International Journal of Food Microbiology 190 (2014) 54–60

Contents lists available at ScienceDirect

International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Phytic acid degrading lactic acid bacteria in tef-injera fermentation Maren M. Fischer a,⁎, Ines M. Egli a,1, Isabelle Aeberli a, Richard F. Hurrell a, Leo Meile b a b

Laboratory of Human Nutrition, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland

a r t i c l e

i n f o

Article history: Received 14 April 2014 Received in revised form 20 July 2014 Accepted 12 August 2014 Available online 20 August 2014 Keywords: Phytic acid Iron Zinc Lactic acid bacteria Tef Injera

a b s t r a c t Ethiopian injera, a soft pancake, baked from fermented batter, is preferentially prepared from tef (Eragrostis tef) flour. The phytic acid (PA) content of tef is high and is only partly degraded during the fermentation step. PA chelates with iron and zinc in the human digestive tract and strongly inhibits their absorption. With the aim to formulate a starter culture that would substantially degrade PA during injera preparation, we assessed the potential of microorganisms isolated from Ethiopian household-tef fermentations to degrade PA. Lactic acid bacteria (LAB) were found to be among the dominating microorganisms. Seventy-six isolates from thirteen different tef fermentations were analyzed for phytase activity and thirteen different isolates of seven different species were detected to be positive in a phytase screening assay. In 20-mL model tef fermentations, out of these thirteen isolates, the use of Lactobacillus (L.) buchneri strain MF58 and Pediococcus pentosaceus strain MF35 resulted in lowest PA contents in the fermented tef of 41% and 42%, respectively of its initial content. In comparison 59% of PA remained when spontaneously fermented. Full scale tef fermentation (0.6 L) and injera production using L. buchneri MF58 as culture additive decreased PA in cooked injera from 1.05 to 0.34 ± 0.02 g/100 g, representing a degradation of 68% compared to 42% in injera from non-inoculated traditional fermentation. The visual appearance of the pancakes was similar. The final molar ratios of PA to iron of 4 and to zinc of 12 achieved with L. buchneri MF58 were decreased by ca. 50% compared to the traditional fermentation. In conclusion, selected LAB strains in tef fermentations can degrade PA, with L. buchneri MF58 displaying the highest PA degrading potential. The 68% PA degradation achieved by the application of L. buchneri MF58 would be expected to improve human zinc absorption from tef-injera, but further PA degradation is probably necessary if iron absorption has to be increased. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Injera, the staple food consumed widely in Ethiopia (Umeta et al., 2005), is a pancake prepared from tef (Eragrostis tef), an ancient cereal, indigenous to Ethiopia (Zegeye, 1997). Traditional preparation of Ethiopian tef-injera has not changed over decades (Stewart and Getachew, 1962; Yetneberk et al., 2004) and involves a fermentation step of 2–3 days based on continuous backslopping, a process in which the new batter is inoculated with a leftover from the previous fermentation, called “ersho” in Amharic. Ersho, tef flour and water are thoroughly kneaded and allowed to rest. Liquid that gets separated on top is discarded and replaced with fresh water. Before the batter is finally poured onto a clay plate and baked, a so-called “absit” is prepared by boiling a portion of the batter with water and adding it back to the

⁎ Corresponding author at: ETH Zurich, Institute of Food, Nutrition and Health, LFV D 16.2, Schmelzbergstrasse 7, 8092 Zurich, Switzerland. Tel.: +41 44 632 43 69. E-mail addresses: maren.fi[email protected] (M.M. Fischer), [email protected] (I.M. Egli), [email protected] (I. Aeberli), [email protected] (R.F. Hurrell), [email protected] (L. Meile). 1 Present address: ETH Board, Haeldeliweg 15, 8092 Zurich, Switzerland.

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.018 0168-1605/© 2014 Elsevier B.V. All rights reserved.

fermenting batter, ensuring the desired textural quality of the baked injera pancake. Due to the use of ersho, the pH drops rapidly (Yigzaw et al., 2004) and reaches values below pH 4 within hours (Baye et al., 2013; Stewart and Getachew, 1962). As would be expected with acidification, microbial analysis of tef-injera batter has reported lactic acid bacteria (LAB) to be the major fermentative microbes accompanied by yeast, Enterobacteriaceae and Bacillus spp., but the latter was detected to a lesser extent (Nigatu and Gashe, 1998). These findings are comparable to other sourdough preparations (Minervini et al., 2014). Tef is rich in phytic acid (PA), myo-inositol hexakisphosphate, which is the main phosphate storage form in most cereals, legumes and nuts. With its 6 phosphate groups, PA has a large number of negatively charged residues over a broad pH range and a strong chelating potential for divalent cations such as Ca2+, Mg2+, Fe2+ and Zn2+ in the human digestive tract (Schlemmer et al., 2009). The PA-mineral chelates are insoluble and prevent the absorption of nutritionally important minerals and trace elements from plant-based foods (Hurrell, 2004). This is a major concern in developing countries, where cereals and legumes are the major source of minerals for large population groups, and where plant based diets are associated with nutritional iron and zinc deficiencies (Ramakrishnan, 2002).

M.M. Fischer et al. / International Journal of Food Microbiology 190 (2014) 54–60

