International Journal of Food Microbiology 142 (2010) 277–285

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Isolation and characterization of folate-producing bacteria from oat bran and

rye flakes
Mirkka Herranen a, Susanna Kariluoto b, Minnamari Edelmann b, Vieno Piironen b, Katja Ahvenniemi b,
Vilja Iivonen b, Hannu Salovaara b, Matti Korhola a,⁎
a
Department of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Finland
b
Department of Food and Environmental Sciences, P.O. Box 27, FIN-00014 University of Helsinki, Finland

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this research was to identify endogenous bacteria in commercial oat bran and rye flake products
Received 7 April 2010 in order to study their folate production capability while maintaining the soluble dietary fibre components in
Received in revised form 24 June 2010 physiologically active, unhydrolyzed form.
Accepted 7 July 2010
Fourty-two bacteria were isolated from three different oat bran products and 26 bacteria from one rye flake
consumer product. The bacteria were tentatively identified by sequence analysis of the 16S rRNA genes. The
Keywords:
Endogenous bacteria
identification results revealed up to 18 distinct bacterial species belonging to 13 genera in oat bran, and 11
Cereal foods species belonging to 10 genera in rye flakes. The most common bacterial genus in oat bran was Pantoea,
Oat followed by Acinetobacter, Bacillus, and Staphylococcus. Pantoea species dominated also in rye flakes. The
Rye extracellular enzymatic activities of the isolates were studied by substrate hydrolysis plate assays. Nearly
Beta-glucan 80% of the isolates hydrolyzed carboxymethylcellulose, whereas starch-degrading activities were surpris-
Folate ingly rare (10%). Beta-glucan was hydrolyzed by 19% of the isolates. Protease, lipase or xylanase activity was
expressed by 24%, 29%, and 16%, respectively, of the isolates. Representatives of the genera Bacillus,
Curtobacterium, Pedobacter, and Sanguibacter showed the highest diversity of enzymatic activities, whereas
members of Janthinobacterium and Staphylococcus possessed no hydrolytic activities for the substrates
studied. Production capability for total folates was analyzed from aerobic cell cultures at the stationary
growth phase. The amount of folates was determined separately for the cell mass and the supernatant by
microbiological assay. For comparison, folate production was also examined in a number of common lactic
acid bacteria. The best producers in oat bran belonged to the genera Bacillus, Janthinobacterium, Pantoea, and
Pseudomonas, and those in rye flakes to Chryseobacterium, Erwinia, Plantibacter, and Pseudomonas.
Supernatant folate contents were high for Bacillus, Erwinia, Janthinobacterium, Pseudomonas, and
Sanguibacter. Compared to the endogenous bacteria, lactic acid bacteria were poor folate producers. The
results of this work provide the first insight into the potential role of endogenous microflora in modulating
the nutrient levels of oat and rye based cereal products, and pave way to future innovations of nutritionally
improved cereal foods.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
Cereal brans provide dietary fibre and many associated bioactive
compounds – vitamins and various phytochemicals – into the diet. Oat
is a valuable source of soluble dietary fibre, mostly composed of (1-3),
(1-4)-β-glucan, that has in clinical tests been shown to lower
cholesterol and attenuate blood glucose level (Braaten et al., 1994;
Mäkeläinen et al., 2007). Accordingly, the use of health claims has
been approved for oat products (FDA, 2002; SNF, 2002; JHCI, 2004;
EFSA, 2009). New innovative oat products have been launched in the
market, such as a yogurt-type oat snack and milk-type oat drinks. In
⁎ Corresponding author. Tel.: + 358 9 191 59212; fax: + 358 9 191 59262.
E-mail address: matti.korhola@helsinki.fi (M. Korhola).
the Nordic countries, rye forms another important source of dietary
fibre. It contains arabinoxylan and beta-glucan that are able to
produce a viscous gel. Rye has been shown to decrease cholesterol
levels (Leinonen et al., 2000) and positively affect glucose and insulin
metabolism (Leinonen et al., 1999; Andersson et al., 2010).
In addition to dietary fibre, outer layers of the grains are rich in
bioactive compounds that partly explain the health benefits of cereals.
Cereals are important sources of folate that are needed to prevent
neural tube defects (Katan et al., 2009). Folate is intensively studied
also for other health-promoting activities, for example decreasing risk
of cardiovascular diseases, stroke, some cancers, and cognitive
disorders (Bazzano et al., 2002; Durga et al., 2007; Seshadri et al.,
2002; Wang et al., 2007; World Cancer Research Fund, 2007). Folate is
concentrated in the bran fractions (Arcot et al., 2002; Liukkonen et al.,
2003; Kariluoto et al., 2010). The folate contents of cereal-based

0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.07.002
278 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285

