production of folate in oat bran fermentation by yeasts isolated from barley and diverse foods

ORIGINAL ARTICLE

Production of folate in oat bran fermentation by yeastsisolated from barley and diverse foodsM. Korhola1, R. Hakonen1, K. Juuti1, M. Edelmann2, S. Kariluoto2, L. Nystr€om2,*, T. Sontag-Strohm2

and V. Piironen2

1 Department of Biosciences, University of Helsinki, Helsinki, Finland

2 Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland

Keywords

barley, folates, lactic acid bacteria, oat bran,

yeast.

Correspondence

Matti Korhola, Department of Biosciences,

University of Helsinki, P.O. Box 56 (Viikinkaari

9), FIN-00014 Helsinki, Finland.

E-mail: [email protected]

*Current address: Institute of Food, Nutrition

and Health, ETH Zurich, Schmelzbergstrasse

9, CH-8092, Zurich, Switzerland

2013/2421: received 3 December 2013,

revised 29 May 2014 and accepted 3 June

2014

doi:10.1111/jam.12564

Abstract

Aims: The focus of the research was to identify yeasts from barley kernels in

order to study their folate production capability while maintaining high

viscosity caused by soluble fibres in oat bran fermentation.

Methods and Results: The 65 isolated yeasts were characterized by API

carbohydrate utilization tests, and assays for extracellular enzyme activities

were the following: amylase, beta-glucanase, cellulase or CMCase, lipase,

protease and xylanase. Yeasts were identified by partial DNA sequencing of

the 25S D1/D2 and ITS1-5.8S-ITS2 regions. They belonged to the genera

Aureobasidium, Cryptococcus, Pseudozyma and Rhodotorula. Folate production

was determined from supernatant and cells grown in a rich laboratory medium

or directly from oat bran solution inoculated with the appropriate yeast. Food

yeasts, Saccharomyces cerevisiae, Candida milleri, Kluyveromyces marxianus and

Galactomyces geotrichum, were used for comparison. Most of the yeasts isolated

from barley destroyed the solid, viscous structure of the oat bran solution,

indicating that they degraded the viscosity-generating soluble fibres, considered

to be nutritionally advantageous. The best folate producers were S. cerevisiae,

followed by Pseudozyma sp., Rhodotorula glutinis and K. marxianus. The yeasts

maintaining high viscosity were used together with lactic acid bacteria (LAB)

Streptococcus thermophilus or Lactobacillus rhamnosus to ferment oat bran

solution. None of the yeasts isolated from barley, contrary to S. cerevisiae and

C. milleri, produced together with LAB significant amounts of folate.

Conclusions: Fermentative yeasts together with LAB are potential for use in

developing novel high folate content healthy foods and snacks from oat bran.

Significance and Impact of the Study: High soluble fibre content and high

natural folate content but low energy content food and snack products with

pleasant fermentation aroma provide possibilities for new developments in the

food industry.

Introduction

Folate (Vitamin B9) is a generic name for a number of

derivatives of pteroylglutamic acid (folic acid) and is nec-

essary for methylation reactions in cell metabolism and for

neural development of foetus during pregnancy. Natural

dietary folates are mostly reduced folates, i.e. derivatives of

tetrahydrofolate.

Folate is obtained especially from cereal foods (Kariluoto

et al. 2004), fruits, dairy products and vegetables

(J€agerstad et al. 2005). Yeasts are a rich source of folate

(Hjortmo et al. 2005; Patring et al. 2006). However, the

diversity of yeasts studied was rather limited: mostly

Saccharomyces spp., some representatives of Candida,

Debaryomyces, Kodamea, Metchnikowia, Wickerhamiella and

a few unidentified yeasts. Physiological growth conditions

Journal of Applied Microbiology 117, 679--689 © 2014 The Society for Applied Microbiology 679

Journal of Applied Microbiology ISSN 1364-5072

and culture medium composition affect the folate contents

of Saccharomyces cerevisiae—minimal medium and respiro-

fermentative growth at high growth rate gave the highest

folate contents (Hjortmo et al. 2007). Clearly, there is a

need to expand the realm of studies on yeasts for folate

production—almost 150 genera and 1500 species of yeasts

are currently known (Kurtzman et al. 2011).

Recently, the European Food and Safety Authority has

stated that ‘Regular consumption of beta-glucans

contributes to maintenance of normal blood cholesterol

concentrations’ with daily consumption of at least 3 g of

b-glucan from nonprocessed or minimally processed

products (EFSA 2009), and specifically, ‘Oat beta-glucan

has been shown to lower/reduce blood cholesterol. Blood

cholesterol lowering may reduce the risk of (coronary)

heart disease’ when consumed at least 3 g of b-glucanper day in a balanced diet (EFSA 2010).

Yeasts have previously been isolated from barley grains,

barley malt or the malting process representing 13 differ-

ent genera (Noots et al. 1998 and references therein) or

16 different genera (Laitila et al. 2006). The most com-

monly found genera in barley grains included Aureobasid-

ium, Bulleromyces, Candida, Cryptococcus, Filobasidium,

Galactomyces (Geotrichum), Rhodotorula and Sporobol-

omyces. Many of these and other yeasts can make extra-

cellular enzymes, such as b-glucanase and amylases (De

Mot 1990; Strauss et al. 2001).