Several strategies are available to overcome the inhibition of PA on iron absorption, including the addition of ascorbic acid or EDTA to meals (Hurrell, 2002), but these strategies do not improve zinc absorption. One promising measure that has been reported to increase both iron and zinc absorption from foods is enzymatic degradation of PA (Egli et al., 2004; Troesch et al., 2009). Phytases hydrolyze inositol phosphates and have been reported in microorganisms, plants and animals, although their activity and optimum conditions for reaction differ widely (Konietzny and Greiner, 2002). The selection of an appropriate enzyme adapted to the processing conditions of a specific food is therefore critical. Phytases are present in cereals (Egli et al., 2002) but have not been reported in tef, although PA has been reported to be partially degraded during tef-injera fermentation (Abebe et al., 2007; Umeta et al., 2005; Urga and Narasimha, 1998). Although PA degradation has been reported to occur to varying extents, the loss is only partial unlike the almost complete reduction reported when wheat is added to injera preparation (Baye et al., 2013). In addition to cereal phytases, microbial phytases can degrade PA during fermentation. Effective PA degradation was achieved by yeasts, isolated from Tanzanian togwa, during togwa fermentation (Hellström et al., 2012). But for the application of microbial phytase producers as starter culture in food production the safety aspect should not be neglected. This is why LAB, which are involved in many food processes and also have been noted in injera fermentations (Gashe, 1985) are of high interest as starter cultures. Selected strains of LAB species originating from European sourdough fermentations for bread production as well as from an African pearl-millet fermentation for gruel making have been reported to be capable of degrading PA (De Angelis et al., 2003; Lopez et al., 2000; Songré-Ouattara et al., 2008). The present study was designed to identify and isolate microorganisms from tef-injera fermentation that have phytase activity and are eligible as starter culture for tef-injera fermentation. LAB, dominating in Ethiopian tef-injera fermentations, were isolated and screened for phytase activity. The LAB isolates showing PA degrading potential were characterized and further tested in model tef fermentations as well as in full scale tef-injera preparation for their ability to degrade PA within the food-matrix. 2. Materials and methods 2.1. Chemicals and flours Unless otherwise specified, chemicals of p.a. grade were purchased from Sigma-Aldrich Chemie GmbH, Switzerland and Merck KGaA, Germany. Media and components for cultivation of microbes were ordered from Becton Dickinson AG, Switzerland, if not stated differently. Tef for fermentations performed at the laboratory in Zurich was bought at a local market in Debre Zeyit (Bishoftu), Ethiopia and milled to wholegrain flour in a mill at the same location. 2.2. Phytase application Aspergillus niger phytase euphoVida™ 20000G was kindly provided by DSM Nutritional Products, Kaiseraugst, Switzerland. To ensure complete PA degradation during 48 h of tef fermentation (see Section 2.7), double the amount of phytase units (380 FTU/g PA) theoretically needed to degrade all the PA was applied to tef flour. The enzyme is inactivated by baking. 2.3. Sampling of Ethiopian tef-injera batter The traditional injera preparation at household level was studied in the Debre Zeyit (Bishoftu) area, which is situated on the elevated plain of central Ethiopia. Families from the urban and surrounding rural areas who were willing to demonstrate their way of tef-injera preparation were selected. The tef flour used by the families was from their own

55

stocks, either bought at a local market or grown on their own land. For later enumeration and isolation of microorganisms, approximately 5 g fermenting batter (before the absit preparation step) was collected in duplicate from each family in a sterile falcon tube prefilled with 1 mL glycerol. The samples were stored on ice during the study-day, before the final storage at −20 °C. 2.4. Enumeration of microbes At ETH Zurich, thawed Ethiopian tef-injera batter samples from 6 households were analyzed in duplicate for total microbial content and a further 7 tef-injera batter samples for LAB only. To evaluate the model fermentations performed at the laboratory in Zurich, samples of about 1 g fresh batter were taken for immediate analysis. All batter samples were diluted tenfold (w/w) in peptone-solution and 0.1 mL of subsequent serial ten-fold dilutions were spread on selective agar media in duplicates for enumeration of bacteria and fungi. Presumptive lactobacilli were selected on MRS agar medium (1.5% agar), incubated anaerobically (AnaeroGen packs, Oxoid, UK) at 37 °C. YM agar medium with chloramphenicol (100 mg/L) against bacterial growth was used to cultivate yeasts at 25 °C. General bacterial counts were performed on aerobic plate count (PC) agar medium at 30 °C as well as presumptive Bacillus spp. spore counts after a heat treatment step at 85 °C for 15 min (Kastner, 2008). Presumptive enterococci were enumerated on KFS agar medium incubated at 43 °C for 48 h. VRBD agar medium was used to detect Enterobacteriaceae after a 48 h incubation at 37 °C. After incubation, cell counts were calculated in colony forming units (cfu) per g of batter. 2.5. Isolation, cultivation and characterization of LAB Dominant, presumptive LAB were picked (2–3 isolates per colony morphology) from MRS agar media, containing between 30 and 300 cfu obtained from diluted injera batter, and were purified by repeated streaking on the same medium. Strains were assessed for Gram-classification (3% KOH), catalase activity (3% H2O2), and by phase contrast light microscopy. Liquid cultures were grown in MRS media and stored at −80 °C in 25% (v/v) glycerol as a cryoprotectant. For DNA extraction from LAB, a single colony was picked and suspended in 0.1 mL buffer and treated further as Goldenberger et al. described in procedure B (Goldenberger et al., 1995). For later amplification, the final DNA preparations were kept at −20 °C. To differentiate the particular LAB strains used, rep-PCR fingerprints were produced by DNA amplification with the (GTG)5 primer (Gevers et al., 2001). PCR products were visualized by UV light on ethidium bromide stained agarose (1.8%) after gel electrophoresis in 1× TAE-buffer (pH 8.0), using 1-kb and 100-bp DNA ladders (Generuler, Fermentas GmbH, Switzerland) as reference. To follow the fate of applied LAB starter strains in fermentations, 2–4 colonies from the two highest injera batter dilutions were randomly picked from MRS agar medium and analyzed by rep-PCR fingerprints as described above. The isolates were identified to the species level by partial 16S rRNA gene sequencing. These regions were amplified with the help of the primer pairs bak4 (5′-AGG AGG TGA TCC ARC CGC A-3′) (Greisen et al., 1994)/bak11w (5′-AGT TTG ATC MTG GCT CAG-3′) (Goldenberger et al., 1997) or 7f (5′-AGA GTT TGA TYM TGG CTC AG-3′)/1510r (5′ACG GYT ACC TTG TTA CGA CTT-3′) (Nielsen et al., 2007) based on a protocol described by Dasen et al. (1998) and Nielsen et al. (2007), respectively. The product was purified (GFX PCR DNA & Gel Band Purification Kit, GE Healthcare, UK) and prepared for standard Sanger sequencing reactions performed at GATC-Biotech (Konstanz, Germany). The resulting sequences were edited in FinchTV (version 1.4, Geospizza Inc., USA), assembled with the help of BioEdit (version 7.0.9, Ibis Biosciences, USA) and compared to sequences reported in GenBank (NCBI, USA), using the blastn algorithm.