products can be further improved by aqueous processing such as Table 1

fermentation (Liukkonen et al., 2003; Jägerstad et al., 2005). Nutrient values (g/100 g) and processing details of oat bran and rye flake products used
in the present study.
Utilization of fibre-rich fractions demands processes where grain
layers are separated. In addition to cereal fibre and other bioactive Natureal® Regular OatWell® 14%, Rye flakes,
compounds, the majority of cereal-associated bacteria are located in OBCN15, Finn oat bran, Swedish Raisio PLC
Cereal Ltd Raisio PLC Oatfibre
the outer layers of the kernel and are thus concentrated in bran
(ICMFS (International Commission on Microbiological Specifications Proteins 19 17 21 10
Carbohydrates 58 46 34 65
for Foods), 2005; Laca et al., 2006). After harvest cereal grains contain
Sugar 1 1,5 – 2,2
bacteria mainly from the families Pseudomonadaceae, Enterobacter- Fat 10 7,5 4 2
iaceae, Microbacteriaceae, Micrococcaceae, Lactobacillaceae, and Bacil- Saturated fat 1,9 1,2 0,9 0,3
laceae (Legard et al., 1994; ICMFS (International Commission on Dietary fibre 32 17 30 13
Microbiological Specifications for Foods), 2005; Yoshida et al., 2006). Beta-glucan 15 6 14 –
Sodium 0,01 0,0003 6 0,005
Contamination of the grains during storage, transport, and processing
Energy 272 320 267 320
further affects the microflora. The number of microbes declines during (kcal/100 g)
milling but the resulting fractions are often contaminated by bacteria Moisture (%) 9 – 5 –
from the milling equipment (Berghofer et al., 2003). The low water Process Heat- Heat- Heat-treatment, Precut rye groats,
treatment, treatment, treatment with steamed and
activity of the products prevents microbial growth, but bacteria and
dry milling dry milling hot ethanol rolled into flakes
especially their spores can remain viable for long periods. When water
content is increased, the microbes are activated and continue growth
in the presence of versatile carbohydrates, nitrogen sources, vitamins,
and minerals provided by the cereal matrix. If not controlled, the (Seward Medical, UK). The homogenates were serially diluted, and
microbial activity eventually leads to spoilage of the product. 100-μl aliquots were spread plated on replicate plates containing
Hydrolytic enzymes excreted by the microorganisms can induce appropriate nutrient agar medium. Plate count agar (PCA, Merck,
undesired chemical and nutritional changes in colloidal food systems Germany) was used as a non-selective medium for heterotrophic
and destroy their health properties. For example, hydration of beta- bacteria, and the plates were incubated under aerobic conditions at
glucan makes it susceptible to enzymatic hydrolysis, which affects its 21 °C or 37 °C, or under anaerobic conditions at 37 °C, for 1 to 7 days.
physical state, such as solubility and viscosity (Salovaara et al., 2003). MRS agar (Merck) and Clostridium selective agar (Merck) were used
On the other hand, microbes are also known to produce beneficial to search for lactic acid bacteria (LAB) and clostridia, respectively, and
bioactive compounds, such as folate and vitamin B12 (Lin and Young, the plates were incubated anaerobically at 37 °C for 1 to 7 days. Single
2000; Kariluoto et al., 2006; Santos et al., 2008), thus offering a colonies with divergent morphology were picked and restreaked
potential for increasing the nutrient level of the product. Especially in twice on fresh medium to obtain pure cultures. After morphological
countries where no mandatory folate fortification is practiced, good examination (cell and colony morphology, spore and Gram staining),
dietary sources of folate and means to enhance natural folate contents the bacterial strains were stored in 15% glycerol at −70 °C.
by food processing need to be studied. Folate biosynthesis has been Bacillus cereus ABM 5122, ABM 5123, Bacillus sp. ABM 5119
studied mainly in lactic acid bacteria, and seems to depend strongly (isolated from OBCN15), and Pantoea agglomerans ABM 5061 (isolated
on species, strain, growth time, and cultivation conditions (Lin and from rye flour; Kariluoto et al., 2006), were used as control strains in
Young, 2000; Crittenden et al., 2002; Sybesma et al., 2003). the analyses. Measurements of folate production were also per-
Little is known about folate production by bacteria found in cereals formed, as a comparison, with eight LAB strains: Bifidobacterium
but we have previously reported that endogenous bacteria in rye flour bifidum BB-12, Lactobacillus acidophilus LA-5, L. rhamnosus ABM 5029,
were able to synthesize folate in significant amounts (Kariluoto et al., L. rhamnosus ATCC 7469, L. rhamnosus LC-705, Lactococcus lactis
2006). Thus, cereal-associated endogenous bacteria may raise folate HAMBI 2345, L. lactis HAMBI 2348, and Streptococcus thermophilus
levels in fermented fibre-rich products. Utilization of bran fractions in ABM 5097.
the development of nutritionally optimized cereal foods requires
processes where levels of bioactive compounds are enhanced while 2.3. DNA extraction, PCR amplification, and sequencing of 16S
keeping the dietary fibre, mainly beta-glucan, in a physiologically active, rRNA genes
unhydrolyzed form. The objective of this study was to identify bacteria
isolated from oat bran and rye flake products; to study their hydrolytic The bacterial strains were identified by sequence analysis of the
enzyme activities; and to examine their folate production capability. 16S rRNA gene. Genomic DNA was isolated from overnight cultures
using Wizard® Genomic DNA Purification Kit (Promega Ltd., UK)
2. Materials and methods according to manufacturer's instructions. Universal PCR primers pA
(5′-AGAGTTTGATCCTGGCTCAG-3′) and pH′ (5′-AAGGAGGTGATC-
2.1. Cereal samples CAGCCGCA-3′) were used to amplify a 1.5 kb fragment of 16S rDNA
(Edwards et al., 1989). Each 50-μl PCR reaction contained 0.5 μM of
Four cereal products were used as sources for microbe isolation. each primer, 200 μM dNTPs, 1 U DyNAzyme™ II DNA polymerase
These included three different oat bran products, Natureal® OBCN15 (Finnzymes Oy, Finland), 1× PCR buffer (10 mM Tris–HCl, pH 8.8,
(Finn Cereal Ltd., Finland), OatWell® 14% (Swedish Oatfibre, Sweden) 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton® X-100) and 1 μl of genomic
and regular oat bran Sydänystävä kauralese (Raisio PLC, Finland), and DNA as a template. The amplifications were performed in GeneAmp®
one rye flake product, Nalle Ruishiutale (Raisio PLC, Finland). The first PCR System 2700 thermocycler (Applied Biosystems, USA) with the
two samples were ordered directly from manufacturers, the latter two following parameters: an initial denaturation step at 94 °C for 3 min,
were purchased from retailers. Table 1 summarizes the nutrient followed by 2 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 75 s,
values and processing methods of the products. two cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 75 s, 30 cycles
at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 75 s, and then a final
2.2. Isolation of bacteria extension step at 72 °C for 5 min. The PCR products were then
partially sequenced by using primer pD′ (5′-GTATTACCGCGGCTGCTG-
Twenty-five grams of each cereal sample was homogenized in 3′, E. coli position 536-518, Edwards et al., 1989) in combination with
225 ml of sterile 0.9% saline by using Stomacher 400 Lab Blender BigDye® Terminator Cycle Sequencing Kit and ABI3130XL Genetic