We have previously shown that certain bacteria isolated

from oat bran or rye flakes (Herranen et al. 2010;

Kariluoto et al. 2010) or found in fermenting rye sour

dough (Kariluoto et al. 2006) are able to synthesize sig-

nificant amounts of folate in rich medium. In the current

study, we isolated yeasts from barley kernels and investi-

gated their ability to synthesize folate in pure culture

alone in rich laboratory medium and in oat bran solution

or in combination with lactic acid bacteria (LAB) in oat

bran solution. One of the LABs was Streptococcus thermo-

philus known to produce folate and the other Lactobacil-

lus rhamnosus unable to synthesize folate (Crittenden

et al. 2003; Sybesma et al. 2003; for a review on LABs

and folate, see Rossi et al. 2011). The objective of this

study was to test whether growth or fermentation by pure

cultures of yeasts alone or together with LABs in oat bran

solution is a feasible approach for increasing food folate

level while maintaining the high viscosity of the product.

Materials and methods

Isolation of yeasts from barley grains and from diverse

foods

The barley Hordeum vulgare, variety Minttu, kernels were

ground by scarification for 20 s periods with an abrasive

mill to five fractions. Whole kernels were soaked for 3 h

in 0�9% NaCl, and each ground fraction was homoge-

nized in stomacher blender for 30 min. Yeasts were

isolated by plating direct and diluted samples on rich

medium (YPD or malt extract agar), with antibiotics

chlortetracycline and chloramphenicol (at 0�01% each)

preventing bacterial growth and with added Triton X-100

(at 0�02%) preventing fungal growth. The plates were

incubated at 18 or 28°C for 4–20 days. Yeast colonies

were picked, purified by restreaking, observed microscop-

ically and tested for carbohydrate utilization using API

32C test strips (BioMerieux Inc., Marcy l’Etoile, France).

The food yeasts were isolated by direct plating on

YPD agar plates. ABM4949 and ABM5103 were isolated

from commercial rye sour dough starter, ABM5031 from

spontaneously fermented apple cider, ABM5032 from spon-

taneously fermented lingonberry jam, ABM5099 from

fermenting soya feed, ABM5102 from food laboratory

air, ABM5130 and ABM5131 from domestic kefir

grains, ABM5136 from fermented milk product viili and

ABM5147 from fermented oat product. Saccharomyces cere-

visiae ALKO743 was used as reference yeast—originally a

commercial baker’s yeast (Cod�on et al. 1998).

DNA extraction, PCR amplification and sequencing of

rDNA coding for 25S D1/D2 and ITS1-5.8S-ITS2 regions

Representative and the control food yeasts were identified

by partial rDNA sequence analysis by utilizing the PCR

primers NL1-NL4 and ITS4-ITS5 described in Kurtzman

and Robnett (2003). Genomic DNA was isolated from

2 days grown YPD shake-flask cultures using Wizard

Genomic DNA Purification Kit (Promega Ltd., Essex,

UK) according to manufacturer’s instructions. The cells

were broken by lyticase (Sigma L2524, Sigma Chemical

Co., St. Louis, MO) treatment or cultures which were

resistant to enzymatic hydrolysis by vigorously shaking

for 3 min with glass beads. Universal

PCR primers NL-1 (50-ATATCAATAAGCGGAGGAAAAG-30) and NL-4 (50-GGTCCGTGTTTCAAGACGG-30)were used to amplify a 0�6 kb fragment of 25S D1/D2

region; ITS-4 (50-TCCTCCGCTTATTGATATGC-30) and

ITS-5 (50-GGAAGTAAAAGTCGTAACAAGG-30) were

used to amplify a 0�4–0�8 kb fragment of ITS1-5.8S-ITS2

rDNA region (Kurtzman and Robnett 2003). Each 50-llPCR reaction contained 0�5 lmol l�1 of each primer,

200 lmol l�1 dNTPs, 1 U DyNAzyme II DNA polymerase

(Finnzymes Oy, Espoo, Finland), 1 9 PCR buffer

(10 mmol l�1 Tris-HCl, pH 8�8, 1�5 mmol l�1 MgCl2,

50 mmol l�1 KCl, 0�1% Triton X-100) and 1 ll of

genomic DNA as a template. The amplifications were per-

formed in GeneAmp PCR System 2700 thermocycler

(Applied Biosystems, Foster City, CA) with the following

Journal of Applied Microbiology 117, 679--689 © 2014 The Society for Applied Microbiology680

Production of folate in oat bran fermentation by yeasts M. Korhola et al.

parameters: an initial denaturation step at 94°C for 5 min,

followed by 36 cycles at 94°C for 1 min, 52°C for 1 min and

72°C for 2 min and then a final extension step at 72°C for

10 min. The PCR products were then partially sequenced

using primers NL-1, NL-4, ITS-4 and ITS-5 in combination

with BigDye Terminator Cycle Sequencing Kit and

ABI3130XL Genetic Analyser (Applied Biosystems). The

nucleotide sequences were checked and edited with CHROMAS

LITE SOFTWARE (ver. 2.01; Technelysium Pty Ltd., South Bris-

bane, Australia) and compared against the sequences in the

National Centre for Biotechnology Information (NCBI) nr-

database using the BLASTN programme. The closest strain

level match (% identity) was considered as the identifica-

tion. The sequence data has been deposited in the EMBL

NUCLEOTIDE SEQUENCE DATABASE (www.ebi.ac.uk) under acces-

sion numbers HG532066–HG532115.

