 nm) and

a X100 1.3-numerical aperture. Fluorescence microscopy revealed adherence pattern

Chapter 4


80

of yeast strains on HT-29 cell line and specificity of labelling (Bunthof and Abee

2002).

Flow cytometry: - Flow cytometry analysis was performed in FACS Caliber flow

cytometer and data analysis was done as per previously described methodology

(Bunthof and Abee 2002). Briefly, forward scatter (FSC), side scatter (SSC) and FL1

fluorescence signal was measured. A band pass filter of 530 nm (515 to 545 nm) was

used to collect the green fluorescence (FL1). All signals were collected at logarithmic

amplification. A combination of FSC and SSC was used to distinguish yeast from the

background signal. Data were analysed using CELLQuest program (Becton

Dickinson) (Bunthof et al. 2001).

4.2.7 In vivo feeding trial for assessment of safe use in mouse model

Yeast feeding trial was performed similarly as described previously for Lactobacillus

sp. (Section 3.2.16). Briefly, the concentration of yeast strains was maintained approx.

to 108 CFU/ml. The yeast strains were administered orally by a gavage.

Taqman Low Density Array for transcript variation in colon

The TaqMan Low-Density Array (TLDA; Applied Biosystems) is a micro fluidic card

designed to detect real time amplification of specified targets. It has been widely used

for large expression profile. These arrays have been preloaded with specific assays or

it can be custom made. In this study, TaqMan Mouse Immune Array (Cat. No.

4367786) was used, which contained 90 target genes and 6 control genes to assess

immune response of BALB/c mice in response to different probiotic treatments. The

data was obtained using Applied Biosystems 7900HT fast real time PCR system. The

reaction condition and procedure briefly includes the following steps. The cDNA was

prepared as per earlier described protocol (Section: 3.2.15). Good quality of cDNA

was ensured before proceeding for this assay. Sample PCR mix was prepared by

adding 0.5 l cDNA (50 ng of 100 ng l-1

) + 49.5l RNase free water (Sigma

Chemicals), b) 50l TaqMan Universal PCR Master mix (2x) (Appliedbiosystems,

Carlsbad, Ca) in a 1.5 ml tube. These tubes were gently vortexed to thoroughly mix

the contents, briefly centrifuged to spin down the contents and eliminate air bubbles.

The micropipette tip loaded 100 µl of sample mix prepared above (held in angled

position) is placed in the fill port in a micro fluidic card and the sample mix is

dispensed slowly to avoid air bubbles. The cards are then centrifuged in

Chapter 4


81

Sorvall/Heraeus Multifuge 3 SR + (Thermo Fisher, Germany) at 1000 rpm for one

minute and then repeated twice to ensure complete loading. The array plates are then

sealed using TaqMan Array Micro Fluidic Card Sealer, then fill strip is trimmed using

scissors and the array card is loaded onto 7900 system. The data obtained was

analysed using Gene Expression (RQ Manager) in an Oracle database.

4.2.8 Aggregation and co-aggregation property of probiotic micro-organism

The extent of aggregation abilities of both lactobacilli and yeast isolates were tested

by spectrophotometric method (Collado et al. 2007). Briefly, lactobacilli and yeast

isolates were grown in MRS and SDB respectively at 37oC for 12-14 h under aerobic

conditions. The cell were harvested by centrifugation (3500 rpm for 5 min), washed

thrice with phosphate-buffered saline (PBS, pH 7.2) and resuspended in the PBS to

get 108

CFU/ml of isolates. The adjusted initial Absorbance ‗AInitial‘ was measured

(equipment name) at 600 nm and mixture was again vortexed and incubated at 37oC

without agitation for 2 h and 4 h. The change in absorbance ‗ATime(2/4)‘ was measured

and the percentage aggregation was determined as [1−(ATime/AInitial)×100].

Co-aggregation between yeasts and lactobacilli isolates was also tested by

spectrophotometric method (Collado et al. 2007; Darilmaz et al. 2012; Xie et al.

