M. Fredrikson Department of Food Science, Chalmers University of Technology, PO Box 5401, S-402 29 Göteborg, Sweden (e-mail: firstname.lastname@example.org).
Aims: To screen micro-organisms for the ability to produce phytase enzyme(s) and to use promising strains for the fermentation of pea flour.
Methods and Results: Two methods using the indirect estimation of phytate degradation were evaluated and both shown to be inadequate. A third method, measuring the inositol phosphate (IP3–IP6) content directly during fermentation, was used instead of the indirect estimations of phytate degradation. In synthetic media, some strains required customized conditions, with no accessible phosphorus sources other than phytate, to express phytase activity. The repression of phytase synthesis by inorganic phosphorus was not detected during fermentation with pea flour as substrate and seemed to be less significant with a higher composition complexity of the substrate. None of the tested lactic acid bacteria strains showed phytase activity.
Conclusions: The methodology for the phytase screening procedure was shown to be critical. Some of the screening methods and media used in previous publications were found to be inadequate.
Significance and Impact of the Study: This paper highlights the pitfalls and difficulties in the evaluation of phytase production by micro-organisms. The study is of great importance for future studies in this area.
Fermentation is widely used to improve the nutritional and functional qualities of food products. Since the characteristics of the micro-organisms are crucial for the fermentation result, great efforts are made to find micro-organisms with specific desired characteristics. This work concentrated on finding micro-organisms with the ability to produce phytase enzyme for phytate degradation. Phytate is a recognized inhibitor of iron and zinc absorption in man (Cheryan 1980; Hallberg et al. 1989; Sandberg et al. 1993; Sandberg et al. 1999) and has also been shown to decrease protein availability in peas (Carnovale et al. 1988). Due to these nutritional consequences, the degradation of phytate in food products by fermentation is desirable.
Screening procedures in synthetic media (SM) were initiated in order to find micro-organisms with a phytase-producing capacity. The application of the most promising strains was then performed using pea (Pisum sativum) flour, where phytate is the most abundant antinutritional factor, as substrate. The focus was primarily on lactic acid bacteria (LAB) strains, but other types of micro-organisms, such as Tempeh fungi, were also evaluated. Lactic acid bacteria were of interest because of the possibility of adding the phytase-producing quality to their previously known positive characteristics. A genetically modified yeast strain (Saccharomyces cerevisiae), constitutively expressing phytase, was also compared with the wild type yeast strain with regard to phytate degradation in pea flour.
A number of papers have been published regarding phytate degradation by fermentation. This paper highlights the pitfalls, difficulties and general aspects of the evaluation of phytase production by micro-organisms.
MATERIALS AND METHODS
Analytical methods and chemicals
Sample preparation and the inositol phosphate (IP3–IP6) analyses were performed according to Skoglund et al. (1997) with some modifications to the sample preparation. Pea flour and semisolid samples (0·5 g) were extracted with 20 ml 0·5 mol l−1 HCl for 3 h. The SM and semiliquid medium (SLM) samples were prepared by adding HCl to a final concentration of 0·5 mol l−1 HCl. The mixtures were then stirred, centrifuged and 400 μl of the supernatant fluid ultrafiltered through centrifugal filters (Microcon YM-30; Millipore, Bedford, MA, USA). The filtrate was then injected into the ion chromatography (HPIC) analysis system, consisting of a biocompatible high performance liquid chromatography pump (Model 626; Waters Associate, Mileford, MA, USA), a 50-μl injector loop, a Carbo-Pac PA-100 (4 × 250 mm) analytical column, a PA-100 (4 × 50 mm) guard column (Dionex, Sunnyvale, CA, USA) and a tuneable absorbance detector (486; Waters Associate). The analyses of inorganic PO4 were performed by centrifuging the liquid sample. The supernatant fluid was ultrafiltered through a centrifugal filter (Microcon YM-30; Millipore) and the filtrate then injected into an ion chromatographic system (4500I; Dionex), equipped with a guard column (PAX-100), analytical column (PAX-100), micro membrane suppresser (AMMS-II) and a conductivity detector. The chromatographic run was performed with a linear gradient of water and 0·2 mol l−1 NaOH in the mobile phase, which also contained isocratic 2% 50/50 isopropanol.