Screening for extracellular hydrolytic enzymes

The ability of yeasts to excrete hydrolytic enzymes was

studied by substrate hydrolysis plate assay method as

described in Herranen et al. (2010). Yeast strains were

first grown on YPD plates (1% yeast extract, 2% tryp-

tone, 2% glucose, 2% agar) for 2 days at 28°C. The yeastswere then streaked on enzyme assay plates and incubated

at 28°C for 3 days. The plates for amylolytic, cellulolytic,

xylanolytic, beta-glucanase or protease activities were glu-

cose-free PCA plates (PCA-G; 0�5% casein peptone,

0�25% yeast extract, 1�5% agar) supplemented with 0�5%(w/v) soluble starch (Merck), 0�5% carboxymethylcellu-

lose (Sigma), 1% xylan (from oat spelt, Sigma), 0�2% oat

beta-glucan or 30% (v/v) skim milk as a substrate,

respectively. The plates for amylolytic activity were

stained with Lugol solution, while those for cellulose,

xylanase or beta-glucanase were stained with 0�2% Congo

Red. Lipolytic activity was determined with modified

Sierra lipolysis agar containing 10 g tryptone, 5 g NaCl,

0�1 g CaCl2, 3 g meat extract, 0�2 g ferric citrate, 15 g

agar and 10 ml of Tween 80 l�1. The distance from the

margin of the colony to the rim of the hydrolysis zone

was measured, and the enzyme activity was expressed as a

function of the distance as follows: + <0�5 cm, ++ 0�5 to

1 cm, +++ 1 to 1�5 cm and ++++ >1�5 cm.

Analysis of total folate

Yeast strains were precultured in 10 ml of YPD broth

overnight at 28°C with agitation at 180 rev min�1. The

overnight cultures were used to inoculate fresh YPD

medium to 5–10 Klett60 units, and the cultures were grown

with agitation at 28°C. 30-ml samples were withdrawn at

the stationary (24–41 h) growth phase. The cell wet weight

(approx. 20% dry weight) yield for different yeasts varied

in the range of 0�4–2�5 g. Cells were harvested by

centrifugation at 4000 9 g for 15 min at room tempera-

ture and washed once with sterile phosphate-buffered sal-

ine (pH 7�1). The supernatants were filtered through 0�45-lm membrane filters (Sarstedt, N€umbrecht, Germany).

Both the cells and supernatants were flushed with nitrogen

gas and stored at 20°C for further analysis.

Total folate contents were determined by a microbiologi-

cal assay on microtiter plates using L. rhamnosus ATCC

7469 as the growth indicator organism (Kariluoto et al.

2004). The sample preparation procedure included heat

extraction followed by deconjugation of folate polygluta-

mates by hog kidney conjugase and treatments with amylase

and protease to liberate folate from the matrix. Method per-

formance was confirmed by analysing a blank sample as well

as certified reference material CRM 121 (wholemeal flour)

or in-house reference in each set of samples.

Oat bran fermentations

Commercial oat bran product OatWell 14% (Swedish

Oat Fiber AB, Bua, Sweden; Herranen et al. 2010) at

3�5% concentration with or without 2% glucose in water

was boiled for 10 min, sterilized by autoclaving at 120°C,divided into 50-ml aliquots in plastic minigrip bags or

Sarstedt tubes and after cooling inoculated with 1 ml of

LAB or yeast overnight culture in YPD. In cases

where LAB (Streptococcus thermophilus ABM5097 or

Lactobacillus rhamnosus LC-705) and yeast were inocu-

lated together, the volume of each culture was 0�5 ml. To

mix the contents, the plastic bags were kneaded by hand,

and the tubes were capped, mixed by inverting 10 times,

then the caps were loosened and incubated at 28°C for 1

or more days. Samples for viable count determinations

on YPD spread plates and for viscosity measurements

were taken after finishing the experiment and mixing the

bag or tube contents as above.

Viscosity determinations of oat bran solution samples

Viscosity properties of the oat bran samples were charac-

terized using a ThermoHaake RheoStress 600 rheometer

(Thermo Electron GmbH, Dreieich, Germany). A flow

curve was obtained using a cone and plate geometry

(35 mm, 2°) over a shear rate range of 0�3–300–0�3 s�1.

All the rheological experiments were performed at 20°C.

