International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

International Journal of Nutritional Science and

Food Technology

Research Article ISSN 2471-7371

In vitro screening and characterization of kefir yeast for probiotic attributes

Renuka Basavaiah, Geethakumari, Madhukar Nagesh, Murali Harishchandra Sripathy, Harsh Vardhan Batra*
Defence Food Research Laboratory, Mysore, Karnataka State, India-570011

Abstract
The yeasts constitute a large and heterogeneous group of microorganisms, currently attracting increased attention from scientists and
industry as probiotics. Till date only Saccharomyces boulardii has been extensively studied for its probiotic effects. Therefore, the current
study aims to characterize the probiotic potential of yeast isolated from kefir, a fermented beverage. Out of 22 yeast isolates screened, 13
isolates could survive (>75%) in simulated conditions similar to the gut (pH 2.0 and 1.0% bile salt). The isolates showed high auto-aggregation
(>85%) ability and cell surface hydrophobicity (>75%) have also expressed high in-vitro adherence (>90%) to HT-29 cells. A simulation of
transit tolerance in the upper human gastrointestinal tract together with auto-aggregation, hydrophobicity, and adherence to HT-29 cells
have been vital in reducing the number of yeast strains to 7 promising probiotics. The probiotic yeast strains showed resistance to
commonly used antibiotics and exhibited a broad spectrum of antagonistic activity against pathogenic microorganisms (E. coli, S.
typhimurium, S. paratyphi-A, S. aureus, S. sonnei, B. cereus and Y. enterocolitica). The results obtained were compared with the reference
culture, Saccharomyces cerevisiae ATCC 7745. Based on 5.8S rRNA gene sequencing the isolates were identified as Pichia kudriavzevii,
Candida xylopsoci, Saccharomyces cerevisiae and Issatchenkia orientalis. Overall these results demonstrated the possible use of these
isolates in the development of novel functional foods with potential probiotic properties.

Keywords: Probiotics; Yeast; Kefir Grains; Beverage; Fermented Foods; Screening

Corresponding author: Harsh Vardhan Batra The microbes responsible for the fermentation of milk to produce
Outstanding Scientist and Director (Retired), Defence Food Research kefir consist of a complex association of lactic acid bacteria (Lactoba-
Laboratory, Mysore, Karnataka State, India-570011 cillus, Lactococcus, Leuconostoc, and Streptococcus spp.) and yeasts
Mobile: +91-9342054402 (Kluyveromyces, Saccharomyces and Torula) that are immobilized on a
E-mail: drharshvardanbatra@gmail.com polysaccharide and protein matrix of the kefir grains to serve as a
starter culture (Garrote et al. 2010; Miguel et al. 2010; Magalhaes et al.
Citation: Harsh Vardhan Batra et al. (2018), In vitro screening and 2011a). Kefir has immuno-modulatory properties as well as antimicrobi-
characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & al, antihypertensive, anti-inflammatory, anticarcinogenic, antiallergic,
Food Tech. 4:8, antioxidant activity, with ability to reduce cholesterol levels, and al-
leviate lactose intolerance (Farnworth et al. 2005; Ahmed et al. 2013).
Copyright: © 2018 Harsh Vardhan Batra et al. This is an open- Regardless of their non-human origin, such non-pathogenic yeastsful-
access article distributed under the terms of the Creative Commons fill the major criteria for probiotic definition. The beneficial features
Attribution License, which permits unrestricted use, distribution, and thus reported therefore, indicate kefir being as a promising source of
reproduction in any medium, provided the original author and source new microbial strains including yeast for the development of function-
are credited al foods.
Received: November 5, 2018 Probiotics are living microorganisms that exert a health benefit to the
Accepted: November 15, 2018 host when administered in sufficient amounts (FAO/WHO 2002). The
Published: xxxx, 2018 most extensively studied probiotics belong to the genera Lactobacil-
lus and Bifidiobacterium (Borriello et al. 2003; Tuohy et al. 2003). De-
Introduction spite the occurrence of yeasts in many dairy related products (Fleet
Yeasts are microorganisms of great economical interest for their nu- et al. 1990; Jakobsen and Narvhus et al. 1996) and in the human gas-
merous applications in traditional and modern biotechnology (Raton, tro intestinal tract (GI) (Knoke et al. 1999; Czerucka et al. 2007), their
2004). The products of yeast form the backbone of many commercial- potential as probiotics has been overlooked. The microbiological and
ly important sectors, including various fermented foods (Yarrow, et chemical composition of kefir indicates that it is a much more complex
al. 1998), beverages, pharmaceuticals and industrial enzymes which probiotic, as the large number of different bacteria and yeast found
are attracting increased attention from scientists and industry. Kefir, a in it distinguishes it from other probiotic products. The studies have
natural fermented probiotic beverage is gaining in popularity because revealed that the yeast component of kefir consists of Kluyveromyces,
of its number of health promoting properties and its distinct flavour, Saccharomyces, Candida and Torulaspora (Angulo et al. 1993; Wyder et
typical of yeast (Farnworth el at. 2005; Lopitz- Otsoa et al. 2006; Mi- al. 1997; Lin et al. 1999; Simova et al. 2002; Loretan et al. 2003). Oth-
guel et al. 2010; Rattray and O’Connel et al. 2011; Magalhaes et al. er yeast that have been less frequently associated with kefir include
2011a; Ahmed et al. 2013). Pichia/Issatachenkia (Latorre-Garcia et al. 2007), Brettanomyces/Dek-

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
4:8,

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International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