56

M.M. Fischer et al. / International Journal of Food Microbiology 190 (2014) 54–60

2.6. Screening for phytase activity Based on a previously described two-step phytase assay (Bae et al., 1999), a screening of PA degradation ability was performed on a modified MRS (mMRS) agar medium, containing 1.5% agar, 1% casein peptone, 0.4% beef extract, 0.2% yeast extract, 1% glucose, 0.82% sodium acetate trihydrate, 0.2% ammonium citrate dibasic, 0.02% magnesium sulfate heptahydrate, 0.0025% manganese sulfate, 2% MOPS, 0.2% calcium chloride, and 0.25% sodium phytate as the sole source of phosphate. The latter two salts were sterile-filtered and added to the autoclaved medium at 55 °C. Due to the precipitation of PA, the media was nontransparent and the formation of clearing zones was the indicator for PA hydrolysis. To avoid false-positive results from clearing zones due to acid dissolution, 5 mL cerium sulfate (5%) was used for reprecipitation of dissolved but non-degraded PA. 2.7. Tef fermentation and injera preparation For the model and full scale tef fermentations, a pre-culture of phytase positive LAB isolates was grown overnight in liquid MRS medium with Tween80 (Biolife, Italy) at 37 °C and shaking at 160 rpm. Optical density (OD600) (BioPhotometer, Eppendorf AG, Switzerland) of the cultures was measured to calculate cell forming units per mL (cfu/mL) based on a standard curve which had been calculated for each isolate. The respective amount of cells was harvested for 7 min at 14,000 ×g (Biofuge pico, Heraeus, Germany) or 15 min at 12,000 ×g in a Beckman centrifuge (JA-12, Beckman Coulter GmbH, Germany) for greater volumes. The cell pellet was resuspended in peptone-solution (0.85% NaCl, 0.1% casein–peptone) and added to autoclaved tap water used for the fermentation. All containers and spoons used during fermentation experiments and for sample collection were autoclaved or disinfected with EtOH (70%). 2.7.1. Model tef fermentation For model fermentations, 9.6 g sterile tap water, 0.4 mL peptonesolution containing the respective starter cultures at 107–108 cfu/g and 6 g flour were mixed and incubated in a McCartney bottle (20 mL) for 48 h at 25 °C in accordance with the maximal average annual temperature in central Ethiopia (Conway et al., 2004). In order to distinguish PA degradation during fermentation caused by microbial activity from any PA degradation from possible endogenous tef phytases, control suspensions of tef flour were held under the same conditions without starter cultures but with the addition of chloramphenicol (200 mg/kg) to prevent bacterial growth and cycloheximide (200 mg/kg) to prevent propagation of fungi. The pH was adjusted to 3.6 after 6 h of incubation with acetic acid. Unless otherwise stated, model fermentation experiments were performed in duplicate. 2.7.2. Full scale tef fermentation Full scale tef fermentation for injera preparation was performed in 600 mL batches. For that, 250 g tef flour and 50 g ersho were blended stepwise with 290 g tap water. When defined starter cultures were added instead of ersho, 275 g of flour and 315 g of liquid, including the respective LAB (107–108 cfu/g batter), were mixed. Thereafter 100 g tap water was carefully added to cover the surface of the batter. The walls of the container were cleaned before the container was covered and incubated for 48 h at RT. After 24 and 48 h of fermentation, liquid present on top of the batter was discarded and replaced by a similar amount of fresh tap water. Before baking injera from the fermented batter, ‘absit’ was prepared as in the traditional process. For this, the fermented batter was first thoroughly mixed with a spoon, before removing and adding a 50 g portion to 150 mL boiling water. The mixture (absit) was stirred, brought back to a boil, cooled to around 60 °C and transferred back to the remaining fermented batter. The batter was incubated for a further 1–2 h at 30 °C until gas production was visible. Portions of around 100 g of batter were then baked to injera

pancakes in a frying pan (Miami 28 cm, Migros, Switzerland) covered with a thin layer of peanut oil (MClassic, Migros, Switzerland). Maximum heat was used until the batter was no longer liquid when heat was reduced to a third to complete the cooking. To imitate the traditional backslopping procedure with ersho, spontaneously fermented batter was prepared from tef and tap water. The fermenting batter was overlaid with water to prevent the formation of mold on the surface. After incubation for 48 h at room temperature (RT), the required amount of fermented batter (‘ersho’) was added to the main fermentation. 2.8. Determination of pH, PA, iron and zinc For pH determination during fermentation, an aliquot of batter was diluted with nanopure water (1:1, v/v) and measured immediately using a pH meter (Metrohm unitrode 6.0258.000, Switzerland). Tef flours, freeze-dried batter and injera samples were analyzed in duplicate or triplicate for PA content, as the sum of inositol-6phosphate and inositol-5-phosphate, according to a modification of Makower's method (Makower, 1970). Inorganic phosphate concentration was determined using a colorimetric assay (Van Veldhofen and Mannaerts, 1987), modified for measurement on 96 well-microtitration plates. Flour was mineralized in triplicates with a mixture of nitric acid and hydrogen peroxide solution by micro-wave heating in closed Teflon® vessels (MLS ETHOSplus, MLS GmbH, Germany). Iron and zinc concentrations in the resulting solutions were measured by graphite furnace (AA240Z, Varian, Australia) and flame (AA240FS, Varian, Australia) atomic absorption spectrometry, respectively. 2.9. Data analysis Data analyses were conducted with IBM SPSS Statistics 19 for Windows and Microsoft Office Excel 2010. PA and pH values obtained from replicated fermentations are given as mean ± standard deviation (SD). 3. Results 3.1. Enumeration of LAB and other microbes in Ethiopian tef-injera fermentation Ethiopian tef-injera batter samples (n(all) = 6, n(LAB) = 13), collected towards the end of the 37 to 75 hour fermentation process, were assessed for microbial composition. Enterococci and Enterobacteriaceae were below the detection limits of 2 lg cfu/g. Spore forming Bacillus species, yeast and mold counts were detected infrequently and were thus considered to be present in numbers around the same detection limit. Aerobic mesophiles were present on PC agar in all samples in a wide range of 3 to 7 lg after a 24 h incubation and increased up to 8 lg cfu/g after 72 h due to faint growth of pinpoint colonies representing potential LAB. The highest counts were observed for presumptive LAB grown on MRS medium with on average 8 lg cfu/g, ranging from 4 to 8 lg cfu/g. 3.1.1. Phytase positive LAB We isolated 76 dominant presumptive LAB from 13 different Ethiopian tef fermentations and screened them in a qualitative assay for their ability to degrade PA on mMRS agar medium, containing PA as the phosphorus supply rather than free phosphate. Twenty eight isolates exhibited phytase activity in the plate assay. They were further analyzed for their rep-PCR fingerprints in order to differentiate identical or highly similar isolates and 13 potentially different strains were identified (Fig. 1). Their phylogenetic affiliation was determined according to 16S rRNA gene sequences (Table 1).