Results

Identification of yeasts

We have isolated from different fractions of barley

kernels altogether 65 pure cultures of yeasts. The viable


M. Korhola et al. Production of folate in oat bran fermentation by yeasts

count of yeasts from whole kernels was 4 9 104 per gram

when incubated at 18°C and 6 9 103 per gram at 28°C.In the different kernel fractions, yeast viable count when

incubated at 18°C varied from 3 9 103 to 4 9 105 g�1

flour and when incubated at 28°C from zero to

2 9 103 g�1. Most of the isolates were first characterized

by observations on colony and cell morphology. API 32C

carbohydrate utilization tests were made for selected

strains isolated from barley grains and for the food yeasts,

and a tentative identification based on API reference

database comparison was made (data not shown). The

barley isolates were assigned to various species in the

genera Candida, Cryptococcus and Rhodotorula and food

yeasts to Candida, Geotrichum and Saccharomyces.

A portion of the isolates was identified by partial

DNA sequencing of the 25S D1/D2 and ITS1-5.8S-ITS2

regions. The identified yeasts (Table 1) isolated from

barley grains belonged to the genera Aureobasidium,

Cryptococcus, Pseudozyma and Rhodotorula, all of which

are assimilative, nonfermentative yeasts. The food yeasts

belonged to the genera Candida, Clavispora, Galactomy-

ces, Kluyveromyces, Pichia, Rhodotorula and Saccharomy-

ces (Table 1). Strain R59 was not sequenced for the D1/

D2 region. ALKO 743 produced overlapping sequences

for the ITS1-5.8S-ITS2 region, indicating that the region

is heterozygous (data not shown). The tentative overall

identification (Table 1) is based on the fact that ITS1-

5.8S-ITS2 region is more discriminatory than D1/D2

region at species level (Kurtzman and Robnett 2003).

Plate assays for hydrolytic enzyme activities amylase,

beta-glucanase, cellulase or CMCase, lipase, protease, and

xylanase showed that the strains exhibited relatively few

Table 1 Identification of yeast strains by DNA sequencing. More than one species is listed if the scores were (nearly) identical

Strain

25S D1/D2

identity Species in NCBI database

ITS1-5.8S-ITS2

identity Species in NCBI database Tentative overall identification

Barley yeasts

R38 556/556 Aureobasidium pullulans/

Kabatiella microsticta

562/562 Aureobasidium pullulans Aureobasidium pullulans

R124 555/555 Kabatiella microsticta 607/607 Aureobasidium pullulans Aureobasidium pullulans

R43 564/564 Cryptococcus adeliensis 542/542 Cryptococcus adeliensis Cryptococcus adeliensis



R133 607/609 Cryptococcus sp. 586/586 Cryptococcus sp. Cryptococcus sp.

R46 564/564 Cryptococcus sp. 566/570 Cryptococcus sp. Cryptococcus sp.

R134 619/619 Cryptococcus laurentii 530/530 Cryptococcus laurentii Cryptococcus laurentii

R59 ND 567/567 Cryptococcus magnus Cryptococcus magnus

R47 638/638 Pseudozyma sp./

Moesziomyces bullatus

683/686 Pseudozyma sp./

Moesziomyces bullatus

Pseudozyma sp.

R45 559/559 Rhodotorula glutinis/

graminis/Rhodosporidium

babjevae

574/574 Rhodotorula glutinis Rhodotorula glutinis

R48 559/559 Rhodotorula glutinis/graminis/

Rhodosporidium babjevae


R63 559/559 Rhodotorula glutinis/graminis/

Rhodosporidium babjevae


R132 564/564 Rhodotorula graminis 585/585 Rhodotorula glutinis Rhodotorula glutinis

R106 583/583 Rhodotorula laryngis 571/571 Rhodotorula laryngis Rhodotorula laryngis

Food yeasts

ABM4949 603/604 Candida humilis 593/599 Candida humilis Candida milleria

ABM5099 603/604 Candida humilis 621/633 Candida humilis Candida milleria

ABM5147 556/561 Clavispora lusitaniae 373/373 Clavispora lusitaniae Clavispora lusitaniae

ABM5136 538/540 Galactomyces geotrichum 347/347 Galactomyces sp. Galactomyces geotrichum

ABM5032 539/539 Kluyveromyces marxianus 678/679 Kluyveromyces marxianus Kluyveromyces marxianus

ABM5130 511/511 Kluyveromyces marxianus 584/585 Kluyveromyces marxianus Kluyveromyces marxianus

ABM5031 560/562 Saccharomyces cerevisiae 441/442 Pichia membranifaciens Pichia membranifaciens

ABM5102 582/582 Rhodotorula pinicola 517/517 Rhodotorula pinicola Rhodotorula pinicola

ABM5103 566/566 Saccharomyces cerevisiae 761/766 Saccharomyces cerevisiae Saccharomyces cerevisiae

ABM5131 591/595 Saccharomyces cerevisiae 826/831 Saccharomyces cerevisiae Saccharomyces cerevisiae