 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285
Analyzer (Applied Biosystems). Strains RB9 and NR13 were se-
quenced by using primer pH′ or pA, respectively. The nucleotide
sequences were checked and edited with Chromas Lite software (v.
2.01, Technelysium Pty Ltd., Australia), and compared against the
sequences in the National Centre for Biotechnology Information
(NCBI) nr-database by using the BLASTN programme. The closest
strain level match (% identity) was considered in the identification.
The sequence data has been deposited in the EMBL databank under
accession numbers FN691962–FN691997.
2.4. Screening for extracellular hydrolytic enzymes
The ability of bacteria to excrete hydrolytic enzymes was studied
by substrate hydrolysis plate assay method. Bacterial strains were first
grown on YPD plates (1% yeast extract, 2% tryptone, 2% glucose, 2%
agar) overnight at 28 °C. The bacteria were then streaked on enzyme
assay plates and incubated at 28 °C for 3 days. The presence of
amylolytic enzymes was determined on glucose-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) as the substrate. After
incubation, the plates were flooded with Lugol solution. A clear
zone around the colonies indicated amylase activity. The presence of
cellulolytic, xylanolytic or beta-glucanase activities was screened on
PCA-G plates containing 0.5% carboxymethylcellulose (Sigma, USA),
1% xylan (from oat spelts, Sigma) or 0.2% oat beta-glucan as a
substrate, respectively. The beta-glucan was extracted from oat bran
(OatWell® 14%) as described in Kivelä et al. (2009). After incubation,
the plates were stained with 0.2% Congo red solution. A clear zone
around the growth indicated hydrolytic activity. Proteolytic activity
was monitored by screening for hydrolysis zones around colonies
growing on PCA-G plates containing 30% (v/v) skim milk. 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 per liter. Opaque halos
surrounding the colonies in an otherwise translucent medium
indicated lipase activity. 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;
+ b0.5 cm, ++0.5 to 1 cm, +++1 to 1.5 cm, ++++ N1.5 cm.
2.5. Analysis of total folate
Bacterial strains were pre-cultured in 10 ml of YPD broth
overnight at 28 °C with agitation at 180 rpm. 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. 50-ml samples were
withdrawn at the exponential (100 Klett60 units) and stationary
(24 h) growth phases. Cells were harvested by centrifugation at
8000 ×g for 15 min at 4 °C, and washed once with sterile phosphate-
buffered saline (pH 7.4) at 4 °C. The supernatants were filtered
through 0.45-μm membrane filters (Sarstedt, Nümbrecht, Germany).
Both the cells and supernatants were then stored at −20 °C under
nitrogen gas for further analysis. LAB strains were grown anaerobi-
cally in static YPD broth at 37 °C for 72 h, after which the cells and
supernatants were separated as described above.
Total folate contents were determined by microbiological 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 polyglutamates by hog kidney or chicken pancreas conjugase as
reported previously (Kariluoto et al., 2004; Piironen et al., 2008) with
the exception that treatments with amylase and protease were found
unnecessary for cell and supernatant samples. Method performance
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.
279
Folate values for samples taken at the stationary phase are
averages of two or three individual incubations whereas values for
samples taken at the exponential phase are, in general, based on
single determination. Folate accumulation into the supernatant was
calculated by subtracting folate content in the exponential phase from
the folate content in the stationary phase.
3. Results and discussion
3.1. Enumeration of bacteria in oat bran and rye flakes
Relatively low levels of bacteria were detected in the oat bran and
wholegrain rye flake products in the present study. The highest viable
counts were measured for the rye flakes, 2.1 × 104 cfu/g. The viable
counts of the regular oat bran, oat bran concentrate OBCN15 and
OatWell 14% were 7.7 × 103 cfu/g, 7.0 × 103 cfu/g and 2.5 × 103 cfu/g,
respectively. The values obtained in this study are similar to or
somewhat lower than the aerobic viable count values published in the
literature for rye and wheat flakes (1.1 × 104 cfu/g; ICMFS (Interna-
tional Commission on Microbiological Specifications for Foods),
2005), wheat bran (1 × 104 cfu/g; Berghofer et al., 2003), rye and
wheat bran (4 × 105 − 2.4 × 107 cfu/g; Hesseltine and Graves, 1966)
and peeled and native rye bran (3 × 105 and 5 × 106 cfu/g respectively;
Katina et al., 2007). Bran is often heavily contaminated, partly not only
because it consists of the most heavily contaminated outer layers of
the grain (Laca et al., 2006), but also because it contains fractions from
several areas of the mill (Berghofer et al., 2003).
3.2. Identification of bacteria in oat bran and rye flakes
A total of 68 bacterial strains were isolated and characterized
during the course of this work (Table 2). Twenty-six of the strains
originated from the rye flakes, and the rest were isolated mainly from
OBCN15 (25 isolates) and regular oat bran (16 isolates). The vast
majority of the isolates were picked from PCA plates incubated
aerobically either at 21 °C or 37 °C (60% vs. 37% of the isolates,
respectively). The bacterial diversity was higher among isolates at
21 °C as compared to that at 37 °C; in fact, only 6 of the strains
growing at 37 °C did not have a counterpart on plates at 21 °C
(Table 2). Only two bacteria, one from rye flakes and one from regular
oat bran, were retrieved from plates incubated anaerobically at 37 °C.
Spore-forming ability was observed for three isolates from OBCN15.
Attempts to isolate clostridia or lactic acid bacteria from our samples
were not successful even though these groups of bacteria are
frequently associated with cereals (Hanis et al., 1988; Vogel et al.,
1999).
In order to identify the bacteria, we subjected each strain to partial
sequencing of the 16S rRNA gene, and searched for similar sequences
in the NCBI database. The sequence analysis revealed up to 26 species
belonging to 20 distinct genera in our research material (Table 2). The
most frequently encountered Gram-negative genus in oat bran and
rye flakes was Pantoea, represented by 50% of the isolates. Twenty
isolates (groups ON1 and NR1) showed 99.6–100% sequence identity
to P. ananatis strain isolated from leaf of tea tree. Fourteen isolates
(ON2, ON3, NR2–NR4) were closely related to strains of P. agglom-
erans. Consistent with our results, Yoshida et al. (2006) reported P.
agglomerans and P. ananatis as the dominant species in the culturable
bacterial community on wheat heads. In a study of Legard et al.
(1994), P. agglomerans was likewise described among the most
common bacteria on wheat heads and phyllosphere, whereas P.
ananatis was considered as only a minor colonist. Besides wheat,
Pantoea species have been reported on numerous other hosts such as
rice (Watanabe et al., 1996; Mano and Morisaki, 2008), maize
(Paccola-Meirelles et al., 2001), barley, buckwheat (Coplin and
Kado, 2001) and onions (Gitaitis and Gay, 1997). Although usually
present as a harmless epiphyte, P. ananatis can also cause severe
280 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285