M.M. Fischer et al. / International Journal of Food Microbiology 190 (2014) 54–60

57

Fig. 1. Footprints of selected LAB after agarose gel electrophoresis of rep-PCR amplicons: lanes 1–3) Lactobacillus buchneri (MF44, MF58, MF61), lanes 4–6) Lactobacillus casei (MF42, MF50, MF54), lane 7) Lactobacillus brevis (MF67), lane 8) Lactobacillus plantarum (MF79), lane 9) negative control without template, lane 10) Lactobacillus fermentum (MF25), lane 11) Lactobacillus crustorum (MF29), and lanes 12–14) Pediococcus pentosaceus (MF32, MF33, MF35); M) Marker (100 bp & 1 kb).

3.2. Model tef fermentations with LAB isolates identified as PA degrading The 13 phytase-positive LAB isolates, originating from Ethiopian tef fermentations, were used as starter cultures for these model fermentations. For comparison, a spontaneous and an antibiotic treated fermentation were performed. The pH recorded in fermented tef after 48 h at 25 °C ranged from 3.36 to 3.83 for the inoculated samples and was 4.08 ± 0.08 in spontaneously fermented tef (Table 2). PA was degraded to a variable extent in all fermentations and also in the control of non-fermented, antibiotic treated batter where the PA was 17% lower after 48 h at 25 °C (0.87 g/100 g dm) than at time zero (1.05 g/100 g dm). However when taking PA in the antibiotic treated approach as the 100% control, the spontaneous fermentation decreased the PA content to 71% of the control value, whereas the LAB inoculated fermentations decreased the PA content to 49–89% of that value. Inoculating with the strains of Lactobacillus buchneri and Pediococcus pentosaceus resulted in the greatest PA degradation and Lactobacillus casei strains in contrast to the lowest. The performance of the starter cultures was monitored by rep-PCR fingerprints. The strains used for inoculation were found to be dominant in all tef fermentations over indigenous bacteria. In tef incubated under antibiotic treatment, roughly 2 lg cfu/g was counted on MRS agar media and 3 lg cfu/g on PC agar media. No growth was observed on YM-chloramphenicol and KFS agar media at the detection limit of 2 lg cfu/g. Table 1 Phylogenetic affiliation after 16S rRNA gene sequence comparison of the 13 phytase active isolates from Ethiopian tef fermentations on MRS medium. Phylogenetic group

Isolate

Closest relative

Sequence identity

Query lengtha

NCBI accession

Lactobacillus brevis

MF67

Lactobacillus buchneri

MF44 MF58 MF61 MF42 MF50 MF54 MF25

L. L. L. L. L. L. L. L. L.

99%/ 99% 99% 99% 100% 100% 99% 100% 99%

1401/ 1401 1389 1354 1434 1442 1423 1153 1414

AY974809.1/ NR_041263.1 CP002652.1 NC_015428.1 NC_015428.1 NC_014334.1 HQ534100.1 NC_014334.1 JF757227.1

99% 100%/ 100% 99% 100% 100%

1289 1449/ 1449 1429 1404 1406

AM285451.1 NR_075041.1/ NR_029133.1 NR_042058.1 NC_008525.1 NC_008525.1

Lactobacillus casei

Lactobacillus fermentum Lactobacillus plantarum Pediococcus pentosaceus a

MF29 MF79 MF32 MF33 MF35

brevis/ harbinensis buchneri buchneri buchneri casei casei casei fermentum

L. crustorum L. plantarum/ L. pentosus P. pentosaceus P. pentosaceus P. pentosaceus

100% coverage for all isolates, besides 99% for MF32.

3.3. Tef-injera production of full scale fermentations with selected starter cultures Based on the model fermentation results, LAB isolates of different PA degradation capability, namely L. buchneri MF58, L. brevis MF67 and L. plantarum MF79, were selected as the starter cultures for full scale tef fermentations followed by the baking of injera pancakes. PA degradation was measured in the fermented batter and compared to tef fermentation with ersho and with ersho plus added phytase (Table 3). PA degradation was not as high in the full scale fermentation as in the model fermentations (Table 2). For example, in the ersho produced batter, PA was 0.77 g/100 g after full scale fermentation compared to 0.61 g/100 g after the model fermentation (Table 2). As in the model fermentations, L. buchneri MF58 had the greatest PA degrading potential reducing the PA to 66% of the content in the ersho fermented batter. L. brevis MF67 and L. plantarum MF79 were again less effective, whereas the addition of purified A. niger phytase degraded PA almost completely. Baking of the fermented batters into injera pancakes resulted in a relatively constant loss of 16–17 mg PA/100 g representing 21–33% Table 2 Influence of selected lactic acid bacteria (LAB) isolates used as starters for model tef fermentation on pH and phytic acid (PA) degradation after 48 h at 25 °C. LAB isolate added