ALKO743 566/566 Saccharomyces cerevisiae ND Saccharomyces cerevisiae

ND, not determined due to mixed peaks in electropherograms.aSuc+ phenotype was taken into account.



activities except for Aureobasidium pullulans which had

all the assayed six activities (Table 2). All the Cryptococcus

as well as Rhodotorula species isolated from barley had

lipase activity and Cryptococcus laurentii and

Cryptococcus. magnus in addition cellulase and the latter

also had protease activity. Pseudozyma sp. exhibited both

amylase and protease activity. Rhodotorula minuta and

R. pinicola both had good b-glucanase activity, while

some of the yeasts (Table 2) made a deep red precipitate

without a halo which may mean some unknown modifi-

cation of the b-glucan substrate.

Folate production and bran viscosity

Folate production by the yeasts and LAB were studied

under conditions which might be applicable to large scale

industrial production. For potential commercial product

development purposes, the preservation of the oat bran

matrix viscosity was viewed as one of the key characteristics.

Folate production, assayed by a microbiological

method, was determined from culture supernatant and

cells grown in a laboratory medium inoculated with the

appropriate yeast or LAB (Fig. 1). Folate contents in the

culture supernatant after subtracting the un-inoculated

control value (YPD 120 ng ml�1) varied from 65 to

230 ng ml�1 or 2–7 lg per 30 ml cultivation (Fig. 1a).

The highest amounts of folate were made by Pseudozyma

sp. R47, Aureobasidium pullulans R38 and Rhodotorula

glutinis R48. Cell biomass from the 30-ml cultivation

amounted on average to 2 g wet weight. Cell-bound

folate assays showed (Fig. 1b) that baker’s yeast

ALKO743 made over 14 lg g�1, Pseudozyma sp. R47 over

12 lg g�1, Rhodotorula glutinis R48 about 11 lg g�1 and

Kluyveromyces marxianus ABM5130 about 9 lg g�1.

Aureobasidium pullulans R38 was among the poor folate

producers with 4 lg g�1, while Rhodotorula laryngis R106

was the worst at 1 lg g�1. When calculated for the total

amount of folate made in the 30-ml cultivations, the best

overall folate producers were baker’s yeast Saccharomyces

cerevisiae ALKO743 at 34 lg, Pseudozyma sp. R47 at

32 lg followed by Rhodotorula glutinis R48 at 26 lg and

Kluyveromyces marxianus ABM5130 at 25 lg. Folate in

the biomass contributed about 78–94% of total folate.

The worst folate producer was Rhodotorula laryngis

R106 at 4 lg total folate. The bacterium Lactobacillus

rhamnosus LC-705 did not make folate, but Streptococcus

thermophilus made a significant amount of cell-bound

folate (Fig. 1b).

Table 2 Production of extracellular hydrolytic enzymes by yeast strains isolated from barley kernels and diverse foodstuffs

Isolate Identification Amylase Cellulase Xylanase Beta-glucanase Protease Lipase

Barley yeasts

R38 Aureobasidium pullulans ++ ++ + + ++++ ++

R124 Aureobasidium pullulans +++ ++++ +++ + ++++ ++

R43 Cryptococcus albidus � � � � � ++

R76 Cryptococcus albidus � � � � � +++

R78 Cryptococcus albidus � � � � � +++

R46 Cryptococcus laurentii � ++ � � � +

R59 Cryptococcus magnus � ++ � � + +++

R47 Pseudozyma sp. + � � (+)a ++ �R45 Rhodotorula glutinis � � � � � ++

R48 Rhodotorula glutinis � � � � � ++

R63 Rhodotorula glutinis � � � � + ++

R106 Rhodotorula laryngis � � � +++ � +

Food yeasts

ABM4949 Candida milleri � � � (++)a � �ABM5099 Candida milleri � � � (++++)a � �ABM5147 Clavispora lusitaniae � � � � � �ABM5136 Galactomyces geotrichum � � � (++)a � �ABM5032 Kluyveromyces marxianus � � � � � �ABM5130 Kluyveromyces marxianus � � � � � �ABM5031 Pichia membranifaciens � � � (+++)a � �ABM5102 Rhodotorula pinicola � � � ++ � �ABM5103 Saccharomyces cerevisiae � � � � � �ABM5131 Saccharomyces cerevisiae � � � � � �ALKO743 Saccharomyces cerevisiae � � � � � �

�, No activity; +, low activity; ++, moderate activity; +++, high activity; ++++, very high activity.

Enzyme activity is expressed relatively as a function of the size of the clearing zone as described in the Materials and methods.aAlthough b-glucanase negative (no clearing zone), the colonies were surrounded by a deep red halo.