Table 2
Bacteria identified from oat bran and rye flake products by 16 S rDNA sequence analysis.

Isolate ID (acc no.) No. of isolates Closest relative strain in NCBI database (acc no.)a Identity %

OAT BRAN
ON1 (FN691962) 11 Pantoea ananatis strain 8 (GQ497892) 100
ON2 (FN691963, FN691964) 3 Pantoea agglomerans strain EQH21 (FJ999950) 99.1–100
ON3 (FN691965) 4 Pantoea agglomerans strain 48b/90 (FJ756354) 100
ON4 (FN691966) 1 Bacillus subtilis strain L520 (FJ906822) 100
ON5b,d (FN691967) 1 Bacillus subtilis strain ES-311-1 (FN393817) 100
ON6b,d (FN691968) 1 Bacillus licheniformis (AB513628) 99.8
ON7 (FN691969) 1 Curtobacterium sp. IK1_62_2 (AB461038) 100
ON8 (FN691970) 1 Pseudomonas sp. CL1.62 (FM173440) 100
ON9b (FN691971) 1 Enterococcus durans strain SS661 (GQ337025) 100
ON10b,d (FN691972) 1 Paenibacillus cookii (AJ438302) 100
RB1 (FN691973) 7 Acinetobacter calcoaceticus strain petra-09 (GQ141870) 100
RB2 (FN691974) 2 Curtobacterium citreum strain Z10zhy (AM411064) 100
RB3b (FN691975) 1 Exiguobacterium sp. RD3 (EF541141) 99.6
RB4 (FN691976) 1 Janthinobacterium sp. Everest-gws-74 (EU584527) 99.6
RB5 (FN691977) 1 Dietzia papillomatosis strain N 1280 (FJ468340) 99.5
RB6 (FN691978) 1 Micrococcus sp. JAM-AC11 (AB526326) 99.1
RB7 (FN691979) 1 Staphylococcus kloosii strain ATCC 43959 (NR_024667) 100
RB8 (FN691980) 1 Staphylococcus sp. TPL10 (EU373380) 99.8
RB9c (FN691981) 1 Propionibacterium sp. 215(113zx) 100
OW1b (FN691982) 1 Staphylococcus pasteuri strain HNL02 (EU373331) 100

RYE FLAKES
NR1 (FN691983, FN691984, FN691985) 9 Pantoea ananatis strain 8 (GQ497892) 99.6–100
NR2 (FN691986) 3 Pantoea agglomerans strain EhY112-9/86 (FJ756356) 99.8
NR3 (FN691987) 2 Pantoea agglomerans strain BBPE014230 (FJ357810) 100
NR4 (FN691988) 2 Pantoea agglomerans strain 48b/90 (FJ756354) 100
NR5 (FN691989) 2 Sanguibacter sp. Everest-gws-55 (EU584523) 100
NR6 (FN691990) 1 Pseudomonas sp. CZ2 (GQ903481) 100
NR7 (FN691991) 1 Chryseobacterium sp. RI39 (DQ530125) 98
NR8 (FN691992) 1 Agrobacterium tumefaciens strain AT108N (FJ666055) 100
NR9 (FN691993) 1 Plantibacter flavus strain P 297/02 (NR_025462) 100
NR10 (FN691994) 1 Pedobacter terrae strain DS-57 (DQ889723) 97.6
NR11 (FN691995) 1 Erwinia persicina strain LMG 2691 (AJ001190) 99.8
NR12c (FN691996) 1 Bacillus licheniformis (AB513628) 99.8
NR13 (FN691997) 1 Sphingomonas sp. PDD-14b-6 (DQ512790) 100
a
In case of multiple matches with the same maximum score value, only the first match was listed.
b
Isolated only from plates at 37 °C.
c
Isolated from anaerobic culture conditions.
d
Strains with spore-forming ability.