kera (Pintado et al. 1996; Wyder et al. 1997), Yarrowia (Loretan et al. elongation at 72 °C for 1 min followed by final extension at 72 °C for
2003), Zygosaccharomyces (Witthuhn et al. 2005), and recently re- 10 min. The amplified products were stored at -20 °C until analyzed.
ported Kazachstaniaaerobia and Lachanceameyersii (Zhou et al. 2009; Aliquots of the amplification products along with DNA ladder mark-
Magalhaes et al. 2011; Gao et al. 2012). The yeasts and bacteriapresent er were analyzed by electrophoresis in horizontal 1.0% (w/v) agarose
in kefir grains have undergone a long association; the resultant micro- gel in 0.5X TBE buffer, stained with ethidium bromide (0.5 µl/ml) at
bial population exhibits many similar characteristics, making isolation a constant current of 90V and visualized under ultraviolet light. The
differentiation of the isolated strains more challenging. PCR products obtained through amplification were purified and sent
The yeast strains recovered from kefir namely, Saccharomyces cer- for sequencing to a commercial sequencing facility. Sequences were
evisiae, Saccharomyces unisporus, Issatchenkia occidentalis, and aligned to 5.8S rRNA gene sequences in the Gen bank database using
Kluyveromyces marxianus have showed acid and bile resistance phe- the BLAST algorithm (Altschul et al. 1997).
notypes and are thus potentially suitable for probiotic purposes(Dios- Probiotic characterization of kefir yeast
ma et al. 2013). But still the only yeast known for its probiotic effects in According to recent Food and Agriculture Organization (FAO) and
humans and often marketed as a dietary supplement (McFarland et al. World Health Organization (WHO) guidelines (FAO/WHO, 2002), pro-
2010) and also employed as a therapeutic agent for the treatment of a biotic organisms used in food must be able to survive passage through
variety of gut disorders to normalize intestinal flora is, Saccharomyces the gut i.e., they must have the ability to resist gastric juices and expo-
cerevisiae var. boulardii (Saccharomyces boulardii) (Szajewska et al. sure to bile. Furthermore, they must be able to proliferate and colo-
2007; Zanello et al., 2009; Saad et al. 2013). Further research into novel nize the digestive tract. Therefore, the yeast isolates were studied for
probiotic yeast isolates is important to satisfy the increasing market their prime probiotic properties.
demand and to obtain highly active probiotic cultures for improved Acid and bile salt tolerance
food products with characteristics that are superior to those present
To determine the acid and bile salt tolerance, 22 selected yeast isolates
on the market. The search for more yeast with probiotic potential
were propagated twice in YPG broth. Initially yeast cultures were incu-
from kefir preparation and with possible application in food industry
bated at 37 oC for 24 h in YPG broth and after 24 h, the yeast isolates
appears to be a promising area of investigation.
were inoculated into sterile YPG broth medium acidified to pH 2.0 with
Materials And Methods 1N HCl and supplemented with 1% ox bile (Syal and Vohra et al. 2013).
Screening of yeast from kefir and growth media Samples were drawn immediately (0 h) and after 4 h of incubation at
The organic milk kefir grains were inoculated (5%; w/v) to the cooled 37 oC, and serial dilutions in saline 0.85% (w/v) was made. Appropriate
pasteurized milk (3% fat Nandini milk; Mysore, Karnataka, India) and in- dilutions were placed on YPGA in order to determine the number of
cubated at 37 °C for 24-48 h. The fermented kefir beverage was filtered viable cells. The survival rate was calculated as the percentage of colo-
to remove the kefir grains. The obtained beverage was serially diluted nies grown on YPGA medium after exposure (4 h) to low pH and high
up to 10-6 dilution and plated on MRS (de Man, Rogosa and Sharpe) bile salt concentration as compared to the initial cell concentrations
agar (Himedia, India) plate by spread plate method and incubated at using standard formula.
37 °C for 24-48 h. The morphology and size of different colonies of % Survival = log number of viable cells survived (CFU/ml) × 100
yeast on MRS agar plates were characterized by microscopic and mac- log number of initial viable cells inoculated (CFU/ml)
roscopic method (Barnett et al. 2000). A total of 22 visually different Tolerance to synthetic gastric juice (SGJ)
colonies were isolated. Survivability of yeast isolates in synthetic gastric juice was determined
The colonies grown on MRS agar plates were further cultured on yeast according to the method of Cotter et al., (2001). The composition of
extract peptone glucose agar (YPGA) and potato dextrose agar (PDA) SGJ per liter is 8.3 g of protease peptone, 3.5 g of glucose, 2.05 g of
to screen suitable media for the growth of kefir yeast. All the 22 iso- NaCl, 0.6 g of KH2PO4, 0.11 g of CaCl2, 0.37 g of KCl, 0.05 g of bile, 0.1 g
lates along with reference culture Saccharomyces cerevisiae ATCC of lysozyme and 13.3 mg of pepsin (pH 2.5). The media was filter ster-
7745 were point inoculated on YPGA, PDA and MRSA plates and incu- ilized (Pedersen et al. 2004) using 0.22µ membrane filter (Millipore,
bated at 37 °C for 24-48 h. In order to determine morphology of India). Samples were drawn immediately (0 h) and after 4 h of incu-
yeasts cells and reproduction type, the yeast isolates were examined bation at 37 oC, and appropriate dilutions in 0.85% (w/v) saline was
microscopically by staining method. inoculated to YPGA by spread plate method in order to determine the
Genotypic characterization number of viable cells and percent survival was calculated using stan-
Total genomic DNA of 22 yeast isolates was extracted using the DNA dard formula.
extraction kit (HiPureATM Bacterial and yeast Genomic DNA Miniprep % Survival = log number of viable cells survived (CFU/ml) × 100
purification spin kit, Himedia, India) following the instructions of the log number of initial viable cells inoculated (CFU/ml)
manufacturer. The extracted DNA was quantified using the Nanodrop Microbial adhesion to hydrocarbons (MATH) assay
(Thermo Scientific Nanodrop 2000 Spectrophotometer, Genesis Bio-
MATH assay was carried out by the method of Syal and Vohra et al.
solutions, and India) and diluted to a concentration of 50ng/ml and
(2013) with slight modification. For the cell surface hydrophobicity,
used for the PCR analysis (Greppi et al. 2013a and 2013b). The amplifi-
the selected yeast isolates were grown in YPG broth at 37 oC for 24 h.
cation of internal transcribed spacer ITS1 and ITS4 with forward and re-
The cells were harvested by centrifugation at 12000 rpm, at 4 oC for
verse universal primers (ITS1 5’-TCCGTAGGTGAACCTGCGG-3’ and ITS4
20 min, washed twice and resuspended yeast pellet in phosphate buf-
5’-TCCTCCGCTTATTGATATGC- 3’) targeted to the 5.8S rRNA gene was
fer saline (PBS; pH 7.0). The absorbance (OD) was measured using an
carried out (Guillamon et al. 1998) in 20 µl of reaction mixture contain-
infinite M200PRO (TECAN) at 600 nm. Aliquots of yeast suspensions
ing 10x PCR buffer (2.0 µl), 25 mM MgCl2 (1.6 µl), 2 mM dNTP (1.0 µl),
were put in contact with hydrocarbons - xylene and toluene, separate-
0.6 µl each of forward and reverse primers, Taq DNA polymerase (0.1
ly (1:3 v/v). The cells were vortexed for 120 sec and the suspension was
µl), autoclaved deionized water (13.1 µl) and genomic DNA (1.0 µl). PCR
kept undisturbed at 37 oC for 30 min to allow phase separation. After
amplification was carried out in a Verti 96 well Thermal cycler (Applied
30 min, the aqueous phase was removed carefully and the absorbance
Biosystems, India) with an initial denaturation at 94 °C for 4 min, fol-
(OD) was measured at 600 nm. The decrease in the absorbance is the
lowed by 35 cycles at 94 °C for 1 min, annealing at 58 °C for 1 min and