Next, the yeasts were inoculated into 50 ml of 3�5% oat

bran solution and incubated for 3 days after which folate

content (data not shown) and viscosity of each sample

was determined. Most of the yeasts isolated from barley

destroyed the solid, viscous structure of the oat bran solu-

tion (Fig. 2), indicating that they degraded the viscosity-

generating soluble fibres. Control oat bran solution had a

thick yoghurt-like viscosity, but at values especially below

50% of control, the yeast-containing samples were very

watery. Some samples showed increased viscosity com-

pared with the control—it remains to be determined

whether that was due to the characteristics of the particu-

lar yeasts S. cerevisiae ABM5103, C. milleri ABM4949,

Cryptococcus sp. R133 and C. laurentii R134.

When some of the best folate-producing yeasts were

incubated alone or together with LAB in oat bran solu-

tion, only C. milleri ABM4949, S. cerevisiae ALKO743,

ABM5103 and ABM5131 made significant amounts of

folates (Fig. 3). There was not much effect on folate

production by the yeasts isolated from barley whether

glucose was added or not, but the fermentative yeast

S. cerevisiae ABM5131 responded to sugar addition

(Fig. 3a). In subsequent experiments, 2% glucose was

added to the oat bran solution (Fig. 3b). Yeast and

L. rhamnosus LC-705 viable count values indicated that

the microbes had grown in the oat bran solution

samples—in the presence of glucose yeasts about 10–20-fold and L. rhamnosus LC-705 about 50-fold. Growth

0 50 100 150 200 250

Lactobacillus rhamnosus LC-705Streptococcus thermophilus ABM5097

Pseudozyma sp. R47Aureobasidium pullulans R38

Rhodotorula glutinis R48Cryptococcus laurentii R134

Cryptococcus sp. R133Rhodotorula glutinis R132

Cryptococcus adeliensis R78Cryptococcus adeliensis R43

Aureobasidium pullulans R124Kluyveromyces marxianus ABM5130

Cryptococcus adeliensis R76Rhodotorula laryngis R106

Saccharomyces cerevisiae ALKO743Rhodotorula pinicola ABM5102

Cryptococcus magnus R59Saccharomyces cerevisiae ABM5103

Pichia membranifaciens ABM5031Candida milleri ABM5099Candida milleri ABM4949

Clavispora lusitaniae ABM5147Saccharomyces cerevisiae ABM5131

Folates, ng ml–1

0 2 4 6 8 10 12 14 16 18

Lactobacillus rhamnosus LC-705Streptococcus thermophilus ABM5097

Saccharomyces cerevisiae ALKO743Pseudozyma sp. R47

Rhodotorula glutinis R48Kluyveromyces marxianus ABM5130Saccharomyces cerevisiae ABM5103

Candida milleri ABM4949Candida milleri ABM5099

Clavispora lusitaniae ABM5147Pichia membranifaciens ABM5031

Aureobasidium pullulans R124Cryptococcus adeliensis R78

Saccharomyces cerevisiae ABM5131Cryptococcus adeliensis R76Cryptococcus adeliensis R43

Cryptococcus magnus R59Aureobasidium pullulans R38

Rhodotorula pinicola ABM5102Cryptococcus sp. R133

Cryptococcus laurentii R134Rhodotorula glutinis R63

Rhodotorula glutinis R132Rhodotorula laryngis R106

Folates, µg g–1

(a)

(b)

Figure 1 Production of folates in rich YPD

medium by the yeasts in 24 h shake-flask

cultures and by two lactic acid bacteria in

3 days static anaerobic cultures. (a) Folates

produced by the micro-organisms into the

culture supernatants (un-inoculated YPD-

medium folates 120 ng ml�1 have been

deducted from each value). (b) Folates found

in wet weight cell mass. Error bars represent

the range.



without glucose was poor—viable counts increased only

2–7-fold.Viscosity determinations showed that many oat bran

samples had lost their viscosity partly or completely. All

the samples containing yeasts isolated from barley kernels

(R38, R47, R48) were either watery or of low viscosity

(data not shown), but most of the samples with yeasts

isolated from diverse foods had retained their high vis-

cosity (Fig. 4). However, viscosity of the oat bran solu-

tion was partially destroyed by the S. cerevisiae ABM5131

and completely destroyed by Galactomyces geotrichum

ABM5136 (Fig. 4).

0

R47 R38 R43R76

*R78

R106

R59

5136

*74

3*R12

4R63

5031

*51

3051

4751

31R13

2

5103

*

4949

*R13

3R13

4R48

50

100

150

200

250

Vis

cosi

ty (

% c

ompa

red

to c

ontr

ol)

Figure 2 Viscosity of 3�5% oat bran solution

after 72 h incubation with different yeasts

compared with un-inoculated control. The

values marked with an asterisk are means of

two independent samples.