economic losses by causing diseases in various agronomically
important crops (Azad et al., 2000; Paccola-Meirelles et al., 2001;
Gitaitis and Gay, 1997; Coutinho and Venter, 2009). Pantoea
agglomerans induces tumorous galls in gypsophila and beet, but was
non-pathogenic on cereals including oat and rye (Azad et al., 2000).
Instead, P. agglomerans has been associated with positive effects on
cereals, such as promotion of growth and increase of grain yield in
wheat and barley (Amellal et al., 1998; Remus et al., 2000).
Furthermore, its anti-fungal and anti-bacterial properties have been
exploited in biological control of various diseases on crop plants
(Wright and Beer 2006), and it is licensed for fire blight management
in the USA, Canada and New Zealand (cited in Völksch et al., 2009).
Oral or transdermal administration of a lipopolysaccharide (IP-PA1)
derived from P. agglomerans has been linked via macrophage activator
effect to several health-promoting effects (Kohchi et al., 2006;
Taniguchi et al., 2009), raising interest for its potential exploitation
as the active ingredient in health foods, skin care cosmetics and
animal feed. The ice-nucleating activity of P. ananatis, in turn, has been
applied in food industry in freezing of foods to obtain the desired
texture (Zasypkin and Lee, 1999), and in freeze-drying of foods
(Watanabe and Arai, 1994). These observations make representatives
of Pantoea interesting players for in situ processing of cereals.
Besides Pantoea, rye flakes contained enterobacteria from the
closely related genus Erwinia; the 16S rDNA gene of pink-pigmented
isolate NR11 shared 99.8% identity to the published sequence for E.
persicina.
Isolate ON8 secreted a distinctive yellow-green fluorescent
pigment upon extended incubation on agar plates, a characteristic
of fluorescent pseudomonads. Based on the 16S rDNA sequence
analysis, this isolate was subsequently affiliated with P. fluorescens
group (Anzai et al., 2000), with 100% sequence identity to grass-
associated saprophytes P. trivialis and P. poae (Behrendt et al., 2003).
Isolate NR6 from rye flakes likewise clustered within this group, by
having identical 16S rDNA sequence to several fluorescent Pseudo-
monas species (e.g. P. fluorescens and P. veronii). Fluorescent
pseudomonads are typical inhabitants of plant phyllosphere and
rhizosphere, and form a significant proportion of the culturable
bacterial community on wheat heads and phyllosphere (Legard et al.,
1994; Yoshida et al., 2006). In food industry, fluorescent pseudomo-
nads are often implicated in food spoilage, especially of milk and other
dairy products (Dogan and Boor, 2003), due to their capacity to
produce thermoresistant proteases and lipases. Many species are
psychrotrophic (also ON8) and may spoil refrigerated foods.
Seven isolates from oat bran (RB1) had identical 16S rDNA
sequences to a number of members of A. calcoaceticus–A. baumannii
complex isolated from both clinical and environmental habitats. In a
study of Legard et al. (1994), A. calcoaceticus was included among the
15 most frequently recovered bacteria in wheat phyllosphere. In
wheat rhizosphere, strains of Acinetobacterium have been shown to
exert plant-growth promoting activity (Huddedar et al., 2002) and
biocontrol activity against pathogens (Sarode et al., 2009). Some
acinetobacteria grow under low temperatures and are thus often
associated with spoilage of refrigerated foods (Kämpfer, 1999).
Isolate RB4 from oat bran shared high 16S rDNA sequence
similarity (99.6%) to Janthinobacterium sp. isolated from glacial
meltwater in Mt Everest. It was also closely related to type strain of
 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285
J. agaricidamnosum, the causal agent of soft rot in Agaricus bisporus
(Lincoln et al., 1999).
Of the remaining Gram-negative isolates from the rye flakes only
Chryseobacterium sp. NR7 with the closest sequence identity to a
strain isolated from soybean rhizosphere (Peterson et al., 2006) and
Pedobacter sp. NR10 which had as closest match P. terrae (Yoon et al.,
2007) were later interesting from folate production point of view.
Both bacteria are normally found in soil.
The Gram-positive isolates in oat bran and rye flakes fell into 10
distinct genera (Table 2). Three isolates (ON5, ON6, ON10) from
OBCN15 were characterized by their spore-forming ability. The 16S
rDNA genes of ON5, and of closely related ON4, were identical to those
of Bacillus subtilis strains isolated from various sources. The sequence
of ON6 was identical to the sequence of NR12 from rye flakes, and the
isolates were identified as Bacillus licheniformis with 99.8% identity.
The third spore-forming isolate, ON10, was identified as Paenibacillus
cookii. Bacillus species are commonly found in soil and in association
with plants, including cereals (Legard et al., 1994; ICMFS (Interna-
tional Commission on Microbiological Specifications for Foods), 2005;
Yoshida et al., 2006). They are among the most frequently encoun-
tered microbes during the milling process and common contaminants
of bran and flour (Hesseltine and Graves, 1966; Berghofer et al., 2003).
Spore-forming bacilli are of particular concern to food processors, as
they may survive heat treatments and induce spoilage in the finished
product even if stored correctly. For example, ropiness is a common
form of bread spoilage caused primarily by B. subtilis and B.
licheniformis (Rosenkvist and Hansen, 1995; Sorokulova et al., 2003).
Isolate ON9 was affiliated to the genus Enterococcus, with 100%
sequence identity to several strains of E. durans, E. faecium and E.
faecalis isolated from fermented food products. Enterococci predom-
inantly inhabit the gastrointestinal tract of mammals and are also
commonly found from environment, plants, and foods, where they are
considered as indicators of low sanitary quality (Giraffa, 2002). They
are, however, recognized as important contributors to the flavor and
texture of several fermented foods, and are also used as probiotics in
commercial products. Moreover, their ability to produce bacteriocins
against food-borne pathogens and spoilage microorganisms has
raised interest for their application in food preservation. Although
enterobacteria have a long history in foods without any health
concerns, their association with nosocomial infections and the
presence of antibiotic resistant strains has raised discussion about
their safe use in food production (Foulquié Moreno et al., 2006).
Three strains from oat bran were phylogenetically affiliated to the
genus Curtobacterium. Isolate ON7 from OBCN15 had identical 16S
rDNA gene sequence with several plant-associated Curtobacterium
strains, including C. flaccumfaciens pv flaccumfaciens type strain DSM
20129. Two isolates (RB2) from regular bran were identified as C.
citreum and were closely related to the type strain DSM 20528,
originally isolated from rice paddy (Komagata and Iizuka, 1964;
Yamada and Komagata, 1972). Curtobacterium species were ranked
among the most abundant culturable bacteria in wheat heads and
phyllosphere (Legard et al., 1994; Yoshida et al., 2006).
Three isolates from oat bran were identified as representatives of
the genus Staphylococcus. Isolate RB7 had identical 16S rDNA
sequence to that of Staphylococcus klosii type strain, isolated from
squirrel skin. Isolate RB8 shared high sequence homology to
endophytic staphylococci from Chinese cabbage and potato plants.
Likewise, homologs of isolate OW1, sharing 100% sequence identity to
S. pasteuri and S. warneri, have been isolated from endophytic
bacterial communities of Chinese cabbage. Various Staphylococcus
species have also been observed in the phyllosphere of wheat (Legard
et al., 1994).
Isolate RB3 was identified as Exiguobacterium sp. with 99.6%
sequence identity to strain RD3 isolated from wastewater contami-
nated soil near textile industry (Dhanve et al., 2009). Isolate RB6 was
closely related to Micrococcus sp., while RB5 showed close relatedness
281
to clinical strains of Dietzia sp., including D. papillomatosis. Isolate RB9
was identified as Propionibacterium sp., the closest relative species
being the cutaneous species P. acnes.
Isolate NR9 from rye flakes showed 100% sequence identity to
Plantibacter flavus. This species, originally isolated from the phyllo-
sphere of grasses (Behrendt et al., 2002), was among the most highly
represented Gram-positive genera in maple sap together with
Staphylococcus and Bacillus (Lagacé et al., 2004). Two isolates (NR5)
representing the genus Sanguibacter had identical 16S rDNA
sequences with S. suarezii strain, isolated from maple sap also.
None of the characterized bacteria are known pathogens in
humans (Directive, 2000), even though four of the genera repre-
sented, Bacillus, Enterococcus, Pseudomonas, Staphylococcus, contain
known pathogenic species. However, some of our isolates are
classified in risk group 2 (TRBA 466 (Technische Regeln für
Biologische Arbeitstoffe), 2006): Acinetobacter calcoaceticus RB1,
Enterococcus durans ON9, Pantoea agglomerans NR2–NR4, ON2, ON3,
Propionibacterium sp. RB9, Sanguibacter sp. NR5, Staphylococcus
pasteuri OW1, Staphylococcus sp. RB8, and the LAB strains of
Lactobacillus rhamnosus used in this study. They can be viewed as
emerging opportunistic pathogens according to this criterium and
some of the species indeed have been isolated from nosocomial
infections or from immunocompromised patients (Bergogne-Bérézin
and Towner, 1996; Giraffa 2002). Pantoea agglomerans was earlier
classified as Enterobacter agglomerans, a known pathogen in humans,
from which only a certain group was transferred to the new genus
(Gavini et al., 1989). Still currently P. agglomerans is viewed as a
heterogeneous species (Delétoile et al., 2009) and it is thus possible
that the plant-associated strains could form a lower risk group. P.
ananatis is risk group 1 and clearly non-pathogenic. Our Pseudomonas
isolates NR6 and ON8 belong to risk group 1 fluorescent species P.
fluorescens, P. poae, or P. trivialis, also found in, for instance, kefir
(Kourkoutas et al., 2006).
Because the oat bran and rye flake products contained substantial
amounts of a wide variety of microorganisms this fact could be
utilized either by fermentation or by aerobic propagation to even
elevate the numbers of microbial cells utilizing these products as raw
materials in aqueous processes. Since folate production is associated
with growth of cells, specific physiological conditions could be used to
favour growth of some desired bacteria over others. Whether the risk
group differences are of real concern depends on the relative growth
efficiency of different bacteria and in case the final product is
sufficiently heat-treated to inactivate all the bacteria, it should be of
no concern.
3.3. Extracellular hydrolytic activities of cereal-associated bacteria
The screen for extracellular hydrolases revealed a wide variety of
enzymatic activities among the bacterial isolates (Table 3). These
enzymes may induce various chemical and nutritional changes in
colloidal food systems, and thereby have a large impact on the
functionality of bran or other cereal products in food applications.
Only five isolates degraded starch, which forms the most abundant
component and storage polysaccharide in cereals. The highest starch-
degrading, i.e. amylolytic activity was observed in Exiguobacterium sp.
RB3 isolated from oat bran. The oat bran isolate B. subtilis ON5 showed
a moderate amylolytic activity, whereas only a weak activity was
observed in the rye flake isolates Sanguibacter sp. NR5, Chryseobacter-
ium sp. NR7 and Pedobacter sp. NR10. Of the control strains, only
Bacillus sp. ABM 5119, originally isolated from OBCN15, hydrolyzed
starch with a moderate activity. Amylase supplementation is routinely
practiced e.g. in industrial bread making, in order to optimize the
amylase activity of the flour and to retard bread staling. However, the
thermostability of microbial enzymes often renders them difficult to
control during the process (Goesaert et al., 2005).
282 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285