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
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International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

measure of the cell surface hydrophobicity. The % hydrophobicity is cultures of the yeast isolates (Christobell et al. 2012). The antibiotic re-
calculated using the equation given below. sistance was assessed against Trimethoprin (TR), Cephotaxim (CTX),
% Hydrophobicity = (OD initial - OD final)/OD initial x 100 Cefixime (CFM), Ofloxacin (OF), Nalidixic acid (NA), Cloramphenicol
Adhesion of yeast isolates to intestinal HT-29 cell lines (C), Amoxyclav (AMC), Oxytetracycline (O), Ceftriaxone (CTR), Tetra-
The colonocytes-like cell line HT-29 was procured from NCCL, Pune, cycline (TE), Gentamycin (GEN), Erythromycin (E), Streptomycin (S),
India and were used to determine the adhesion ability of the yeast Ampicillin (AMP), Vancomycin (VA), Polymyxin–B (PB), Pencillin-G (P),
isolates. The culture and maintenance of the HT-29 cell lines were Co-trimoxazole (COT), Azithromycin (AZM), Doxycycline Hydrochlo-
carried out following standard procedures (Sanchez et al. 2010) using ride (DO) and Rifampicin (RIF). The antibiotic discs were placed on the
DMEM (Dulbecco's Modified Eagle's medium) supplemented with surface of agar and the plates were incubated at 37 °C for 24 h. The
FBS (Fetal Bovine Serum, Sigma). Intestinal cells were seeded in 24- zone size (mm) interpretative chart for antibiotics was measured ac-
well tissue culture plate and cultivated until a confluent differentiated cording to performance standards.
state was reached. For adhesion experiments, 9±1 day-old cellular Antimicrobial activity
monolayers were used. Yeasts were cultured for 24 h and after The antimicrobial activity of yeast isolates against enteric pathogens
washing twice with phosphate buffer solution (PBS), they were re- (Escherichia coli ATCC 10536, Salmonella typhimurium MTCC 1251, Sal-
suspended in the corresponding cell-line media without FBS. Cellular monella paratyphi-A ATCC 9150, Staphylococcus aureus ATCC 700699,
monolayers were also carefully washed with PBS and yeast Shigella sonnei ATCC 25931, Bacillus cereus ATCC 14579, and Yersinia
suspensions (108 CFU/ml) were added at a ratio of about 10:1 (yeast: enterocolitica ATCC 23715) was performed by using the agar well dif-
eukaryotic cell). Adhesion experiments were carried out for 1 h at 37 ° fusion method, as described by Tatsadjieu et al. (2009). The cultures
C, 5% CO2. After incubation, wells were gently washed to release filtrate of the 24-48 h old yeast isolates was inoculated to the wells of
unattached yeast before proceeding with the lysis of cellular YPGA plate containing pathogens. The plates were incubated at 37 oC
monolayer using 0.25% Trypsin–EDTA solution (Sigma). Dilutions of for 24 h and the diameter of zone of inhibition was measured in
samples, before and after adhesion were made in 0.85% NaCl solution millimeter (mm). The zone of inhibition around the wells indicates pos-
and yeast counts were performed in YPGA plates. The percent of itive (+) for antimicrobial activity, while the absence of zone indicates
yeast adhering to the intestinal epithelial cells was calculated as, a negative (-) result.
% = CFU adhered yeasts /CFU added yeasts Results And Discussion
Auto-aggregation assay Screening of yeast from kefir and growth media
Auto-aggregation assay was performed as described by Collado et al. Out of 26 isolates from kefir, 22 strains were confirmed as yeast based
(2008) with minor modifications. Yeast isolates were grown for24–48 on their colony morphology on MRS agar plates. Colonies were off
h at 37 ºC in YPG broth. The cells were harvested by centrifugation white in colour, circular in shape with slightly irregular margins, con-
at 12000 rpm for 10 min, washed twice and resuspended in PBS (pH vex elevated with opaque opacity and smooth texture. It was further
7.0). Cell suspensions (5 ml) were mixed by vortexing for 10 sec and confirmed by lacto-phenol cotton blue staining and observed under
auto-aggregation was determined after 3 h and 20 h of incubation at microscope. The screened isolates were preserved on MRS agar plate
37 ºC. An aliquot (100µl) of the upper suspension of PBS after incuba- for further study.
tion was transferred to another tube with 3.9 ml of PBS and the absor- The growth profile of yeast strains on YPGA, MRSA and PDA media
bance (A) was measured at 600 nm. The auto-aggregation percentage is shown in Fig. 1 and it revealed that the culture media had great in-
is expressed as: 1-(At/A0) X 100. Where, At represents the absorbance fluence on growth. It was observed that irrespective of the culture
at time t = 3 h or 20 h. A0 the absorbance at t = 0 h. medium employed and the conditions maintained, the growth of each
All the experiments were performed in triplicates. strain is found to be diverse (Table І). Based on the result obtained,
Antibiotic susceptibility YPGA is considered suitable media for the rapid growth of yeast strains
The antibiotic resistance of yeast isolates was analyzed using various when compared with that of MRSA and PDA media. Therefore, YPGA
antibiotic discs (HiMedia, India) on YPGA plate seeded with 24 h active medium is selected for further study.

Figure 1 : Yeast strains on PDA, MRSA, and YPGA plate

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
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International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

GenBank Accession Diameter of the yeast cultures

Yeast strains numbers (cm)

YPGA MRSA PDA

1y - Pichiakudriavzevii MF685411 5.3 3.1 0.9
2y - Pichiakudriavzevii- MF685412 4.5 2.7 0.7
3y - Pichiakudriavzevii MF685413 3.9 2.0 1.1
4y - Saccharomyces cerevisiae MF685414 4.6 3.0 1.2
5y- Saccharomyces cerevisiae MF685415 4.2 2.8 0.8
6y - Issatchenkiaorientalis MF685416 3.5 2.3 1.4
7y- Issatchenkiaorientalis MF685417 3.2. 2.2 0.7
8y - Pichiakudriavzevii MF685418 3.0 2.2 1.1
9y - Pichiakudriavzevii MF685419 3.7 1.7 1.2
10y - Issatchenkiaorientalis MF685420 3.9 2.4 0.8
11y - Issatchenkiaorientalis MF685421 3.8 1.5 0.7
12y - Candida xylopsoci MF685422 4.0 2.0 0.9
13y - Pichiakudriavzevii MF685423 3.5 2.4 1.3
14y - Candida xylopsoci MF685424 4.3 1.9 1.1
15y - Candida xylopsoci MF685425 5.0 2.4 0.7
16y - Candida xylopsoci MF685426 3.4 2.5 0.9
17y -Saccharomycescerevisiae MF685427 3.2 2.7 0.7
18y - Pichiakudriavzevii MF685428 5.0 2.5 0.8
19y - Issatchenkiaorientalis MF685429 4.5 2.4 0.6
20y - Pichiakudriavzevii MF685430 4.1 2.8 0.9
21y - Pichiakudriavzevii MF685431 3.6 1.7 1.2
22y - Issatchenkiaorientalis MF685432 5.0 2.1 1.0