0

Contro

l51

31 R38 R47 R48

LC-7

0550

97

5131

& L

C-705

5131

& 5

097

R38 &

LC-7

05

R38 &

509

7

R47 &

LC-7

05

R47 &

509

7

R48 &

LC-7

05

R48 &

509

7

20

40

60

80

100

120

140

Fol

ates

, ng

g–1

Contro

l

LC-7

05

743

& 509

7

5103

& 5

097

5131

& 5

097

5136

& 5

097

743

& LC-7

05

5147

& L

C-705

5136

& L

C-705

5131

& L

C-705

5103

& L

C-705

4949

& L

C-705

5147

& 5

097

4949

& 5

097

5097

5147

5136

5131

5103

494974

30

20

40

60

80

100

120

140

Fol

ates

, ng

g–1

(a)

(b)

Figure 3 Folate production ng per gram in

3�5% oat bran solution yeast fermentation

and together with lactic acid bacteria

Lactobacillus rhamnosus LC-705 and

Streptococcus thermophilus ABM 5097. (a)

No added glucose ( ) or with 2% added

glucose ( ); Yeasts isolated from barley

kernels: Aureobasidium pullulans R38,

Pseudozyma sp. R47, Rhodotorula glutinis

R48; Saccharomyces cerevisiae ABM 5131

was used as control. (b) With 2% added

glucose( ); Yeasts isolated from diverse

foodstuffs: Candida milleri ABM 4949,

Clavispora lusitaniae ABM 5147,

Galactomyces geotrichum ABM 5136,

Saccharomyces cerevisiae ABM 5103, ABM

5131, ALKO 743.



Discussion

Yeasts previously isolated from barley kernels have been

found to belong to ascomycetous genera Aureobasidium,

Candida, Debaryomyces, Geotrichum, Hansenula, Kloeckera,

Saccharomyces, Torulopsis, Williopsis and to basidio-

mycetous genera Bulleromyces, Cryptococcus, Filobasidium,

Rhodotorula, Sporobolomyces and Trichosporon (Noots et al.

1998; Laitila et al. 2006). We found representatives of

mostly basidiomycetous yeasts in our barley kernel samples

(Table 1). The total yeast colony counts 6 9 103–4 9 104 g�1 were in close agreement to those found earlier

—5 9 102–4�4 9 103 g�1 (Tuomi et al. 1995), 7 9 104–2 9 105 g�1 (Laitila et al. 2006) and 1�9–4�7 9 103 g�1

(Petters et al. 1988). We did not observe any significant

difference in the yeast counts between surface and deeper

layers contrary to Laca et al. (2006) who found higher cell

counts at the surface layers compared with deeper layers in

the grain.

As a new yeast in barley, we found a representative of

still another basidiomycetous genus Pseudozyma; how-

ever, identification to the species level needs confirmation

by other criteria.

The yeasts isolated from diverse foods were identified

by rDNA sequencing (Table 1) to genera and species

often found in the corresponding matrixes. Rye sour

dough is known to contain mainly Candida milleri, but

Saccharomyces cerevisiae or Saccharomyces exiguus yeasts

are also often present (M€antynen et al. 1999). Candida

humilis has been reported to be dominant in wheat sour

dough (Gullo et al. 2002). We found that both C. milleri

ABM4949 and S. cerevisiae ABM5103 were present in the

same rye sour dough sample. Candida milleri ABM5099

was found in spontaneously fermenting soya bean process-

ing feed product, which contained also LAB and thus

resembled sour dough fermentation. Kefir is known to

contain Brettanomyces anomalus, Candida holmii, C. incon-

spicua, C. krusei, C. lipolytica, C. lambica, C. maris,

Cryptococcus humicolus, Geotrichum candidum (teleomorph

Galactomyces geotrichum), Kluyveromyces marxianus

(anamorph C. kefyr), Pichia fermentans, Saccharomyces

cerevisiae, S. exiguus, S. humaticus, S. turicensis, S. unispo-

rus, Torulaspora delbrueckii and Zygosaccharomyces sp.

yeasts depending on the origin of the kefir (Wyder et al.

1997, 1999; Simova et al. 2002; Witthuhn et al. 2004,

2005; Latorre-Garcia et al. 2007; Wang et al. 2008). Our

kefir sample contained both K. marxianus ABM5130 and

S. cerevisiae ABM5131 as the dominant species. Finnish

viili contained Galactomyces geotrichum ABM5136, but the

anamorph name Geotrichum candidum only is used by the

commercial producers (Merilainen 1984). Spontaneously

fermented apple cider contained Pichia membranifaciens

ABM5031, which species has also been found in pilot fer-

mentations and in cider plant must in Spain (Cabranes

et al. 1990) as well as in tequila fermentation (Lachance

1994). Spontaneously fermenting lingonberry jam con-

tained Kluyveromyces marxianus ABM5032 which was

unexpected, as lingonberry juice is difficult to ferment

due to high content of benzoic acid (Visti et al. 2003),

and Kluyveromyces yeasts are not particularly resistant

to benzoic acid (Warth 1988). A commercial fermented

oat product contained the yeast Clavispora lusitaniae

ABM5147 which seemed to be moderately fermentative

in our tests with glucose and grew in the API32C tests

well on rhamnose, which latter characteristic earlier has

been considered as a potential diagnostic test for this

0

100

200

300

400

500

600

700A

ppar

ent v

isco

sity

mP

as a

t 10

1/s

Contro

l

LC-7

05

743

& 509

7

5103

& 5

097

5131

& 5

097

5136

& 5

097

743

& LC-7

05

5147

& L

C-705

5136

& L

C-705

5131

& L

C-705

5103

& L

C-705

4949

& L

C-705

5147

& 5

097

4949

& 5

097

5097

5147

5136

5131

5103

494974

3Figure 4 Dynamic viscosity of oat bran

solution after 24 ( ) and 72 h ( )

fermentation by yeasts and lactic acid

bacteria alone and in combinations. Error bars

represent the standard deviation.