Table 3
Production of extracellular hydrolytic enzymes by bacterial strains isolated from rye flakes and oat bran.

Isolate Tentative identification Amylasea Cellulasea Xylanasea Beta-glucanasea Proteasea Lipasea

OAT BRAN
ON1 Pantoea ananatis − +(8)/++(3) − −b − −
ON2 Pantoea agglomerans − −(1)/+(1)/++(1) − − − −
ON3 Pantoea agglomerans − +(3)/++(1) − − − −
ON4 Bacillus subtilis nd +++ ++++ ++ ++ +
ON5 Bacillus subtilis ++ ++ nd ++ ++ −
ON6 Bacillus licheniformis − + nd ++ ++ +
ON7 Curtobacterium sp. − + + + ++ −
ON8 Pseudomonas sp. − − − − ++ −
ON9 Enterococcus durans − − − − + −
ON10 Paenibacillus cookii nd nd nd − − ++
RB1 Acinetobacter calcoaceticus − − − − − +
RB2 Curtobacterium citreum − + +++ ++(1)/+++(1) ++ +
RB3 Exiguobacterium sp. +++ − − − ++++ −
RB4 Janthinobacterium sp. − − − − − −
RB5 Dietzia papillomatosis − + − − − +
RB6 Micrococcus sp. − − − − ++ −
RB7 Staphylococcus kloosii − − − − − −
RB8 Staphylococcus sp. − − − − − −
OW1 Staphylococcus pasteuri − − − − − −

RYE FLAKES
NR1 Pantoea ananatis − +(4)/++(4) − −b − −
NR2 Pantoea agglomerans − + − −b − −
NR3 Pantoea agglomerans − +(1)/++(1) − −b − −
NR4 Pantoea agglomerans − + − −b − −
NR5 Sanguibacter sp. + ++ +++ ++ − −
NR6 Pseudomonas sp. − − − − ++++ +
NR7 Chryseobacterium sp. + − − − +++ +
NR8 Agrobacterium tumefaciens − + − + − −
NR9 Plantibacter flavus − ++ + −b − −
NR10 Pedobacter terrae + + + + ++ ++
NR11 Erwinia persicina − + − −b − −
NR12 Bacillus licheniformis nd ++ + ++ + +