Table 1: Growth profile of yeast strains on YPGA, MRSA, and PDA

plate

Genotypic characterization orientalis and C. xylopsoci has not been previously reported in kefir as
The 22 yeast isolates screened were identified as Pichia kudriavzevii probiotics. Overall, these results confirm the importance of these
(9), Issatchenkia orientalis (6), Candida xylopsoci (4), and Saccharo- genera for kefir production. Irrespective of the possible failures in the
myces cerevisiae (3) based on the ITS region of 5.8S ribosomal RNA isolation and identification of the yeast in the grains, the results ob-
gene. The sequence of rRNA gene from all the yeast strains was ho- tained in the present study confirms the high microbial heterogeneity
mologous to an extent of 99% with that of other strains. In the cur- in kefir grains. Further, these yeast strains were characterized for their
rent study, P. kudriavzevii, being the most prevalent species during probiotic properties. Because the complexity of intestinal flora where
fermentation of kefir. S. cerevisiae comprised less than 15% of the total popular yeast is supposed to serve as a probiotic requires a clear
isolated microflora in kefir. Previously S. cerevisiae has been reported definition of the selection criteria based on which, it could be
to be one of the predominant yeast species in fermented products classified as a new target-specific or site-specific probiotic strain
(Aditi Sourabh et al. 2012). The fermentation of kefir is often initi- (Collins et al. 1998; Klaenhammer and Kullen et al. 1999; Gueimonde
ated by I. orientalis, C. xylopsoci. The presence of P. kudriavzevii, I. and Salminen et al. 2006).

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
4:8,
International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

Resistance at low pH more than 80% survivability was comparable with the reference cul-
Ability of the yeast strains to survive at low pH (2.0) for 4 h at 37 °C is ture S. cerevisiae ATCC 7745 which showed 85% survivability. The rea-
shown in Fig. 2. The acidic environments encountered in food and in sons of the factors responsible for this could be cell size, composition
the gastrointestinal tract provide a significant survival challenge for of cell wall, extrude protons etc (Czerucka et al. 2007). Exposure of the
intestinal flora. Hence, acid tolerance is accepted as one of the indis- yeast cells to environmental stresses like low pH and high bile concen-
pensable properties used to select potentially probiotic strains. The tration (Arino et al. 2010) can possibly triggers biochemical and gene
result obtained indicates that, all the strains were tolerant to pH 2.0 expression changes (Gasch et al. 2000) and may also causes immedi-
for 4 h despite variation in their degree of viability. Of the 22 strains ate changes in the cytosolic calcium, an important second messenger
tested, 15 yeast strains showed significant viable count [10 isolates (S. in eukaryotic cells. The survivability of kefir yeast at low pH reported
cerevisiae - 4y; I. orientalis – 6y, 7y, 11y, 19y; P. kudriavzevii - 9y, 13y; by Katarzyna Rajkowska and Styczynska, et al. (2010) and Diosma et
C. xylopsoci - 12y, 14y, 15y) showed more than 80% and 5 isolates (C. al. (2013) was found to be 50% and 90% after 4 hours and 3 hours of
xylopsoci - 16y; S. cerevisiae - 17y; P. kudriavzevii - 18y, 20y; I. orientalis - incubation respectively, which is significantly less when compared to
22y) showed 75% survivability]. The other yeast isolates showed less the survival rate (>80%) of P. kudriavzevii, S. cerevisiae, I. orientalis and
than 75% tolerance after 4 h of incubation. The yeast isolates showed C. xylopsoci in the current study.

100
90
80
70
% Survivability

60
50
40
30
20
10
0

Yeast isolates

Figure 2: Survivability of yeast isolates at low acid (pH-2.0)

Tolerance to high ox-bile S. cerevisiae ATCC 7745. However, other yeasts (P. kudriavzevii - 1y; S.
Once the yeast passes through the acidic stomach condition, it is im- cerevisiae - 4y, 5y; I. orientalis - 6y, 10y, 11y, C. xylopsoci - 16y) showed
portant for the yeast to survive in the high bile salt environment of good survivability (>75%) after 4 hour of incubation (Fig. 3). The differ-
the small intestine for growth, colonization and metabolic activity in ence in the level of bile tolerance of yeast strains in the present study
the host’s gut (Liong and Shah et al. 2005). The small intestine and may probably be due to the differences in their ability to grow and
colon of humans and animals contain relatively high concentrations colonize the intestinal tract (Usman and Hosono et al. 1999; Kheadr
of bile salt, which can inhibit growth or kill many bacteria. Therefore, et al. 2006). The kefir yeasts reported by Rajkowska and Kumicka-Sty-
it is essential that probiotic bacteria, to be effective, should be ableto czynska (2010) showed least bile salt tolerance in 1% bile salt concen-
grow in 0.3-1.0% ox-bile (Goldin and Gorbach et al. 1992). The 22 yeast tration. The yeast strains investigated in the current study displayed
strains were tested for their tolerance to 1% ox-bile concentrations for good resistance to 1% ox-bile as all could replicate and hence survive
4 h at 37 oC. In the current study, a total of 16 yeast strains survived at the exposure to bile salts. There was considerable variability in resis-
1.0% ox-bile concentration with the survival rate of more than 75%. tance to bile salts between the different species of yeasts, supporting
The % survivability (>80%) exhibited by I. orientalis - 7y, 19y, 22y; C. the importance of assessing the bile tolerance of isolates in selecting
xylopsoci - 12y, 15y; S. cerevisiae - 17y; P. kudriavzevii - 9y, 18y, 21y was potential probiotics. Hence the yeast strains survived >80% were con-
highly comparable with the survival rate (86%) of the reference culture sidered as the most bile salt tolerant strains.

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
4:8,
International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

100
90
80
70
% Survivability 60
50
40
30
20
10
0

Yeast isolates

Fig 3: Survivability of yeast isolates at high bile salt concentration (1% ox bile)

Tolerance to simulated gastric condition have been very important in decreasing the number to 9 strains (I. ori-
The effects of simulated gastric condition on the viability of yeast entalis - 7y, 19y, 22y; P. kudriavzevii - 9y, 18y; C. xylopsoci - 12y, 15y, 16y,
strains are depicted in Fig. 4. Tolerance to simulated gastric juice is an S. cerevisiae - 17y).
important trait of probiotic microorganisms because the probiotics The possible use of yeasts as probiotics is encouraged by the observa-
entering the gastro intestinal tract must be resistant to local stresses tion of the ability of S. cerevisiae members to survive passage through
such as the presence of gastro intestinal enzymes besides pH and bile the intestinal tract (Lourens-Hattingh and Viljoen et al. 2001). The kefir
salt. The results showed that the 13 yeast strains (P. kudriavzevii - 2y, yeast strains demonstrated high tolerance to simulated gastric envi-
3y, 9y, 18y, 21y; I. orientalis - 7y, 19y, 22y; C. xylopsoci - 12y, 14y, 15y, 16y; ronment and thus they offer a better source of potential probiotics,
S. cerevisiae - 17y) survived (>75%) under simulated gastric condition. apart from lactic acid bacteria. This finding suggests that these strains
Yeast strains able to survive conditions mimicking the gastro intestinal have the potential to survive the passage through the stomach, small
environment together with low pH and high bile salt concentration, and large intestine.

