ABSTRACT Phytase activity was studied in natural sourdough bread starters to determine physicochemical characteristics (phytic acid hydrolysis, dough rising capacity and pH) in the flour and during sourdough fermentation. Fermentation microorganisms (yeasts and lactic acid bacteria) were also characterized. Results showed a decrease of phytic acid in sourdoughs started with traditional starters, and wide variation in phytase activity. Microorganism counts were high at the end of fermentation, indicating higher fermenting activity of the starters. Yeast populations showed wide variation and lactic acid bacteria had high counts in the fermentation. Phytase activity was demonstrated in starter cultures made of lactic acid bacteria and yeast isolates, the most interesting of which were Saccharomyces cerevisiae combined with Lactobacillus plantarum and Leuconostoc mesenteroides.

Bread is a staple food in most developing countries. Bread consumption in Morocco is an estimated 210.44 kg/year in grain equivalent. Baker’s yeast is now more frequently used than traditional starter, with a consequent loss of some of the nutritional properties of the bread. It is assumed that most of the minerals are bound by phytates in cereals due to the high chelating reaction of the myo-inositol hexakis dihydrogenphosphate (IP6) molecule.

The bioavailability of some important minerals is lowered by phytates [1] and the digestibility of proteins [2], starch [3] and lipids [4] is reduced. We have previously demonstrated that traditional sourdough starters in Morocco may contain two kinds of microorganisms, lactic acid bacteria and yeasts [5]. Faid et al. has demonstrated that controlled fermentation of sourdough bread inoculated with selected strains of lactic acid bacteria and yeasts has characteristics similar to bread made with traditional starters (the strains used were isolated from the traditional starters) [6].

There is a need to study the nutritional properties of natural sourdough starters and phytate destruction and the bioavailability of minerals to more clearly understand the role of mixed fermentation in bread-making by yeasts and lactic acid bacteria. In the present study we examined phytase activity in sourdough bread started with both traditional starters and selected isolates of lactic acid bacteria and yeasts.

We propagated 16 samples of traditional sourdough bread starters on wheat flour in sterilized glass flasks. We mixed 10–20 g of each sample with 100 g of wheat flour and 35–40 mL of distilled water. The mixture was well-kneaded with a wooden spoon and the flasks were incubated at 30 °C for 6 hours.

Doughs to be analysed were prepared by mixing 50 g of the starter with 200 g of wheat flour and 100 mL of water. The mixture was kneaded manually and the resultant dough introduced into a 500 mL graduated cylinder. Each assay was prepared in duplicate, one sample to determine the rising capacity of the dough and the second to determine pH, phytase activity and microbial count. The cylinders were placed in a water bath at 30 °C.

A pH-meter (Crison micro-pH 2000) was used to measure the pH of a 10 g sample mixed with 10 mL of distilled water. Dough rising capacity was determined according to the method described by Faid et al. [6].

All glassware was rinsed with nitric acid (10%) and with distilled water prior to use. Precisely measured aliquots of the samples were introduced into 50 mL conical flasks. Nitric acid was added and the flasks were shaken for 4 hours. The mixture was centrifuged at 2000 g and the supernatant used for the determination of iron.

All reagents were analytical grade and all solutions were prepared just before use. The samples and the calibration curve were prepared as follows: to 0.5 mL of the supernatant, 1 mL of ammonium iron sulfate [NH4Fe(SO4)] (0.5 mmol) and 1.4 mL of distilled water were added in tubes. The mixture was heated in a boiling water bath for 20 minutes and allowed to cool to room temperature, after which 5 mL of amyl alcohol and 0.1 mL of ammonium thiocyanate (10%) were added. The optical density of the red complex in the upper layer was checked at 465 nm in a spectrophotometer (C-Cil Instruments Type CE 303). The calibration curve was established using sodium phytate (2 mmol) (Merck, Germany) as a reference standard. The range was 0–320 µL.

Yeasts and lactic acid bacteria were evaluated in the fermented sourdough to determine the origin of the phytases. Yeasts were counted on potato dextrose agar and the lactic acid bacteria counted on MRS (Merck, Germany) for lactobacilli. Leuconostoc were determined on the medium of Garvie (Merck, Germany). Pediococci were counted on APT agar (Merck, Germany), incubated at 40 °C.