yeast (Lachance and Phaff 1998). Clavispora lusitaniae,

which has been determined to be one of the dominating

species on agave plants, was not significant in tequila

fermentation (Lachance 1994) but was one of the three

main yeasts in mescal fermentation (Escalante-Minakata

et al. 2008). Clavispora lusitaniae is also involved in

cheese ripening (Kaminarides and Anifantakis 1989;

El-Sharoud et al. 2009) and in whey and carrot-lemon

juice fermentations (Sahota et al. 2010).

The plate tests for extracellular hydrolytic activities

(Table 2) were designed to simulate degradation of the

fermentation matrix components but seem not to be as

sensitive as measurement of loss of viscosity in oat bran

solution (Fig. 2). From control experiments (data not

shown), we know that bacterial a-amylase treatment

decreases the viscosity of oat bran solution by up to

50–80% and treatment with fungal b-glucanase by up to

50–90%. Thus, one would expect that amylases, cellulases

and b-glucanases, if produced by the yeasts, should sig-

nificantly reduce the viscosity of the oat bran solution.

Only A. pullulans R38 and R124 strains had all the tested

hydrolytic enzyme activities (Table 2) in agreement with

recent literature (Li et al. 1993, 2007; Laitila et al. 2006;

Ma et al. 2007; Liu et al. 2008), and reduced viscosity of

3�5% oat bran solution was consistently observed (Fig. 2).

Also Pseudozyma sp. R47 with amylase, C. magnus R59

with cellulase and R. minuta R106 with b-glucanase activ-ity reduced the oat bran solution viscosity. Surprisingly

also C. adeliensis R43, R76 and R78, and R. glutinis R 48

and R63 reduced the oat bran solution viscosity (Fig. 2)

even though the plate tests did not show any amylase,

cellulase or b-glucanase activity (Table 2). However, it is

possible that the physiological conditions on a solid plate

are not as favourable as growth in liquid culture for the

extracellular hydrolytic enzymes production. It is known

that at least one strain of R. glutinis produced an extra-

cellular endo-b-glucanase enzyme (Oikawa et al. 1998)

and one C. adeliensis a xylanase (Scorzetti et al. 2000).

Also the baker’s yeast S. cerevisiae ALKO743 reduced the

viscosity of the oat bran solution but did not show in

plate assays any enzymatic activity. Later work has indi-

cated that the viscosity-reducing activity by ALKO743 is

best expressed in shake-flask cultures with 1–3�5% oat

bran but without added glucose (data not shown).

The yeasts and LAB used in mixed culture fermenta-

tions were chosen in view of possible larger scale applica-

tions under industrially feasible conditions: growth at

28°C, nonaerated fermentation conditions and time of

fermentation 1–3 days.

None of the assimilatory yeasts isolated from barley

kernels made significant amounts of folate either alone or

together with S. thermophilus or L. rhamnosus in oat bran

solution (Fig. 3a). The fermentative yeast S. cerevisiae

ABM5131 made folate when glucose was added to the oat

bran solution. We recently reported results on oat and

barley fermentations by pure cultures of the food yeasts

ALKO743, ABM4949, ABM5131 and ABM5147 (Kariluoto

et al. 2014). Significant increase in folate production was

found with added glucose compared with plain matrix.

S. thermophilus ABM5097 lowered the pH about 0�5 units

less than L. rhamnosus LC-705 and consequently allowed

better growth and folate production by the yeasts

A. pullulans R38 and R. glutinis R48. If a daily dose of oat

bran solution was 200 g similarly to yoghurt, it would

mean that in the most favourable case a folate intake of

20 lg, or 10% of the recommended daily intake. The

average folate content in oat bran solution with the

best producers was 65 ng g�1, similar to the highest

folate concentration 69 ng ml�1 obtained in Tanzanian

fermented maize porridge togwa (Hjortmo et al. 2008).

Many assimilative yeasts, isolated from barley, pro-

duced considerable amounts of folate but destroyed the

solid, viscous structure of the oat bran solution, indicat-

ing that they degraded the viscosity-generating soluble

fibres, considered to be nutritionally advantageous. Many

fermentative food yeasts also produced folate and did not

reduce the viscosity or reduced it less radically (Figs 2

and 4)—they might be useful for further studies aiming

at even higher folate concentrations.

Acknowledgements

This research was funded by the Academy of Finland as

part of the project “FOLAFIBRE—Aqueous processing of

oats and barley: In situ enhancement of folate and associ-

ated bioactive compounds while maintaining soluble

dietary fibre physiologically active”.

Conflict of Interest

No conflict of interest declared.

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