100
90
80
70
% Survivability

60
50
40
30
20
10
0

Yeast isolates

Fig 4: Survivability of yeast isolates in simulated gastric juice

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
4:8,
International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

Hydrophobicity, auto-aggregation and adhesion ability to intestinal to be active and such abilities favour colonization in the gastrointes-
HT-29 cell line tinal tract (Venkatasatyanarayana Nallala and Jeevaratnam 2015). In
One of the important properties of probiotic microorganisms is their the present study, I. orientalis - 7y, 19y and 22y; P. kudriavzevii – 9y and
ability to adhere to the target sites for their colonization in the gut for 18y, C. xylopsoci - 16y; S. cerevisiae - 17y showed strong (>85%)
expressing optimal functionality. The yeast strains (P. kudriavzevii - 2y, hydrophobicity and auto-aggregation properties. Microbial adhesion
3y, 9y, 18y, 21y; I. orientalis - 7y, 19y, 22y; C. xylopsoci - 12y, 14y, 15y, 16y; is initially based on non-specific physical interactions between two
S. cerevisiae - 17y) showed more than 75% survivability in simulated surfaces, which then enable specific interactions between adhesins
gastric condition was undertaken with the objective to elucidate the (usually proteins) and complementary receptors. Adhesion scores of
adherence potential of yeasts under in vitro conditions based on their I. orientalis (7y, 19y and 22y), P. kudriavzevii (9y and 18y), C. xylopsoci
ability to adhere HT-29 cells, hydrocarbonsand auto-aggregation (Fig. (16y) and S. cerevisiae (17y) were more than 90% on HT-29 cell lines
5). Auto-aggregation and hydrophobicity value of 67.59 ± 0.27% and which is highly comparable with the reference culture (84%) and there-
58.21 ± 1.09% reported for S. cerevisiae (Sourabh et al. 2011) falls fore, these isolates can be regarded as strongly adhesive to HT-29 cell
between the auto-aggregation (%) and hydrophobicity values in the line. These strains demonstrated their ability to adhere to epithelial
present study which is in the range of 51.11% to 91.30% and 40.74% to cell and exhibited strong hydrophobicity under in vitro conditions, and
86.79% respectively. Strains possessing high hydrophobicity and au- thus could have better prospects to colonize the gut with extended
to-aggregation ability have been more strongly associated to adhesion transit. Hence simulation of transit tolerance in the upper human gas-
property (Del Re et al. 2000; Pan et al. 2006; Rahman et al. 2008) since, trointestinal tract, together with auto-aggregation and hydrophobic-
adhesion is a prerequisite for colonization (Yongchen Zheng 2013). ity, has been decisive in reducing the number of promising probiotic
Aggregation between the cells of same strains (auto-aggregation) is yeast. Therefore, out of 13 yeast strains, only 7 isolates (7y, 9y, 16y, 17y,
of considerable importance in the human gut where probiotics are 18y, 19y and 22y) could fulfill the preliminary in vitro selection criteria
for being designated as probiotic.

Fig 5: Adhesion and auto-aggregation of yeast isolates

Antibiotic susceptibility There have been a number of studies carried out to determine the
Antibiotics taken during illness not only kill the disease causing micro- benefit of taking a probiotic supplement to help reduce antibiotic-as-
organisms but also disrupt the normal microbial balance of the gut sociated diarrhoea, of which an encouraging number have yielded a
leading to a number of side effects and encouraging the patients to positive result. In double-blind placebo-controlled randomized stud-
restore their natural gut microflora with the intake of probiotics (Natt ies, probiotic S. boulardii (Surawicz et al. 1989; McFarland et al. 1995;
and Garcha 2011). The results showed that all the probiotic yeasts (P. D’Souza et al. 2002) significantly decreased the incidence of diarrhea
kudriavzevii, C. xylopsoci, S. cerevisiae, and I. orientalis - 7y, 9y, 16y, 17y, in healthy subjects and patients treated with antibiotics. Most of the
18y, 19y and 22y) were resistant to most of the commonly used probiotic microorganisms are bacteria and many of them are not able
antibiotics except Polymyxin-B (Fig. 6). This could be due to the sur- to resist or tolerate antibiotics, whereas, yeasts have a natural
face active bactericidal and fungicidal Polymyxin-B altered the cell en- resistance against antibiotics. Thus in the present study, the probiot-
velope by binding to the lipid-A portion of lipopolysaccharide and also ic yeasts showed resistance to antibiotics could be used for patients
to phospholipids component of the membrane and caused cell lysis undergoing antibiotic treatment, indicating their potential to be used
(Schwartz et al. 1972). in therapeutics.

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International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

Fig 6: Probiotic P. Kudriavzevii and S. cerevisiae showing antibiogram against polymyxin-B

Antimicrobial property and translocation in the intestinal epithelial cells, production of factors
One of the most desirable properties of probiotic yeasts is the antimi- that neutralize bacterial toxins and modulation of the host signaling
crobial activity against pathogens that penetrate various mucosasites pathway with proinflammatory response during bacterial infection
(Syal and Vohra et al. 2013). The use of antagonistic bacteria to inhibit (Czerucka et al. 2007; Martins et al. 2011; Tiago et al. 2012).
pathogenic bacteria has been studied extensively over the years, while A study of Andreas et al., (2010) showed that the yeast strains isolated
little attention has been given to yeasts in a similar role. Therefore, the from feta cheese (S. cerevisiae) and infant’s faeces (S. boulardii and I.
probiotic yeast strains exhibited the spectrum of antimicrobial activi- orientalis) had no antibacterial or antagonistic activity against the se-
ty against few of the food borne pathogens (E. coli, S. typhimurium, lected food borne pathogens. It has been proposed that the most ad-
S. paratyphi-A, S. aureus, S. sonnei, B. cereus, and Y. enterocolitica) was hering Lactobacillus strains inhibit the S. typhimurium attachment and
determined. The inhibitory action was observed as a clear zone of cell entry to human enterocyte-like Caco-2 cells (Gorbach and Newton,
10mm-30mm (Table 2) around the colonies of the pathogen against 1996). Since the eukaryote microbes have ten times surface area than
the lawn of the growth of probiotic yeasts (Fig. 7). The probiotic yeasts bacteria, greater protection to intestinal cell walls is expected. The an-
showing inhibition zones with, at least, more than 10 mm of diameter tagonistic activity exerted by probiotic yeasts (P. kudriavzevii-9y, C. xy-
were considered mycocin-producing strains and the latter the yeast lopsoci-16y, S. cerevisiae-17y, and I. orientalis-19y), which showed the
demonstrated a strong antimicrobial activity against tested patho- best adhesion properties (>90%) to HT-29 cells, could be the most im-
gens. The mechanisms involved in yeasts antibacterial property to act portant inhibitor of S. typhimurium attachment. Likewise the adhesion
against enteric pathogens are the prevention of bacterial adherence capacity of the probiotic yeast would have inhibited the attachment of
other pathogens studied.