pHa

Uninoculatedd Uninoculated + ABe L. brevis

4.08 3.39 n.d.f 3.83 3.70 n.d. n.d. 3.44 n.d. 3.36 3.52 3.52 3.67 3.65 3.36

L. buchneri

L. casei L. L. L. P.

crustorum fermentum plantarum pentosaceus

MF67 MF44 MF58 MF61 MF42 MF50 MF54 MF29 MF25 MF79 MF32 MF33 MF35

PA [g/100 g dm] remainingb,c ± 0.08 ± 0.01 ± 0.03 ± 0.03

± 0.01 ± ± ± ± ± ±

0.00 0.01 0.01 0.02 0.00 0.01

0.62 0.87 0.59 0.45 0.43 0.52 0.64 0.74 0.77 0.67 0.63 0.62 0.47 0.51 0.44

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.01 0.00 0.03 0.03 0.03 0.08 0.05 0.00 0.00 0.00 0.00 0.02 0.03 0.06

a Mean from duplicate fermentations started with LAB isolates and four replicates of the uninoculated tef, respectively ±SD. b Mean from fermentations performed at least in duplicates ± SD; mean relative SD from the assay, performed at least in duplicates for each sample, was 4.5%. c Of 1.05 g PA/100 g dry matter (dm) in tef flour. d Spontaneously fermented. e Under antibiotic (AB) treatment; pH adjusted after 6 h to pH 3.6. f n.d., not determined.

58

M.M. Fischer et al. / International Journal of Food Microbiology 190 (2014) 54–60

Table 3 Influence of fermentation starter and additive on phytic acid (PA) degradation and PA:mineral molar ratios in fermented batter and baked injera. Starter for fermentation

Additive

Ershod Ershod L. buchneri MF58 L. brevis MF67 L. plantarum MF79

–e Phytase – – –

PA [g/100 g dm] remaininga,b

PA:mineral molar ratios in baked injerac

In batter

In baked injera

Iron

Zinc

0.77 0.01 0.51 0.59 0.66

0.61 0.01 0.34 n.d.f 0.52

± 0.06 ± 0.00 ± 0.02

7:1 b1 4:1

22:1 b1 12:1

± 0.02

6:1

18:1

± ± ± ± ±

0.04 0.04 0.03 0.07 0.06

a

Of 1.05 g PA/100 g dry matter (dm) in tef flour. Means from triplicate measurements ± SD. With 2.8 mg zinc/100 g dm native in the tef flour and 7.6 mg iron/100 g flour, adapted from USDA National Nutrient Database for Standard Reference (2012). d Spontaneously fermented flour was used as ersho. e –, no additive. f n.d., not determined: no injera was baked from that batter. b c

of the PA in the respective fermented batters. The PA level in the injera pancake made from the L. buchneri MF58 fermented tef was 0.34 g/100 g. This represents 57% of the PA content remaining in the ersho fermented injera and 32% of the native PA in tef flour (1.05 g/100 g). The fermented batter made with L. brevis MF67 as a starter culture was not baked due to a surface contamination with molds. While no formal sensory or textural analysis of the injera pancakes was made, there was a clear difference in visual appearance. Pancakes made from batter fermented with ersho, ersho plus phytase, and with L. buchneri MF58 showed the expected three-dimensional structure with holes or “eyes” formed during the baking of good quality injera (Yetneberk et al., 2004). The injera from L. buchneri showed larger and more evenly distribute holes than the ersho injera (Fig. 2). The injera made from tef batter fermented with L. plantarum MF79, on the other hand, was dense with few holes. 3.4. Molar ratios of PA:iron and PA:zinc The tef flour used for injera preparation contained 28 mg zinc and 383 mg iron per kg. PA:mineral molar ratios were calculated according to the determined PA content and the native zinc content, while the amount of iron was corrected due to soil contaminations predicted in the Ethiopian flour by using a published iron value of 7.6 mg/100 g for the iron content of presumptive non-contaminated tef, taken from the US National Nutrient Database (U.S. Department of Agriculture and N.D.L.H.P., 2012) (Table 3). According to the PA levels, the PA:molar ratios were also highest in the traditional injera fermentation. 4. Discussion Earlier microbial investigations of fermented tef-injera batter (Gashe, 1985; Nigatu and Gashe, 1998; Umeta and Faulks, 1989) and

similar sorghum-kisra fermentations (Mohammed et al., 1991) have reported the predominance and importance of LAB in the fermentation process. Our studies confirmed the predominance of LAB, low numbers of Bacillus, yeast and mold species, and the absence of enterococci or Enterobacteriaceae. The high numbers of LAB are assumed to be responsible for the relatively high lactic and acetic acid production and thereby outcompeting non-acid-tolerant species. The fall in pH below 4, reported from different Ethiopian injera fermentations (Baye et al., 2013; Yigzaw et al., 2004) was similarly observed in our model set-up. Certain LAB have previously been reported to be capable of degrading PA (Corsetti and Settanni, 2007) and we have thus screened 76 presumptive LAB isolated from Ethiopian tef-injera fermentations for this property. All thirteen PA degrading isolates have been classified into LAB species or groups that have frequently been described to occur in different European and African sourdoughs used for bread production (De Vuyst and Neysens, 2005; Scheirlinck et al., 2007). Furthermore, except for the single isolate identified as Lactobacillus crustorum, all the detected species can be found on the latest qualified presumption of safety (QPS) list, published by EFSA (European Food Safety Authority), supporting their safe use as starter culture (EFSA, 2013). As the matrix surrounding the substrate has a strong impact on the extent of PA degradation by phytases (Bohn et al., 2007; Brejnholt et al., 2011) model fermentations with tef flour were used to test the PA degradation potential of the different LAB species isolated from the Ethiopian fermented tef-injera batter. The highest PA degradation in the model fermentations with selected LAB was seen after incubation with the L. buchneri and P. pentosaceus strains which showed roughly three times higher degradation than observed in the antibiotic treated approach, reducing the PA to less than half of the level in native tef. This is in agreement with previous reports from other plant sources. Camacho et al. (1991) reported that a strain of L. buchneri decreased PA by more than 55% in average, after 12 h of lupine flour fermentation and several P. pentosaceus strains from sourdough have been shown to exhibit PA degradation capacity on modified MRS agar medium (Raghavendra et al., 2010). Numerous studies have also reported L. plantarum strains to be phytase positive (Lopez et al., 2000; Songré-Ouattara et al., 2008; Sreeramulu et al., 1996; Tang et al., 2010) although more detailed studies revealed an extracellular dephosphorylating enzyme rather unspecific for sodium PA but with a high affinity to acetyl phosphate (Zamudio et al., 2001). In our study, teffermentation with the single L. plantarum isolate MF79 led to an intermediate PA reduction, not different from the spontaneous fermentation. The lowest PA reduction was observed in fermentations started with the L. casei isolates. In contrast to the partial PA degradation achieved by application of selected starter cultures, the addition of purified A. niger phytase showed the possibility for complete enzymatic PA turnover during tef fermentation although processing conditions are not optimal for respective enzyme activity. The contribution of endogenous tef phytases to the PA degradation during fermentation is unclear. Cereals such as wheat and rye are reported to exhibit high phytase activity (Egli et al., 2002) and in wheat