2012). Bacterial suspensions and yeasts suspensions were prepared as described

above, equal volumes of yeasts and lactobacilli cells (1:1, v/v) were mixed and the

initial Absorbance ‗AInitial‘ was measured. The mixture was again vortexed and

incubated at 37oC without agitation for 2 h and 4 h. The change in absorbance of the

above mixture was measured at 600 nm. Percentage co-aggregation was determined

as [(Alac + Ayeast)/2−(Amix)/ (Alac + Ayeast)/2]×100, where Alac and Ayeast represent the

A600 of lactobacilli and yeast strains after incubation for 2/4 h, respectively.

4.2.9 Stability of probiotic strains in Kutajarista

The stability of probiotic lactobacilli and yeasts in Kutajarista was evaluated by

enumerating the viable organisms over a period of 16 days. Yeasts and lactobacilli

isolates were grown in MRS and SDB respectively at 37oC for 12-14 h. After growth

cells were harvested and washed twice with PBS. Suspension of cells was added to

Kutajarista to get 107 of yeast cells and 10

8 of Lactobacillus per ml of Kutajarista.

Sufficient number of aliquots of each isolates were prepared for 16 days studies and

incubated at room temperature. Viability of the isolates were checked on alterative

Chapter 4


82

days till the completion of studies using MRS for lactobacilli and SDB for yeasts

enumeration, and incubation conditions was 37oC for 48 h for both yeasts and

Lactobacillus isolates.

The effect of co-aggregation on the survivability of both probiotic yeast and

Lactobacillus isolates were evaluated by adding both yeast and lactobacilli together to

the Kutajarista. Their stability was checked by enumerating the viable organisms over

a period of 16 days. Bacterial and yeast samples were prepared as described above

and added together to the Kutajarista to get a final cell density of 107 yeast cells and

108 Lactobacillus sp. ml

-1 Kutajarista. Sufficient number of aliquots of each isolates

were prepared and incubated at room temperature. The viability of isolates were

checked on alterative days till the completion of studies using MRS supplemented

with Amphotericin B (100 µg ml-1

) for enumeration of lactobacilli and SDB

supplemented with Ampicillin (50 µg ml-1

) for yeasts and incubation conditions of

37oC for 48 h for both yeasts and Lactobacillus isolates.

4.3 Results

4.3.1 Molecular characterisation of yeast isolates

Yeast species were isolated from samples during the fermentation of Panchakolasava

(PA), Varunadi Kwatha (BQ) and Kutajarista (VQ). Four yeast species were purified

and selected from these formulations. All of the isolates showed its homology with S.

cerevisiae with 99% of the similarity (Table 4.2a). Moreover, the sequence similarity

among the strains was more than 98% (Table 4.2b).

Table 4.2a) BLAST hit results of yeast species b) Similarity Matrix based on ITS

sequencing and alignment

a)

S.No. Isolate

Name

BLAST Hit Accession

number

Percentage

Similarity (%)

1 PA Saccharomyces cerevisiae JN093147.1 99

2 BQ Saccharomyces cerevisiae JN093147.1 99

3 VQ Saccharomyces cerevisiae JN093147.1 99

4 SAB Saccharomyces cerevisiae HQ026726.1 100

Chapter 4


83

b)

PA_ITS BQ_ITS VQ_ITS SAB_ITS

PA_ITS 100 98.8281 100 99.6063

BQ_ITS 98.8281 100 98.8281 98.4252

VQ_ITS 100 98.8281 100 99.6063

SAB_ITS 99.6063 98.4252 99.6063 100

To further differentiate S. cerevisiae at strain level, microsatellite typing method was

employed. The microsatellite locus for strain typing was adopted from previously

published scheme (Hennequin et al. 2001). For preliminary strain differentiation, the

size of microsatellite was determined on PAGE. Fig 4.1a to Fig 4.1c shows the

difference in sizes which clearly indicates the change in number of polymorphic sites

among these strains. All the microsatellite amplicons were cloned into pGEMT easy

vector and then sequenced. The number of repeats were counted at polymorphic sites

and compared to S. cerevisiase S288c. All the different strains found are designated in

Table 4.3. No two strains isolated from two different fermentative processes were

genotypically identical i.e. all the strain gave a pattern distinguishing from other

strains. However, S. boulardii SAB and DCB, taken from two different sources are

indistinguishable at strain level.