Fig 7: Probiotic yeasts showing zone of inhibition against pathogens

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
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International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

Zone of inhibition (mm)

Probiotic yeasts E. coil S. paratyphi-A S. aureus B. cereus S. typhimurium S. sonnei Y. enterocolitica

ATCC 10536 ATCC 9150 ATCC 700699 ATCC 14579 MTCC 1251 ATCC 25931 ATCC 23715

28 22 19 28 26 23 12
I. orientalis-7y
29 31 18 18 24 22 14
P. kudriavzevii-9y
20 26 17 23 24 24 16
C. xylopsoci-16y
17 19 13 22 30 20 18
S. cerevisiae-17y
17 15 14 24 28 18 14
P. kudriavzevii-18y
21 14 13 26 26 21 15
I. orientali-19y
18 16 14 15 14 17 13
I. orientalis-22y
17 19 19 16 15 14 10
S. cerevisiae ATCC 7745

Table 2: Antimicrobial activity (mm) of probiotic yeast against food borne pathogens

Conclusion Conflicts of interest

Kefir grains are considered as an excellent source of beneficial pro- The authors declare that there is no conflict of interest.
biotics and a logical natural product to investigate for new probiot-
References
ic strains. Although, lactic acid bacteria and bifidobacteria group are
1. Ahmed Z, Wang Y, Ahmad A, Khan ST, Nisa M, Ahmad H (2013) Ke-
most widely studied for probiotic properties, the use of yeast as a
fir and health: a contemporary perspective. Crit Rev Food Sci Nutr 53:
probiotic food supplement is gaining relevance (Fleet and Balia2006).
422-434.
Saccharomyces boulardii is a strain of yeast which has been extensive-
ly studied for its probiotic effects. Research into novel probiotic strains 2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
is important to satisfy the increasing market demand and to obtain Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of
highly active probiotic cultures for improved products (Bertazzoni et protein database search programs Nucleic Acids Res 25: 3389-3402.
al. 2004) with probiotic characteristics that are superior to those 3. Andreas V (2010) Antimicrobial peptides: The ancient arm of the hu-
presently on the market. Therefore an attempt has been made to man immune system. Virulence 1(5): 440-464.
identifying and characterizing the probiotic potential of kefir yeast. In 4. Angulo L, Lopez E, Lema C (1993) Microflora present in kefir grains
summary, I. orientalis (7y, 19y and 22y) P. kudriavzevii (9y and 18y), C. of the Galician region (North-West of Spain). J Dairy Res 60:263-267.
xylopsoci (16y), S. cerevisiae (17y) exhibited promising probiotic prop- 5. Arino J (2010) Integrative responses to high pH stress in S. cerevisiae,
erties such as excellent pH and bile tolerance, cell surface traits like OMICS: A J Integrative Biol 14(5):517-523.doi:10.1089/omi.2010.0044
hydrophobicity, aggregations, and suppression of pathogen growth
6. Barnett JA, Payne RW, Yarrow D (2000) Yeasts: Characteristics and
under in vitro conditions. Moreover, these probiotic yeast strains
Identification. Cambridge, UK; New York, NY, USA: Cambridge Univer-
were sensitive only to Polymyxin-B. It is interesting to note that even
sity Press, 1139 pages. Book; Illustrated 13. https://trove.nla.gov.au/
after several decades of investigation, the potential of yeast,especial-
work/5635020
ly those of kefir origin, has not yet been fully exploited for probiotic
properties. Hence, more research is needed to exploit other potential 7. Bertazzoni Minelli E, Benini A, Marzotto M, Sbarbati A, Ruzzenente
probiotic and functional properties of these strains. Further, in vivo O (2004) Assessment of novel probiotic Lactobacillus casei strains for
trials are needed to determine whether they function as probiotics in the production of functional dairy foods. Int Dairy J 14: 723-736.
ideal therapeutic conditions. 8. Borriello SP, Hammes WP , Holzapfel W, Marteau P, Schrezenmeir J,
Acknowledgement Vaara M (2003) Safety of probiotics that contain lactobacilli or bifido-
bacteria. Clin Infect Dis 36: 775-780.
The authors thank Director, Defence Food Research Laboratory My-
sore, India for providing the facilities. Renuka B is thankful to UGC, 9. Christabell M, Budambula N, Kiiru J and Kariuki S (2012) Character-
New Delhi, India for the financial support in the form of Post-Doctoral ization of antibiotic resistance in environmental enteric pathogens
Fellowship. The authors specially acknowledge the help of Dr. Hitesh from Kibera slum in Nairobi-Kenya. J Bacteriol Res 4(4): 46-54.
and Dr. Banerjee for providing the kefir grains. 10. Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggrega-
tion properties of probiotic and pathogen strains. Eur Food Res Tech-

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
4:8,
International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