Fig. 2. Texture of baked injera started with ersho (A), started with ersho and added phytase (B), with L. buchneri MF58 as starter culture (C) and with L. plantarum MF79 as starter culture (D).

M.M. Fischer et al. / International Journal of Food Microbiology 190 (2014) 54–60

and rye sourdough acidification during fermentation is reported to activate wheat and rye phytases and lead to PA degradation (Leenhardt et al., 2005; Reale et al., 2007). It is possible therefore that the 17% reduction of PA in the antibiotic treated fermentation was due to tef phytases, however the contribution was minor compared to the PA degradation caused by spontaneous fermentation or by LAB inoculation. Thus the ability to express enzymes for PA degradation could be especially advantageous for LAB adapted to the tef environment. PA degradation during tef fermentation for injera preparation was comparable to the model fermentations. Additional PA degradation was observed during baking due to thermal hydrolysis (Mahgoub and Elhag, 1998). But not all fermentation approaches yielded in injera with typical “eyes”-structure. The different appearance of the injera pancakes might be due to more intense CO2 production by L. buchneri (Fig. 2C), an obligatory heterofermentative species of LAB, compared to L. plantarum (Fig. 2D), a facultative heterofermentative species, and undefined mixtures of LAB (Fig. 2A & B) in spontaneously fermented dough (Barrangou et al., 2011). The Ethiopian tef flour used in this study was found to have a very high iron content, which most probably results, at least in part, from soil contamination, introduced during the threshing procedure (Besrat et al., 1980). This soil iron is not expected to be available for absorption in humans as it does not dissolve in the gastrointestinal tract (Hallberg and Bjorn-Rasmussen, 1981). We therefore used a published value of 7.6 mg/100 g for the iron content of presumptive non-contaminated tef, taken from the US National Nutrient Database (U.S. Department of Agriculture and N.D.L.H.P., 2012) and the determined 2.8 mg/100 g for zinc in order to calculate molar ratios of PA to iron and zinc, respectively, as predictor for human absorption (Hurrell, 2004; Lönnerdal, 2002). The PA:iron molar ratio in the baked injera with L. buchneri MF58 as starter culture was lower compared to when ersho was used (Table 3) but still above the threshold of 1 or preferably 0.4 — above which iron absorption is assumed to be poor (Hurrell, 2004). Considering the achieved decrease of the molar ratio below 6, iron absorption could be improved if the injera were consumed in meals with small amounts of meat or vegetables (Hurrell and Egli, 2010), however this is not the normal practice in Ethiopia as injera is often consumed with pulses, free of ascorbic acid and also high in PA. In order to improve iron absorption substantially from the tef-injera based diets, PA needs to be further decreased during the tef-injera fermentation or the diet needs to be complemented with iron absorption enhancing foods. In the case of zinc, using L. buchneri MF58 as a starter culture for tef-injera production would increase zinc availability from low (PA:zinc molar ratios N 15) to moderate (PA:zinc molar ratios in the range of 5–15) and therefore would be expected to result in a modest but useful increase in zinc absorption (FAO and WHO, 2004). The present study shows for the first time that LAB isolated from fermented Ethiopian tef-injera batter degrade PA during fermentation within the food matrix. The power of our study was limited by the use of non-sterile flour, but as LAB starter cultures dominated the lactic acid fermentation, this constant microbial background was not expected to greatly influence the overall findings. Another potentially unfavorable aspect was that fermentations were performed in Zurich not in Ethiopia but since we used Ethiopian tef flour, the outcome of our ersho mediated tefinjera was estimated to be closely related to traditional Ethiopian injera production. The model fermentation set-up was established to evaluate the ability of LAB to degrade PA directly in the tef-matrix, although contribution of unspecific acid phosphatases, whether extra- or intracellular enzymes, on PA turnover could not be distinguished by this model. In conclusion, we have shown that certain strains belonging to different LAB species, isolated from tef-injera fermentation, partially degrade PA. Injera pancakes prepared with L. buchneri MF58 fermented tef contained substantially less PA than those prepared by traditional fermentation and could help to improve zinc absorption. PA levels, however, are still relatively high and further PA degradation is probably needed for increased iron absorption.