Figure 4.1a PAGE for microsatellite differentiation A= 057 microsatellite for strains

(Left to right) VQ, DCB, SAB, PA, BQ; L= 10 bp ladder, B=072 microsatellite VQ,

DCB, SAB, PA, BQ

Chapter 4


84

Figure 4.1b PAGE for microsatellite differentiation A= 289 microsatellite for strains

(Left to right) VQ, SAB, DCB, PA, BQ; L= 10 bp ladder, B=172 microsatellite VQ,

SAB, DCB, PA, BQ

Figure 4.1c PAGE for microsatellite differentiation A= YLR microsatellite for strains

(Left to right) SAB, DCB, VQ, PA, BQ; L= 10 bp ladder, B=139 microsatellite SAB,

DCB, VQ, PA, BQ

Table 4.3 Microsatellite repeat of different allelic locus tested for S. cerevisiae strains

used in this study, ND-Not determined

MICROSAT 172 O72 177 289 O57 YLR 139

S. cerevisiae

(s288c) 10 13 10 11 14 31 8

BQ 8 9 4 7 ND 12 7

DCB 9 10 ND 8 7 5 ND

PA 8 9 8 10 10 ND 7

SAB 9 10 11 8 7 5 ND

VQ 4 9 8 10 10 ND 7

Chapter 4


85

4.3.2 Probiotic characterisation of yeast isolates

pH tolerance

Acid tolerance test for all the strains of S. cerevisiae also showed pH tolerance ability

in comparison to S. boulardii. At pH 2, there was a slight decrease in cell viability of

around 1 CFU ml-1

in all the strains including the S. boulardii (Fig 4.2a). At pH 4,

there was no loss in cell viability, as all the strains maintained constant CFU/ml for

three hours (Fig 4.2b). Moreover, pH 6 was found to be favourable for all the strains,

as there was 0.5 CFU/ml increase in 3 h for all the tested strains (Fig 4.2c).

Figure 4.2 a) Viability of S. cerevisiae (BQ, PA, VQ) strains including two standard

strains of S. boulardii (SAB, DCB) at pH 2

Figure 4.2 b) Viability of S. cerevisiae (BQ, PA, VQ) strains including two standard


Chapter 4


86

Figure 4.2 c) Viability of S. cerevisiae (BQ, PA, VQ) strains including two standard


Figure 4.2 d) Viability of S. cerevisiae (BQ, PA, VQ) strains including two standard

strains of S. boulardii (SAB, DCB) with 0.3% bile salts

Bile tolerance

All the selected strains of S. cerevisiae showed comparative tolerance to 0.3% bile

concentration (Fig 4.2d). Interestingly there is slight increase of 0.1 CFU/ml in all the

strains of S. cerevisiae isolated from herbal formulations. However, commercial strain

of S. cerevisiae did not show any loss or gain in cell viability.

Chapter 4


87

Gastric juice tolerance

All the strains of yeast showed similar pattern of tolerance of gastric juice as observed

earlier during acidic and bile tolerance tests. One of the yeast strain i.e. PAMRS

showed marginal increase in tolerance by increase of 0.2 CFU ml-1

(Fig 4.3).

Moreover, none of the strain showed any loss of cell viability. In concordance to

earlier report, S. cerevisiae strains under investigation also showed no loss in cell

viability i.e. all strains showed nearly 100% cell viability, during the incubation of 60

min (Fietto et al. 2004).

Figure 4.3 Viability of S. cerevisiae (BQ, PA, VQ) strains including two standard

strains of S. boulardii (SAB, DCB) for tolerance to simulated gastric juice for 60 min

Adhesion of yeast to HT-29 cell line

Selected yeast strain (VQ) and a standard strain were labelled with CFSE as per the

previously described methodology. Confocal microscopy revealed that strain VQ

showed higher adherence in comparison to other tested strains (Fig 4.4). To further

confirm its adhesive capacity, Flow cytometry was used to compare the adherent

CFSE labelled yeast on HT-29 cell line. FACS analysis revealed that 50% VQ yeast

were adherent to cell line while 59% of adherence was observed in case of

commercial yeast strain, SAB (Fig 4.5).