nol 226: 1065-1073. 29. Guillamón JM, Sabaté J, Barrio E, Cano J, Querol A (1998) Rapid
11. Collins JK, Thornton G, Sullivan GO (1998) Selection of probiotic identification of wine yeast species based on RFLP analysis of the ri-
strains for human applications. Int Dairy J 8: 487-490. bosomal internal transcribed spacer (ITS) region. Arch Microbiol 169:
12. Cotter PD, Gahan CG, Hill C (2001) A glutamate decarboxylase sys- 387–392
tem protects Listeria monocytogenes in gastric fluid. Mol Microbiol 30. Jakobsen M, Narvhus J (1996) Yeasts and their possible beneficial
40: 465-475. and negative effects on the quality of dairy products. Inter Dairy J 6:
13. Czerucka D, Piche T, Rampal P (2007) Review article: yeast as probi- 755-768.
otics – Saccharomyces boulardii. Alimentary pharmacol Therapeutics 31. Kheadr EE (2006) Impact of acid and oxgall on antibiotic suscep-
26: 767-778. doi:10.1111/j.1365-2036.2007.03442.x tibility of probiotic lactobacilli. African J Agri Res 1(5): 172-181. http://
14. Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, www.academicjournals.org/AJAR
autoaggregation and hydrophobicity of 13 strains of Bifidobacterium 32. Klaenhammer TR and Kullen MJ (1999) Selection and design of pro-
longum. Letters App Microbiol 31: 438-442. biotics. Int J Food Microbiol 50: 45-57.
15. Diosma G, Romanin DE, Rey-Burusco MF, Londero A, Garrote GL 33. Knoke M (1999) Gastrointestinal microecology of humans and Can-
(2013) Yeasts from kefir grains: isolation, identification, and probi- dida. Mycoses, 42: 30-34.
otic characterization. World J Microbiol Biotechnol 30(1): 43-53. DOI 34. Latorre-Garcia L, del Castillo-Agudo L. & Polaina J (2007) Taxonomi-
10.1007/s11274-013-1419-9 cal classification of yeasts isolated from kefir based on the sequence of
16. D'Souza AL, Rajkumar C, Cooke J, Bulpitt CJ (2002) Probiotics in their ribosomal RNA genes. World J Microbiol Biotechnol 23: 785-791.
prevention of antibiotic associated diarrhoea: meta-analysis. British 35. Lin CW, Chen HL, Liu JR (1999) Identification and characterization
Micro J 324:1361. of lactic acid bacteria and yeasts isolated from kefir grains in Taiwan.
17. FAO/WHO. Guidelines for the Evaluation of Probiotics in Food, Joint Australian J Dairy Tech 54: 14-18.
FAO/WHO Working Group Report on Drafting Guidelines for the Evalu- 36. Liong MT, Shah NP (2005) Bile salt deconjugation ability, bile salt
ation of Probiotics in Food London, Ontario, Canada: April 30 and May hydrolase activity and cholesterol co-precipitation ability of lactobacilli
1, 2002. strains. Int Dairy J 15: 391-398.
18. Farnworth ER (2005) Kefir-a complex probiotic. Food Sci Technol 37. Lopitz-Otsoa F, Rementeria A, Elguezabal N, Garaizar J (2006) Ke-
Bulletin 2(1): 1-17. DOI: 10.1616/1476-2137.13938. fir: a symbiotic yeasts-bacteria community with alleged healthy capa-
19. Fleet GH, Balia R (2006) The Public Health and Probiotic Signifi- bilities. Rev Iberoam Micol 23: 67-74.
cance of Yeasts in Foods and Beverages. In: Querol A, Fleet G (eds). 38. Loretan T, Mostert JF, Viljoen BC (2003) Microbial flora associated
Chapter 12. The yeast handbook. Yeasts in food and beverages, Spring- with South African household kefir. South African J Sci 99(1-2): 92-94.
er, Heidelberg, 386-397. DOI: 10.1007/978-3-540-28398-0 https://hdl.handle.net/10520/EJC97578
20. Fleet GH (1990) Yeasts in dairy products. J Applied Bacteriol68: 39. Lourens-Hattingh A, Viljoen C (2001) Yogurt as probiotic carrier
199-211. food. Int Dairy J 11:1-17.
21. Gao J, Gu F, Abdella NH, Ruan H, He G (2012) Investigation on cul- 40. Magalhaes MS, Salminen S, Anna P, Marchelli R, Ferreira CL, Tom-
turable microflora in tibetan kefir grains from different areas of china. mola J. (2011) Terminology: Functional Foods, Probiotics, Prebiotics,
J Food Sci 77: M425-433. Synbiotics, Health claims, Sensory evaluation of foods, Molecular gas-
22. Garrote GL, Abraham AG, De Antoni G (2010) Microbial Interac- tronomy. In English-portuguese-italian edition. Magalhaes MS, Salmi-
tions in Kefir: A Natural Probiotic Drink. In: (editors) Mozzi F, Raya nen S, Anna P, Marchelli R, Ferreira CL, Tommola J.(Eds) Funct. Foods
RR, Vignolo GM. Biotechnology of Lactic Acid Bacteria: Novel Ap- Forum, University of Turku (Ed), Finland.Magalhaes KT, Pereira GVM,
plications. Iowa: Wiley-Blackwell Publishing. 327-340. https://doi. Campos CR, Dragone G, Schwan RF (2011a) Brazilian kefir: structure,
org/10.1002/9780813820866.ch18 microbial communities and chemical composition. Brazilian J Microbi-
23. Gasch AP, Spellman PT, Kao CM (2000) Genomic expression pro- ol 42(2): 693-702.
grams in the response of yeast cells to environmental changes. Mol 41. Martins FS, Elian SD, Vieira AT, Tiago FC, Martins AK, Silva FC, Souza
Biol Cell 11(12): 4241- 4257. EL, Sousa LP, Araujo HR, Pimenta PF, Bonjardim CA, Arantes RM, Teix-
24. Goldin BR, Gorbach SL (1992) Probiotics for humans. In: Probiotics. eira MM, Nicoli JR (2011) Oral treatment with Saccharomyces cerevisi-
The Scientific Basis (Fuller, R. ed.), Chapman & Hall, London, U. K. 355- ae strain UFMG 905 modulates immune responses and interferes with
376. DOI: 10.1007/978-94-011-2364-8_13 signal pathways involved in the activation of inflammation in a murine
model of typhoid fever. Inter J Med Microbiol 301: 359-364.
25. Gorbach SL, Newton SI (1996) The discovery of Lactobacillus GG.
Nutri Today (Supplement1) 31: 5S. doi:10.1097/00017285-199611001- 42. McFarland LV, Surawicz CM, Greenberg RN, Elmer GW, Moyer KA,
00002 Melcher SA (1995) Prevention of beta-lactam-associated diarrhea by
Saccharomyces boulardii compared with placebo. American J Gastro-
26. Greppi A, Rantsiou K, Padonou W, Hounhouigan J, Jespersen L,
enterol 90: 439-448.
Jakobsen M, L Cocolin. 2013a. Determination of yeast diversity in ogi,
mawè, gowé and tchoukoutou by using culture-dependent and inde- 43. McFarland LV (2010) Systematic review and meta-analysis of Sac-
pendent methods. Inter J Food Microbiol 165(2): 84-88. charomyces boulardii in adult patients. World J Gastroenterol 16: 2202-
2222.
27. Greppi A, Rantisou K, Padonou W, Hounhouigan J, Jespersen L, Ja-
kobsen M, L Cocolin (2013b) Yeast dynamics during spontaneous fer- 44. Miguel MGCP, Cardoso PG, Lago LA, Schwan RF (2010) Diversity
mentation of mawè and tchoukoutou, two traditional products from of bacteria presenting milk kefir grains using culture-dependent and
Benin. Inter J Food Microbiol 165(2): 200-207. culture-independent methods. Food Res Int 43: 1523-1528.
28. Gueimonde M, Salminen S (2006) New method for selecting and 45. Natt NK, Garcha S (2011) Antibiotic sensitivity of acid stressed
evaluating probiotics. Digestive Liver Dis 38: S242-S247. Lactobacillus acidophilus NCDC 291. Inter J Microbiol 9(2): DOI-
10.5580/1004.