CONTROL STRAINS
ABM5061 Pantoea agglomerans − ++ − −b − −
ABM5119 Bacillus sp. ++ + − − + −
ABM5122 Bacillus cereus − − nd − +++ +
ABM5123 Bacillus cereus − − nd − +++ +
a
Enzyme activity is expressed relatively as a function of the size of the clearing zone as described in Materials and methods. −, no activity; +, low activity; ++, moderate activity;
+++, high activity; ++++, very high activity; nd, not determined. Subscript numbers in parenthesis describe the number of strains giving the reported result.
b
Although beta-glucanase negative (no clearing zone), the colonies were surrounded by a deep red halo.

Dietary fibres in cereals are composed of non-starch polysacchar-
ides and include soluble beta-glucan, partially soluble hemicelluloses
xylan and arabinoxylan, and insoluble cellulose. Nearly 80% of all
isolates were capable of degrading carboxymethylcellulose (Table 3).
Bacillus subtilis ON4 from oat bran showed high hydrolytic activity
against cellulose, whereas the cellulase activities in the other isolates
ranged from weak to moderate. Notably, this was the only hydrolytic
activity observed in E. persicina NR11 and in all strains of Pantoea. Nine
of the cellulase-positive isolates also showed extracellular hydrolysis
of xylan. High to very high xylanase activities were present in oat bran
isolates B. subtilis ON4 and C. citreum RB2, and in Sanguibacter sp. NR5
originating from rye flakes. Curtobacterium sp. ON7, Pedobacter sp.
NR10 and Plantibacter flavus NR9 showed weak activity against xylan.
With the exception of P. flavus, all xylanase positive isolates also
hydrolyzed beta-glucan. Moderate beta-glucanase activity was ob-
served in C. citreum RB2, Sanguibacter sp. NR5 and in all four strains of
Bacillus (ON4–ON6, NR12). Curtobacterium sp. ON7, Pedobacter sp.
NR10 and A. tumefaciens NR8 showed weak beta-glucanase activity.
The functionality of dietary fibres is strongly related to their molecular
mass and can thereby be modulated by even small amounts of
hydrolytic enzymes. From a nutritional point of view, enzymatic
breakdown of beta-glucan is highly undesirable, as the health-
promoting effects of beta-glucan are lost along with a decrease in its
viscosity. On the other hand, certain microbial endoxylanases are
utilized in various food and feed applications, e.g. in bread making,
gluten–starch separation, and animal feed to improve processing and
the quality of products (Goesaert et al., 2005).
Oat bran and rye flakes also contain proteins and fats (see Table 1).
Exiguobacterium sp. RB3 showed very strong proteolytic activity
against casein. High protease activity, in combination with weak
lipase activity, was also observed for Chryseobacterium sp. NR7 and
Pseudomonas sp. NR6 originating from rye flakes, whereas Pseudomo-
nas sp. ON8 from oat bran showed merely moderate protease activity.
Weak to moderate protease activity was also observed for Enterococ-
cus durans ON9, Micrococcus sp. RB6, Pedobacter sp. NR10, and all
representatives of Curtobacterium and Bacillus. Weak to moderate
lipase activity was present in Pedobacter sp. NR10, C. citreum RB02,
Paenibacillus cookii ON10 and in most Bacillus strains. In A.
calcoaceticus RB1, weak lipase activity was the only hydrolytic activity
detected. Proteases generally speaking are important for formation of
colour, flavour, and aroma whereas lipases are useful for anti-staling
effect and quality of baked products. However, it remains to be
determined what role these groups of enzymes have in oat bran
products having high protein and lipid contents (see Table 1).
Taken together, the most significant producers of hydrolytic
exoenzymes were strains of Bacillus, Curtobacterium and Pedobacter,
whereas strains of Staphylococcus and Janthinobacterium were
inactive against all substrates tested. Considering the role of cereal
products as the main source of dietary fibres and beta-glucan, the
most important notion was that beta-glucan was not degraded by
 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285 283