Chapter 4


88

Figure 4.4:- Cell adhesion studies of different strains of S. cerevisiae A) PA B) BQL

C) SAB D) VQ on HT-29 cell line, (Left to right:- Phase contrast, fluorescent,

merged)

Chapter 4


89

Figure 4.5 Flow cytometric analysis of S. cerevisiae VQ in comparison to S.

cerevisiae DCB on HT-29 cell line A) positive control of only labelled cells B) VQ C)

SAB

4.3.3 Taqman Low Density Array for transcript variation in colon

TLDA (Taq low density array) has been used to assess immunomodulatory property

of probiotic isolates in different systems like use of in vitro and in vivo models.

Feeding trial with strains of our interest showed differential expression of cytokines

and chemokines in colon. Few selected chemokines and cytokines are highlighted in

Fig 4.6. In comparison to control, mice fed with Lactobacillus plantarum VR1 and

Chapter 4


90

Saccharomyces cerevisiae VQ showed similar trend in the expression of ccl19, il2,

il12, il2, il3, il4 and il9.

Figure 4.6 Transcript variation for few selected cytokines with different treatment

group of S.cerevisiae DCB, S. cerevisiae VQ and L. plantarum VR1

4.3.4 Aggregation property

4.3.4.1 Auto-aggregation

The probiotic yeast showed good aggregation property in comparison to bacterial

strain. Yeast isolated from herbal fermentation showed better autoaggregation

capacity in comparison to the commercial probiotic yeast of Saccharomyces

boulardii. Among bacterial isolates commercial bacterial strain Lactobacillus

rhamnosus GG showed better aggregation (17%) than other bacterial strains (11-12%)

(Table 4.4). The capacity of aggregation increased after 4 h of incubation for all the

strains of bacteria and yeast. Moreover, yeast showed marked increase of 15-20%

during 4 h of incubation. While bacterial isolates showed 5-7% increase in percentage

aggregation.

4.3.4.2 Co-aggregation

In order to estimate the aggregation of Lactobacillus and yeast strains, co-aggregation

capacity of selected strains were estimated. L. plantarum VR1 was used to estimate

the co-aggregation capacity with selected yeast strains i.e. S. cerevisiae DCB and S.

Chapter 4


91

cerevisiae SAB. L. plantarum VR1 was found to be 9% aggregative to S. cerevisiae

DCB, 13% aggregative to S. cerevisiae SAB strain.

Table 4.4:- Aggregation capacity of different strains taken at two time points of

incubation at 2 h and 4 h, LGG- Lactobacillus rhamnosus GG, VR1 Lactobacillus

plantarum, VSA Lactobacillus plantarum, VM2, DCB- S.boulardii, VQ- S. cerevisiae

Probiotic

strains

% Auto aggregation after 2h % Auto aggregation after 4h

LGG 17.33±1.22 24.66±1.92

VR1 11.45±1.82 19.30±1.58

VSA 12.63±2.04 19.28 ±0.54

VM2 11.79±2.35 18.35±0.86

DCB 59.9±3.46 79.26±3.95

VQ 72.84±2.07 87.45±2.82

4.3.5 Stability of probiotic strains in Kutajarista

The bacterial and yeast strains isolated from herbal fermentation showed higher

stability in comparison to commercial strains. L. plantarum VR1 strain isolated from

Kutajarista showed stability for longer duration (14 days) in comparison to

commercial strain of L. rhamnosus GG. In the case of L. rhamnosus GG, no growth

was observed after 8 days of incubation. Yeast strains also showed similar trend as

bacterial isolates. Commercial strain of S. boulardii showed no growth after 14 days

of incubation while S. cerevisiae VQ showed only a slight change (2 log fold) even

after 14 days of incubation.

To evaluate the effect of co-culture on stability of L. plantarum VR1 and S. cerevisiae

VQ in Kutajarista. Samples were taken at same time periods as taken for individual

treatments. As observed in individual treatment group, yeast maintained its high

viability of 5 CFU ml-1

log units while there was slight improvement in viability of L.

plantarum VR1 till 16 days of stability test at room temperature.