The final biomass yield of fungi in SM3a–SM3d was quantified by collecting 50 ml medium for each sample. The samples were membrane filtered and washed with 0·9% NaCl before determination of the dry weight. The microbial growth in pea flour samples was estimated as colony-forming units (cfu) on agar plates. The agar media plates were customized according to the type of strain and method of precultivation. Duplicate samples were analysed and all results correspond to the dry weight of the sample, unless otherwise stated.
Deionized water was purified by a water system (Millipore) to a specific resistance of 18 M cm−1 or greater. Sodium phytate (Aldrich Chemical, Milwaukee, WI, USA) was used as the IP6 source, unless otherwise stated. Calcium phytate and potassium phytate were obtained from Sigma Chemical (St Louis, MO, USA). All other reagents used were of analytical grade and obtained from commercial sources.
Screening methods and culture conditions
Three methods were used for the phytase activity screenings of micro-organisms. The first was based on the papers by Howson and Davis (1983) and Shieh and Ware (1968) who used opaque agar plates containing calcium phytate (media SM1a and SM1b, see below). The formation of transparent zones around the microbial colonies during growth was supposed to indicate phytase activity. Strains able to produce transparent zones were then fermented in the liquid media without agar, which were then analysed for IP3–IP6 content.
The second method was based on the work of Shirai et al. (1994), where the growth of micro-organisms during fermentation was measured as optical density (O.D.) at 610 nm (media SM2a and SM2b, see below). The increasing O.D. during fermentation was proportional to the microbial growth. The strains were evaluated by comparing the growth rate in media containing phytate as the sole phosphorus source with media containing inorganic phosphorus. Fermentations where strains were able to grow well on phytate media were then analysed for IP3–IP6 content.
The third method was a modification of the second, with the medium further customized for the study of phytate degradation (SM3). The IP3–IP6 content was measured during fermentation instead of using an indirect estimation of phytate degradation.
Solid synthetic media.
Synthetic solid medium 1a (SM1a) for fungi contained (g l−1): glucose, 15; Ca-phytate, 5; NH4NO3, 5; MgSO4.7H2O, 0·5; KCl, 0·5; FeSO4.7H2O, 0·01; MnSO4.H2O, 0·01 and agar, 15–20. Synthetic medium 1b (SM1b) was modified to suit LAB and was similar to SM1a but with the addition of 10 g peptone and 5 g yeast nitrogen base. Control agar plate media were made by replacing Ca-phytate with Ca3(PO4)2. An additional control medium was made by replacing Ca-phytate with 2 g K2HPO4 and 2 g KH2PO4. This medium was transparent and used only for the comparison of growth ability. SM1 was autoclaved at 121°C for 20 min.
Liquid synthetic media.
Synthetic medium 2a (SM2a) consisted of (g l−1): demineralized whey, 10; Na-acetate, 5; K-phytate, 0·6 and glucose, 10. In SM2b used for LAB, the glucose was replaced by 10 g lactose. SM2 was autoclaved at 121°C for 20 min.
Synthetic medium 3 (SM3) was prepared according to Tynkkynen et al. (1989), with some modifications. To reduce contamination of inorganic phosphorus, the buffer was excluded, the phytate content minimized and the phytate solution filter sterilized (0·22 μm) instead of autoclaving. This medium was called SM3a. To further evaluate certain strains, three additional variations of the SM3 medium with different IP6 and/or PO4 contents were used: SM3a, 0·8 mmol l−1 IP6 as the sole phosphorus source; SM3b, combination of 0·8 mmol l−1 IP6 and 26 mmol l−1 PO4 (as K2HPO4 and KH2PO4); SM3c, 6·7 mmol l−1 IP6 as the sole phosphorus source and SM3d, 26 mmol l−1 PO4 (as K2HPO4 and KH2PO4) as the sole phosphorus source.
These SM3a–SM3d fermentations were performed for 10 d and, in addition to IP6 degradation, the final biomass yield was also estimated. This experiment was only performed with single samples, hence the results are not given as absolute numbers.
Pea flour media.
Semiliquid pea flour medium SLMa was prepared by mixing approx. 5 g pea flour with 100 ml water, followed by autoclaving for 30 min at 121°C; glucose was then added to a final concentration of 1%. Semiliquid pea flour medium SLMb was prepared in the same way but with the addition of KH2PO4, corresponding to approx. 10 mmol l−1 PO4, before autoclaving. The reference samples were not inoculated. The inocula were added at levels corresponding to 0·1 O.D. in the pea flour samples, except for fungal strains D192 and D642 which formed spherical pellet colonies in the preculture, making O.D. determination impossible. Instead, colonies were picked one by one, washed and then added to the pea flour samples.