59

Acknowledgments We are deeply grateful for the families who gave us the possibility to collect samples from their private tef-fermentations. Furthermore we thank Susanna Wenk (M. Sc.) for her contribution in the study and Medicor Foundation Liechtenstein for supporting this project financially. References Abebe, Y., Bogale, A., Hambidge, K.M., Stoecker, B.J., Bailey, K., Gibson, R.S., 2007. Phytate, zinc, iron and calcium content of selected raw and prepared foods consumed in rural Sidama, Southern Ethiopia, and implications for bioavailability. J. Food Compos. Anal. 20, 161–168. Bae, H.D., Yanke, L.J., Cheng, K.J., Selinger, L.B., 1999. A novel staining method for detecting phytase activity. J. Microbiol. Methods 39, 17–22. Barrangou, R., Lahtinen, S., Ouwehand, F., 2011. Genus Lactobacillus, In: Von Wright, A. (Ed.), Lactic Acid Bacteria: Microbiological and Functional Aspects, 4 ed. CRC Press, pp. 77–91. Baye, K., Mouquet-Rivier, C., Icard-Verniere, C., Rochette, I., Guyot, J.P., 2013. Influence of flour blend composition on fermentation kinetics and phytate hydrolysis of sourdough used to make injera. Food Chem. 138, 430–436. Besrat, A., Admasu, A., M., O, 1980. Critical study of the iron content of tef (Eragrostis tef). Ethiop. Med. J. 18, 45–52. Bohn, L., Josefsen, L., Meyer, A.S., Rasmussen, S.K., 2007. Quantitative analysis of phytate globoids isolated from wheat bran and characterization of their sequential dephosphorylation by wheat phytase. J. Agric. Food Chem. 55, 7547–7552. Brejnholt, S.M., Dionisio, G., Glitsoe, V., Skov, L.K., Brinch-Pedersen, H., 2011. The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin. J. Sci. Food Agric. 91, 1398–1405. Camacho, L., Sierra, C., Marcus, D., Guzman, E., Campos, R., Vonbaer, D., Trugo, L., 1991. Nutritional quality of lupine (Lupinus albus cv. Multolupa) as affected by lactic acid fermentation. Int. J. Food Microbiol. 14, 277–286. Conway, D., Mould, C., Bewket, W., 2004. Over one century of rainfall and temperature observations in Addis Ababa, Ethiopia. Int. J. Climatol. 24, 77–91. Corsetti, A., Settanni, L., 2007. Lactobacilli in sourdough fermentation. Food Res. Int. 40, 539–558. Dasen, G., Smutny, J., Teuber, M., Meile, L., 1998. Classification and identification of propionibacteria based on ribosomal RNA genes and PCR. Syst. Appl. Microbiol. 21, 251–259. De Angelis, M., Gallo, G., Corbo, M., McSweeney, P., Faccia, M., Giovine, M., Gobbetti, M., 2003. Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1. Int. J. Food Microbiol. 87, 259–270. De Vuyst, L., Neysens, P., 2005. The sourdough microflora: biodiversity and metabolic interactions. Trends Food Sci. Technol. 16, 43–56. EFSA, 2013. Panel on Biological Hazards (BIOHAZ): scientific opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2013 update). EFSA J. 11, 3449. Egli, I., Davidsson, L., Juillerat, M.A., Barclay, D., Hurrell, R.F., 2002. The influence of soaking and germination on the phytase activity and phytic acid content of grains and seeds potentially useful for complementary feeding. J. Food Sci. 67, 3484–3488. Egli, I., Davidsson, L., Zeder, C., Walczyk, T., Hurrell, R., 2004. Dephytinization of a complementary food based on wheat and soy increases zinc, but not copper, apparent absorption in adults. J. Nutr. 134, 1077–1080. FAO, WHO, 2004. Vitamin and Mineral Requirements in Human Nutrition: Report of a Joint FAO/WHO Expert Consultation, Bangkok, Thailand, 1998, 2nd ed. Gashe, B.A., 1985. Involvement of lactic acid bacteria in the fermentation of TEF (Eragrostis tef) an Ethiopian fermented food. J. Food Sci. 50, 800–801. Gevers, D., Huys, G., Swings, J., 2001. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol. Lett. 205, 31–36. Goldenberger, D., Perschil, I., Ritzler, M., Altwegg, M., 1995. A simple universal DNA extraction procedure using SDS and proteinase-K is compatible with direct PCR amplification. PCR Methods Appl. 4, 368–370. Goldenberger, D., Kunzli, A., Vogt, P., Zbinden, R., Altwegg, M., 1997. Molecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing. J. Clin. Microbiol. 35, 2733–2739. Greisen, K., Loeffelholz, M., Purohit, A., Leong, D., 1994. PCR primers and probes for the 16S ribosomal-RNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal-fluid. J. Clin. Microbiol. 32, 335–351. Hallberg, L., Bjorn-Rasmussen, E., 1981. Measurement of iron-absorption from meals contaminated with iron. Am. J. Clin. Nutr. 34, 2808–2815. Hellström, A.M., Almgren, A., Carlsson, N.G., Svanberg, U., Andlid, T.A., 2012. Degradation of phytate by Pichia kudriavzevii TY13 and Hanseniaspora guilliermondii TY14 in Tanzanian togwa. Int. J. Food Microbiol. 153, 73–77. Hurrell, R.F., 2002. Fortification: overcoming technical and practical barriers. J. Nutr. 132, 806s–812s. Hurrell, R.F., 2004. Phytic acid degradation as a means of improving iron absorption. Int. J. Vitam. Nutr. Res. 74, 445–452. Hurrell, R.F., Egli, I.M., 2010. Iron bioavailability and dietary reference values. Am. J. Clin. Nutr. 91, 1461s–1467s. Kastner, S., 2008. The Inoculum of the Cassava Product Attiéké: Microbiological Diversity, Impact on Product Quality and Safety, and Development of a Controlled Fermentation Process. Eidgenössische Technische Hochschule Zürich, Switzerland.