Chapter 4


92

a)

b)

c)

Figure 4.7 Stability of a) L.plantarum VR1 and L. rhamnosus LGG b) S. cerevisiae

VQ and S. cerevisiae var boulardii DCB c) Effect of co-culture (S. cerevisiae VQ &

Chapter 4


93

L. planatarum VR1) in Kutajarista product, Viability of strains were checked at

alternative days till 14 days at room temperature

4.4 Discussion

The probiotic properties are strain specific, therefore reliable identification of strain is

very important. Molecular tools provide rapid and reliable identification for microbial

species. ITS sequencing has extensively been used for fungal identification. However,

for strain level identification alternative techniques like Pulse field gel Electrophoresis

(PFGE), randomly amplified polymorphic (RAPD), Mitochondrial DNA restriction

analysis and microsatellite typing have been utilized (Kourelis et al. 2010a).

Moreover, rDNA ITS of S. boulardii showed very less intraspecies variability in

comparison to S. cerevisiae (Fietto et al. 2004). These molecular studies concluded

that S. boulardii is subtype of S. cerevisiae. Gel based typing methods like PFGE,

RAPD or RFLP, have been extensively been used for strain typing but it lacks

reproducibility, is very laborious intensive and has inter lab variability. Recently,

microsatellite typing has emerged as discriminatory tool for yeast strain

differentiation. Moreover, the pattern observed is portable and have been extensively

employed for strain differentiation for epidemiological studies. It also makes it very

handy tool for comparing the strains at global level. Microsatellites used in this study

clearly differentiated yeast strain originated from fermented formulations, with S.

boulardii a commercial probiotic strains. In support of previous studies, this study

also highlights the fact that S. boulardii is subspecies of S. cerevisiae as it also

showed amplification with S. cerevisiae specific primer sets (Hennequin et al. 2001).

As probiotic properties are strain specific, preliminary strain selection could assist in

further selection of strains for probiotic attributes.

Ability to tolerate low pH is one of the important characteristics of probiotic

microorganisms. Despite of genomic differences between S. cerevisiae and S.

boulardii they were found to have similar response to lower pH treatment (van der Aa

Kuhle et al. 2005; Edwards-Ingram et al. 2007). S. boulardii and S. cerevisiae

maintained its cellular viability similar to control at pH 2 and pH 2.5 respectively

(Edwards-Ingram et al. 2007; Pennacchia et al. 2008). S. cerevisiae strains used in

current study have capacity to tolerate pH 2 and pH 6 could be beneficial for their

growth. In an another study it was found that the growth of S. boulardii was unaltered

Chapter 4


94

at pH 6.8 (Graff et al. 2008). This study further supports the hypothesis that the acid

tolerance is one of the easily recoverable properties in yeasts.

In addition to tolerance to acidic conditions, probiotic microorganism should be able

to tolerate bile salts. Moreover, concentration of bile varies across the intestine.

Different studies have highlighted the use of varied concentration of bile from 0.15%

to 0.3% (Pennacchia et al. 2008; Roopashri and Varadaraj 2009). S. cerevisiae has

showed to tolerate bile salts and yeast strains under investigation showed very little

changes in cell viability (Kumura et al. 2004; van der Aa Kuhle et al. 2005). Probiotic

microorganism has to pass through gastrointestinal tract, it would be important to

determine the survival of yeast in gastric environment. S. cerevisiae are also known to

have good stress tolerance capacity (Fietto et al. 2004; Pennacchia et al. 2008).

Intestinal cell lines like Caco2 and IPEC-J2 have been used to study cell adhesion

property of yeast strains (Kumura et al. 2004; van der Aa Kuhle et al. 2005). Unlike

Lactobacilli, yeast spp. are known to possess less adhesive capacity. S. boulardii have

also shown to be less adhesive in vitro model systems (van der Aa Kuhle et al. 2005;

Edwards-Ingram et al. 2007). Confocal microscopy also revealed that the adherence

of S. cerevisiae strains showed very low adherence to HT-29 cell line. S. cerevisiae

strain VQ showed higher number of adhesive cells per field in comparison to other

yeast strains. Flow cytometry with S. cerevisiae VQ and S. boulardii also proved that

there is similar binding of labelled yeast cells in this cell line.