Table 4 cell folate contents (N12 μg/g) in oat bran were Pseudomonas sp. ON8,
Folate levels a) in cell biomass and b) folate production in supernatants for Bacillus subtilis ON4, and Janthinobacterium sp. RB4, and those in rye
monocultures of bacteria grown in YPD medium.
flakes Pseudomonas sp. NR6, E. persicina NR11, Chryseobacterium sp.
Isolate Tentative identification Folate levels in Folate production NR7, and P. flavus NR9. For these bacteria, the increase in folate
cell biomass after in supernatantb content from exponential to stationary phase was 30 to 110%, except
24-h incubation a
for Chryseobacterium sp. NR7 that had high cellular folate content
OAT BRAN already at the exponential phase (data not shown).
ON1 Pantoea ananatis + +
In general, folate excretion into the medium was moderate and
ON2 Pantoea agglomerans ++ +
ON3 Pantoea agglomerans ++ + independent of the folate content in the cells. Interestingly, some of
ON4 Bacillus subtilis +++ + the best folate producers – E. persicina NR11, Janthinobacterium sp.
ON5 Bacillus subtilis + +++ RB4, and Pseudomonas sp. NR6 – also released folate. For these
ON6 Bacillus licheniformis + − bacteria the net increase in the folate content of the spent medium
ON7 Curtobacterium sp. + ++
varied from 0.19 μg/g to 0.38 μg/g. On the other hand, there were
ON8 Pseudomonas sp. +++ +
ON9 Enterococcus durans + ++ bacteria with high folate concentration in cells but low concentration
ON10 Paenibacillus cookii + ++ in the spent medium (for example, B. subtilis ON4 and Pseudomonas
RB1 Acinetobacter calcoaceticus + + sp. ON8) as well as bacteria that had low folate concentration in cell
RB2 Curtobacterium citreum + +
but released folate to the medium (for instance, B. subtilis ON5). The
RB3 Exiguobacterium sp. ++ +
RB4 Janthinobacterium sp. +++ +++ control strains had folate contents from moderate to good. P.
RB5 Dietzia papillomatosis ++ + agglomerans ABM 5061 had similar folate levels as in the isolates
RB6 Micrococcus sp. ++ + from oat bran, and the Bacillus species seemed to both synthesize
RB7 Staphylococcus kloosii ++ ++ folate and release it to the growth medium.
RB8 Staphylococcus sp. + +
The folate production in cells, in supernatant, or in both offers
RB9 Propionibacterium sp. + ++
OW1 Staphylococcus warneri + − technological alternatives for exploiting different microorganisms
characterized in this study to make various novel food products.
RYE FLAKES Natural mixed culture of a certain raw material could be propagated
NR5 Sanguibacter sp. ++ +++
under suitable physiological conditions or pure culture inocula could
NR6 Pseudomonas sp. +++ +++
NR7 Chryseobacterium sp. +++ ++
be used either alone or in sequence or simultaneously with traditional
NR9 Plantibacter flavus +++ ++ LAB starters. Enhanced folate content breads, drinks, porridges,
NR10 Pedobacter terrae ++ ++ snacks, and soups could be envisioned as final products.
NR11 Erwinia persicina +++ +++ Folate contents in cells of lactic acid bacteria at the stationary
NR12 Bacillus licheniformis + +
phase were rather low, up to 5.6 μg/g, and well in line with previous
NR13 Sphingomonas sp. + ++
results (Kariluoto et al., 2006). Most of the lactic acid bacteria seemed
LAB to consume folate from the medium. Bifidobacterium bifidum BB-12
Bifidobacterium bifidum BB-12 + + and S. thermophilus ABM5097 were exceptions as they released folate
Lactobacillus acidophilus LA-5 + −
into the medium (from 0.05 to 0.1 μg/g). Streptococcus thermophilus
Lactobacillus rhamnosus ABM 5029 + −
Lactobacillus rhamnosus ATCC 7469 + −
has in several studies been reported to produce folate in significant
Lactobacillus rhamnosus LC-705 + − amounts, approximately 0.03 to 0.05 μg/g including both intra- and
Lactococcus lactis HAMBI 2345 + − extracellular folate (Crittenden et al., 2002; Holasová et al., 2004; Lin
Lactococcus lactis HAMBI 2348 + − and Young, 2000), and a large proportion of its folate is excreted
Streptococcus thermophilus ABM + ++
(Sybesma et al., 2003). Pompei et al. (2007) also reported that most of
5097
the folate produced by bifidobacteria was excreted, with maximum
CONTROL STRAINS excretion of 0.082 μg/ml by B. pseudocatenulatum.
Pantoea agglomerans ABM 5061 ++ + The results of this study are in good agreement with our previous
Bacillus sp. ABM 5119 +++ ++ study reporting folate production and excretion by two endogenous
Bacillus cereus ABM 5122 ++ +++
Bacillus cereus ABM 5123 ++ ++
bacteria isolated from rye flour (Kariluoto et al., 2006). Folate
a
production capability has been studied mainly in milk-based products
+ 0 to 6 μg/g FW, ++ 6 to 12 μg/g FW, +++ N 12 μg/g FW.
b using lactic acid bacteria, propionibacteria, and bifidobacteria, but
− b 0 μg/g, + b0.1 μg/g, ++ 0.1 to 0.19 μg/g, +++ N 0.19 μg/g.
differences in strains and cultivation conditions make results
controversial and difficult to interpret. To our knowledge, folate
strains of Acinetobacter, Chryseobacterium, Dietzia, Enterococcus, production or consumption by endogenous bacteria in cereals has not
Erwinia, Exiguobacterium, Janthinobacterium, Micrococcus, Pantoea, been investigated before. Our study is the first large-scale screening of
Plantibacter, Pseudomonas, or Staphylococcus. endogenous cereal bacteria, some of which are capable of producing
significant amounts of folate.
3.4. Folate levels in cells and supernatants
4. Conclusions
There were substantial differences in the folate levels in bacteria
(Table 4). Folate levels in cells of Sphingomonas sp. NR13, E. durans In this study we showed that oat bran and rye flake products
ON9, Paenibacillus cookii ON10, Propionibacterium sp. RB9, P. ananatis contain a wide variety of bacteria with different hydrolytic activities,
ON1, Curtobacterium sp. ON7, C. citreum RB2, A. calcoaceticus RB1, and which are potentially important in aqueous processing of grains and
most of the Bacillus and Staphylococcus species were low after a 24- foods. There were substantial differences in the folate levels of
hour incubation, and for P. ananatis ON1, B. subtilis ON5, B. bacteria and the degree of excretion of folate into medium. The best
licheniformis NR12, and Propionibacterium sp. RB9 even lower than producers could easily exceed folate levels reported for S. thermo-
at the exponential growth phase (data not shown). Moderate folate philus or other lactic acid bacteria. Thus, better understanding and
producers included Sanguibacter sp. NR5, Pedobacter sp. NR10, P. characterization of endogenous microorganisms in cereals could
agglomerans ON2 and ON3, Exiguobacterium sp. RB3, D. papillomatosis facilitate the development of folate-rich cereal-based foods. Even
RB5, S. kloosii RB7, and Micrococcus sp. RB6. Bacteria with the highest though microorganisms may not be intentionally added, they may as
284 M. Herranen et al. / International Journal of Food Microbiology 142 (2010) 277–285
natural affiliates affect technological and nutritional features espe-
cially in aqueous processing of cereals. The most promising bacteria –
Erwinia, Janthinobacterium, and Pseudomonas – yielded relatively high
levels of folate in the cell and the supernatant and lacked beta-glucan-
degrading activity.
Acknowledgments
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 associated bioactive compounds while
maintaining soluble dietary fibre physiologically active”.
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