Isolates of lactic acid bacteria were collected from the samples, purified and studied for their morphological (Gram reaction and shape) and physiological characteristics (growth at 45 °C, 30 °C and 15 °C, arginine deaminase and carbon dioxide formation). Yeast isolates were also collected from the samples and broadly checked for their morphological and physiological characteristics. The isolates were first screened for their rising capacity.

Isolates of each group, including Lactobacillus and Leuconostoc, were grown on MRS broth. The cultures were incubated 24 hours at 30 °C for Lactobacillus and 48 hours at 26 °C for Leuconostoc. Yeast isolates were grown on yeast extract glucose broth (yeast extract 5 g; glucose 20 g; distilled water 1 L). The biomass was harvested by centrifuging at 2800 g for 20 minutes and stored at 4 °C until use.

Soft wheat flour (200 g) was mixed with 100 mL distilled water in 500 mL wide-mouthed glass flasks and inoculated with a suspension of the isolates to be studied to obtain a final concentration of 106 cells/g in the dough. The flasks were incubated at 30 °C. The dough rising capacity, pH and phytates were determined as indicated above.

The physicochemical properties of the starters were also evaluated to study their characteristics in bread fermentation. It should be emphasized here that the technological factors are widely related to the nutritional characteristics. The pH was decreased in almost all the trials involving the traditional starters (Table 1). Values ranged between 3.02 (T2, A4) and 3.71 (L1). This was most probably due to the active microbiota (lactic acid bacteria). Low pH value in sourdough bread has become an area of increasing interest over the past few decades and a number of studies have been undertaken [7].

The dough rising capacity (DRC) was evaluated to provide accurate information about fermentation time at 30 °C and about the physical properties (rheology) of the dough. DRC ranged from 20 mL (A4) to 130 mL (A2), indicating a wide variation among the samples (Table 1). The DRC was directly related to the nature of the fermenting yeasts derived from the starters.

Apart from phytate destruction activity in traditional starters, the physicochemical properties of the starters, including pH and DRC, are also of interest. Sourdough fermentation may take longer than baker’s yeast fermentation, possibly because of the time required for growth of the lactic acid bacteria. Sourdough fermentation at home or in bakeries may involve a normal fermentation with active starters that can be maintained easily by regenerating them after each kneading operation.

It has previously been reported that lactic acid fermentation can reduce phytate levels in bread fermentation [8,9]. Khetarpaul and Chauhan have reported that mixed cultures of yeasts and lactobacilli may reduce the phytate content of bread [10].

The phytase activity found in the strains of lactic acid bacteria isolated from those starters showing high phytate reduction rates points to the existence of active strains of these microorganisms in traditional starters.

The microbiota of traditional sourdough starters is complicated and in most cases not well understood. Control of fermentation may be achieved by using pure culture starters as a substitute for traditional starters to direct chemical reactions through a defined biotechnological process. Zyta [11] has reported on the use of mould phytases in the food industry and Wang et al. [12] have reported on their application in fermented oriental foods.

The use of sourdough starters with high phytase activities, high DRC and low pH is highly suited to the bread-making process. The reduction of phytic acid by the traditional starter culture during fermentation is more practical and easier to maintain and to apply in bakeries than is the use of the more complicated two starters and enzymes.

Bread fermentation is better served by using species of lactic acid bacteria and yeasts not only for the organoleptic and gustatory benefits, but also for the enhanced nutritional properties such as phytate destruction and nutrient formation in the dough during the fermentation process.

Some inherent factors in fermentation, including pH and time of fermentation, may affect the destruction of phytates [13,14]. Some representative unidentified isolates of Lactobacillus, Leuconostoc and yeasts were inoculated in pure cultures separately and in combination with wheat flour doughs to study their hydrolyzing activity on phytic acid. In our results, phytic acid hydrolysis and dough characteristics showed evidence of net phytase activity in some combined cultures of the lactic acid bacteria and yeasts. The combination of Lactobacillus and Leuconostoc strains with the yeast strains showed a high phytate reduction (85.4%). This correlated with the traditional sourdough starters and is similar to the activity of these starters since the three microorganisms are combined in the pure culture starters to simulate the existing microorganisms in the traditional starter.

14. Tangkongchitr U, Seib PA, Hoseney RC. 1981. Phytic acid II. Its fate during bread making. Cereal chemistry, 1981, 58: 229–34.