60

M.M. Fischer et al. / International Journal of Food Microbiology 190 (2014) 54–60

Konietzny, U., Greiner, R., 2002. Molecular and catalytic properties of phytate-degrading enzymes (phytases). Int. J. Food Sci. Technol. 37, 791–812. Leenhardt, F., Levrat-Verny, M.A., Chanliaud, E., Remesy, C., 2005. Moderate decrease of pH by sourdough fermentation is sufficient to reduce phytate content of whole wheat flour through endogenous phytase activity. J. Agric. Food Chem. 53, 98–102. Lönnerdal, B., 2002. Phytic acid — trace element (Zn, Cu, Mn) interactions. Int. J. Food Sci. Technol. 37, 749–758. Lopez, H.W., Ouvry, A., Bervas, E., Guy, C., Messager, A., Demigne, C., Remesy, C., 2000. Strains of lactic acid bacteria isolated from sour doughs degrade phytic acid and improve calcium and magnesium solubility from whole wheat flour. J. Agric. Food Chem. 48, 2281–2285. Mahgoub, S.E.O., Elhag, S.A., 1998. Effect of milling, soaking, malting, heat treatment and fermentation on phytate level of four Sudanese sorghum cultivars. Food Chem. 61, 77–80. Makower, R., 1970. Extraction and determination of phytic acid in beans (Phaseolus vulgaris). Cereal Chem. 47, 288–295. Minervini, F., De Angelis, M., Di Cagno, R., Gobbetti, M., 2014. Ecological parameters influencing microbial diversity and stability of traditional sourdough. Int. J. Food Microbiol. 171, 136–146. Mohammed, S.I., Steenson, L.R., Kirleis, A.W., 1991. Isolation and characterization of microorganisms associated with the traditional sorghum fermentation for production of Sudanese kisra. Appl. Environ. Microbiol. 57, 2529–2533. Nielsen, D.S., Teniola, O.D., Ban-Koffi, L., Owusu, M., Andersson, T.S., Holzapfel, W.H., 2007. The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Int. J. Food Microbiol. 114, 168–186. Nigatu, A., Gashe, B.A., 1998. Effect of heat treatment on the antimicrobial properties of tef dough, injera, kocho and aradisame and the fate of selected pathogens. World J. Microbiol. Biotechnol. 14, 63–69. Raghavendra, P., Rao, T.S.C., Halami, P.M., 2010. Evaluation of beneficial attributes for phytate-degrading Pediococcus pentosaceus CFR R123. Benefic. Microbes 1, 259–264. Ramakrishnan, U., 2002. Prevalence of micronutrient malnutrition worldwide. Nutr. Rev. 60, S46–S52. Reale, A., Konietzny, U., Coppola, R., Sorrentino, E., Greiner, R., 2007. The importance of lactic acid bacteria for phytate degradation during cereal dough fermentation. J. Agric. Food Chem. 55, 2993–2997. Scheirlinck, I., van der Meulen, R., van Schoor, A., Huys, G., Vandamme, P., de Vuyst, L., Vancanneyt, M., 2007. Lactobacillus crustorum sp nov., isolated from two traditional Belgian wheat sourdoughs. Int. J. Syst. Evol. Microbiol. 57, 1461–1467.

Schlemmer, U., Frolich, W., Prieto, R.M., Grases, F., 2009. Phytate in foods and significance for humans: food sources, intake, processing, bioavailability, protective role and analysis. Mol. Nutr. Food Res. 53, S330–S375. Songré-Ouattara, L.T., Mouquet-Rivier, C., Icard-Vernière, C., Humblot, C., Diawara, B., Guyot, J.P., 2008. Enzyme activities of lactic acid bacteria from a pearl millet fermented gruel (ben-saalga) of functional interest in nutrition. Int. J. Food Microbiol. 128, 395–400. Sreeramulu, G., Srinivasa, D.S., Nand, K., Joseph, R., 1996. Lactobacillus amylovorus as a phytase producer in submerged culture. Lett. Appl. Microbiol. 23, 385–388. Stewart, R., Getachew, A., 1962. Investigations of the nature of injera. Econ. Bot. 16, 127–130. Tang, A.L., Wilcox, G., Walker, K.Z., Shah, N.P., Ashton, J.F., Stojanovska, L., 2010. Phytase activity from Lactobacillus spp. in calcium-fortified soymilk. J. Food Sci. 75, M373–M376. Troesch, B., Egli, I., Zeder, C., Hurrell, R.F., de Pee, S., Zimmermann, M.B., 2009. Optimization of a phytase-containing micronutrient powder with low amounts of highly bioavailable iron for in-home fortification of complementary foods. Am. J. Clin. Nutr. 89, 539–544. U.S. Department of Agriculture, N.D.L.H.P., 2012. USDA National Nutrient Database for Standard Reference. Release 25 [Online]. Available at http://www.ars.usda.gov/ba/ bhnrc/ndl (Accessed: June 2013). Umeta, M., Faulks, R.M., 1989. Lactic acid and volatile (C2–C6) fatty acid production in the fermentation and baking of tef (Eragrostis tef). J. Cereal Sci. 9, 91–95. Umeta, M., West, C.E., Fufa, H., 2005. Content of zinc, iron, calcium and their absorption inhibitors in foods commonly consumed in Ethiopia. J. Food Compos. Anal. 18, 803–817. Urga, K., Narasimha, H.V., 1998. Phytate:zinc and phytate × calcium:zinc molar ratios in selected diets of Ethiopians. Bull. Chem. Soc. Ethiop. 12, 1–7. Van Veldhofen, P., Mannaerts, G., 1987. Inorganic and organic phosphate measurements in the nanomolar range. Anal. Biochem. 161, 45–48. Yetneberk, S., de Kock, H.L., Rooney, L.W., Taylor, J.R.N., 2004. Effects of sorghum cultivar on injera quality. Cereal Chem. 81, 314–321. Yigzaw, Y., Gorton, L., Solomon, T., Akalu, G., 2004. Fermentation of seeds of teff (Eragrostis teff), grass-pea (Lathyrus sativus), and their mixtures: aspects of nutrition and food safety. J. Agric. Food Chem. 52, 1163–1169. Zamudio, M., Gonzalez, A., Medina, J.A., 2001. Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase. Lett. Appl. Microbiol. 32, 181–184. Zegeye, A., 1997. Acceptability of injera with stewed chicken. Food Qual. Prefer. 8, 293–295.