Citation: Harsh Vardhan Batra et al. (2018) , In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci & Food Tech.
4:8,
International Journal of Nutritional Science and Food Technology Volume 4 Issue 8, November 2018

46. Pan WH, Li PL, Liu Z (2006) Food microbiology: The correlation 59. Surawicz CM, Elmer GW, Speelman P, McFarland LV, Chinn J, van
between surface hydrophobicity and adherence of Bifidobacterium Belle G (1989) Prevention of antibiotic-associated diarrhea by Saccha-
strains from centenarians’ faeces. Anaerobe 12: 148-152. romyces boulardii: A prospective study. Gastroenterol 96: 981-988.
47. Pedersen C, Lindberg E, Roos S (2004) Microbiological characteri- 60. Syal P, Vohra A (2013) Probiotic potential of yeast isolated from
zation of wet wheat distillers’ grain with focus on isolation of Lactoba- traditional Indian fermented foods. Inter J Microbiol Res 5(2): 390-398.
cilli with potential as probiotics. Appl Environ Microbiol 70:1522-1527. 61. Szajewska H, Skorka A, Dylan M (2007) Meta-analysis: Saccharomy-
48. Pintado ME, DaSilva JAL, Fernandes PB, Malcata FX, Hogg TA ces boulardii for treating acute diarrhoea in children. Alim Pharmacol
(1996) Microbiological and rheological studies on Portuguese kefir Therap 25: 257-264.
grains. Inter J Food Sci Technol 31: 15-26. 62. Tatsadjieu NL, Njintang YN, Sonfack TK, Daoudou B, Mbofung CM
49. Rahman MM, Kim WS, Kumura H, Shimazaki KI (2008) Autoaggre- (2009) Characterization of lactic acid bacteria producing bacteriocins
gation and surface hydrophobicity of bifidobacteria. World J Microbiol against chicken Salmonella enterica and Escherichia coli. African J Mi-
Biotechnol 24: 1593-1598. crobiol Res 3(5): 220-227. http://www.academicjournals.org/ajmr
50. Rajkowska K, Kunicka-Styczynska A (2010) Probiotic properties of 63. Tiago FC, Martins FS, Souza EL, Pimenta PF, Araujo HR, Castro IM,
yeasts isolated from chicken feces and kefirs. Pol J Microbiol 59(4): Brandão RL, Nicoli JR (2012) Adhesion onyeast cell surface as a trap-
257-263. ping mechanism of pathogenic bacteria by Saccharomyces probiotics.
51. Raton TO (2004) Metodos moleculares de identification de levadu- J Med Microbiol 61(9): 1194-1207.
ras de interes biotecnologico. Rev Iberoam Micol 21: 15-19. 64. Touhy KM, Probert HM, Smejkal CW, Gibson GR (2003) Using pro-
52. Rattray FP, O’Connell MJ (2011) Fermented Milks Kefir. In: (editor) biotics and prebiotics to improve gut health. Drug Discovery Today 8:
Fukay JW. Encyclopedia of Dairy Sciences. 2nd ed. Academic Press; 692-700.
San Diego, USA: 518-524. https://doi.org/10.1016/B978-0-12-374407- 65. Usman, Hosono A (1999) Bile tolerance, taurocholate deconjuga-
4.00188-6 tion, and binding of cholesterol by Lactobacillus gasseri strains. J Dairy
53. Saad N, Delattre C, Urdaci M, Schmitter JM, Bressollier P (2013) Sci 82: 243-248.
An overview of the last advances in probiotic and prebiotic field. LWT 66. Venkatasatyanarayana Nallala, Jeevaratnam K (2015) Molecular
Food Sci Technol 50:1-16. Characterization of Bacteriocinogenic, Antifungal and Probiotic Lactic
54. Sánchez B, González-Tejedo C, Ruas-Madiedo P, Urdaci MC, Mar- Acid Bacteria Isolated from Chicken Gastrointestinal Tract. Advances
golles A (2010) Lactobacillus plantarum extracellular chitin-binding Microbiol 5: 644-660.
protein and its role in the interaction between chitin, Caco-2 cells, and 67. Witthuhn RC, Schoeman T, Britz TJ (2005) Characterisation of the
mucin. Appl Environ Microbiol 77: 1123-1126. microbial population at different stages of Kefir production and Kefir
55. Schwartz SN, Medoff G, Kobayashi GS, Kwan CN, Schlessinger D grain mass cultivation. Inter Dairy J 15: 383-389.
(1972) Antifungal Properties of Polymyxin-B and Its Potentiation of 68. Wyder MT, Spillmann H, Meile L, Puhan Z (1997) Investigation of
Tetracycline as an Antifungal Agent. Antimicro Agents Chemo 2(1): 36- the yeast flora in dairy products: A case study of kefyr. Food Technol
40. Biotechnol 35(4): 299-304
56. Simova E, Beshkova D, Angelov A, Hristozova T, Frengova G, 69. Yarrow D (1998) Methods for the isolation, maintenance and
Spasov Z (2002) Lactic acid bacteria and yeasts in kefir grains and kefir identification of yeasts. The Yeasts: a Taxonomic Study. 4th edn. CP
made from them. J Indian Microbiol Biotechnol 28:1-6. Kurtzman, JW Fell (eds). Elsevier, Amsterdam, the Netherlands. 77-100
57. Sourabh A, Kanwar SS, Sharma OP (2011) Screening of indigenous https://doi.org/10.1016/B978-044481312-1/50014-9
yeast isolates obtained from traditional fermented foods of Western 70. Yongchen Zheng, Yingli Lu, Jinfeng Wang, Longfei Yang, Chenyu
Himalayas for probiotic attributes. J Yeast Fungal Res 2(8):117-126. Pan, Ying Huang (2013) Probiotic properties of Lactobacillus Strains
http://www.academicjournals.org/JYFR Isolated from Tibetan Kefir Grains. Plos One 8(7): e69868
58. Sourabh A, Kanwar SS, Sharma OP (2012) In vitro characterization Zanello G, Meurens F, Berri M, Salmon H (2009) Saccharomyces bou-
of Saccharomyces cerevisiae HM535662 obtained from an indigenous lardii effects on gastrointestinal diseases. Curr Issues Mol Biol 11: 47-58.
fermented food Bhaturu of Western Himalayas. African J Biotechnol Zhou JZ, Liu XL, Jiang HH, Dong MS (2009) Analysis of the microflora
11(52):11447-11454. http://www.academicjournals.org/AJB in Tibetan kefir grains using denaturing gradient gel electrophoresis.
Food Microbiol 26: 770-775.

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