The intestinal immune system undergoes complex regulatory processes while major

function includes elimination of pathogenic micro-organisms and maintaining

tolerance towards food antigens and endogenous flora (Delcenserie et al. 2008). The

expression profile of different treatment group suggests that the Th1/Th2 is skewed

towards Th2 response. It has also been proposed that this skew of balance favours

tolerance and IgA production. For example, L. acidophilus and L. rhamnosus HNOOI

are well characterised for skewing immune balance to Th2 (Delcenserie et al. 2008).

In an another study, oral administration of L. rhamnosus GG for 5 weeks affected the

systemic cellular immune response (Ghosh et al. 2004).

In conclusion, S. cerevisiae strains of this study have genetically close proximity with

commercially used S. boulardii strain. Tolerance of yeast to different conditions like

pH, bile and gastric juice, suggests that it can be used as potential probiont. Further, in

Chapter 4


95

vitro cell line studies confirmed the same pattern observed in comparison to

commercial strain of S. boulardii. In vivo studies also proved that these strains are

safe in use when administered orally for 7 days.

Capacity to aggregate is another desirable property of probiotic microorganisms. It

helps establishing probiotic microorganisms in adequate number to confer health

benefits to host (Darilmaz et al. 2012). Dairy strains of Propionibacterium

freudenreichii subsp. shermanii JS strain ranged from 7.7% to 17.4% after 2 h of

incubation (Collado et al. 2008). In this study, there is marked increase in aggregation

capacity after 4 h of incubation. Other studies also supports this finding of increased

aggregation with time (Darilmaz et al. 2012). Aggregation phenotype can also exert

protective effect on colitis (Castagliuolo et al. 2005).

Recently, co-aggregation between L. plantarum and S. cerevisiae strains have been

reported to play important role in traditional fermentation processes like sake, soy

sauce, wine, lambic beer, whiskey, kefir etc. (Furukawa et al. 2011; Furukawa et al.

2012). These reports also suggest that the interaction between L. plantarum and S.

cerevisiae is mediated by mannose or lectin specific cell surface proteins. L.

plantarum ML11-11 and S. cerevisiae Y11-43 showed co-aggregation within 5 min of

mixing (Furukawa et al. 2011). In our study L. plantarum VR1 and S. cerevisiae VQ

strains of herbal origin showed higher co-aggregation capacity in comparison to

widely used strains of L. rhamnosus GG and S. boulardii. Co-aggregation property

also helps in increased survival of microorganism in hostile environment

(Castagliuolo et al. 2005). Co-aggregation of Yeast and Lactobacillus sp. are known

to be very important for mixed biofilm formation and which can be further utilized for

low cost designing of continuous fermentation processes (Furukawa et al. 2012).

Taking in account the origin of these potential probiotic strains, the stability of these

strains were evaluated in Kutajarista. As described in chapter 2.2.1, Kutajarista has

unique therapeutic properties for gastrointestinal disorders and it could be an excellent

vehicle for the incorporation of these strains. Additionally, herbal extracts based

probiotic products are gaining lot of interest worldwide because of its proven health

benefits, particularly as an antioxidant and nervous system stimulant (Lima et al.

2012). In Kutajarista, the viability of L. plantarum VR1 and S. cerevisiae VQ were

considerably more in comparison to standard strains. However, L. plantarum VR1

Chapter 4


96

could not survive more than 14 days which probably needs further standardisation in

terms of storage condition and temperature. The present study also suggest that yeast

strains were better adapted to Kutajarista matrix as there was 40% viable cells even

after 14 days of incubation. Moreover, yeasts strains have known to play important

role in these fermentation processes. When yeast and L. plantarum cultures were

mixed together, there was increase in lactobacilli viability, suggesting that yeast might

assist lactobacilli survival in hostile environment. Increase in survival of lactobacilli

by yeast has been attributed to specific interaction and it varies across strains (Liu and

Tsao 2009).

Incorporation of ingredients like xanthenes, polyphenol and other antioxidant

components in herbal extracts may enhance its benefits and larger consumer

acceptance (Lima et al. 2012). There are various methods which have been practised

for increasing the lactobacilli viability like addition of prebiotics and nutrients, stress

adaptation, use of protectants and microencapsulations (Liu and Tsao 2009). Sensory

analysis along with other functional tests could lead to better designing of functional

foods.

chapter:4 selection of yeast for probiotic attributes and...

Documents