Semisolid medium (SSM) with equal parts pea flour and water was also used. The samples were autoclaved at 121°C for 20 min before inoculation, the total sample size was 100 g and the reference samples were not inoculated.
Saccharomyces cerevisiae (SKQ, YS18 and YS-pPHO5) was precultivated in yeast extract peptone dextrose (YPD) medium (Difco, Detroit, MI, USA) at 30°C. Rhizopus microsporus var. oligosporus (D192) was precultivated in potato dextrose (Difco) and YPD media at 25°C. Aspergillus ficuum (D642) and Geotrichum candidum (D648) were precultured in 2% Malt Extract (ME) medium (Difco) at 30°C. The ME medium consisted of (g l−1): malt extract, 20; Bacto peptone, 1 and glucose, 20. Escherichia coli (E998) was precultivated in LB broth (Difco) at 30°C and LAB strains in De Man Rogosa Sharpe (MRS) broth (Oxoid, Basingstoke, UK) at 30°C. The precultivations were performed until sufficient cell densities were obtained.
The inocula were washed twice in 0·9% NaCl before addition to different media or pea flour samples. Experimental cultures were stirred in a rotary shaker at 200 rev min−1 except in the SSM experiments. In addition to the strains listed in Table 1, approx. 30 LAB strains were evaluated for phytase synthesis in SM and pea flour media. A majority of the LAB originated from food products, such as beer, fermentation of olives, tomatoes, bread, beestings and cereals. One strain was isolated from humans and some were of commercial origin (Chr. Hansen A/S, Copenhagen, Denmark).
Table 1. The phytase-producing micro-organisms used in experiments*
Evaluation of screening methods
When using the method of Howson and Davis (1983) and Shieh and Ware (1968) no correlation could be found between the formation of transparent zones in opaque agar plates and phytate degradation. By reproducing this assay in this study it was demonstrated that, by autoclaving the media, calcium phytate was hydrolysed resulting in reduced levels of IP6 and, accordingly, the release of a corresponding amount of inorganic phosphorus (Table 2). The release of inorganic phosphorus resulted in the formation of calcium phosphate complexes which kept the agar plates opaque. The formation of transparent zones was, therefore, a result of micro-organisms consuming the calcium phosphate complexes instead of calcium phytate, as previously suggested. Thus, the formation of transparent zones cannot be used as evidence of phytate degradation. Control samples, with added Ca3(PO4)2 instead of calcium phytate, gave an identical visual effect on the agar plates with respect to the formation of clear zones around colonies. The proportion of IP6 hydrolysed by autoclaving may vary depending on the type of medium, batch size, concentration of IP6 and the autoclaving time and temperature (Table 2 shows two examples).
Table 2. The hydrolysis of IP6 by autoclaving (121°C for 20 min) of synthetic medium
Synthetic liquid media assay.
When using transparent media and monitoring the growth by measurement of O.D., the data obtained were consistent with the first method, i.e. the growth of LAB in SM2 over 48 h did not correlate with degradation of phytate. The LAB consumed phosphorus originating as contamination from media ingredients and the hydrolysis of IP6 during autoclaving. The LAB strains used all inorganic phosphorus but never succeeded in utilizing phytate as a phosphorus source (Table 3). In contrast, the E. coli and A. ficuum strains succeeded in degrading the phytate, which also resulted in an increased amount of inorganic phosphorus. It was necessary to use medium SM3a with a filter-sterilized phytate solution to minimize the growth made possible by phosphorus contamination. The fermentations in SM3a were performed for 72 h and phytase activity was detected for fungal strains D192, D642, D648 and E. coli E998. The phytate degradation by strains D192 and D648 was insignificant and difficult to quantify and, therefore, the lower inositol phosphates (IP5–IP3) were analysed. Escherichia coli E998 was used as a control strain as it was known to be a phytase producer (Greiner et al. 1993).
Table 3. The concentration of IP6 and PO43− before and after fermentation in synthetic media
Strains D192, D642 and D648 were chosen for fermentation for 10 d in media SM3a–SM3d in order to further study their phytase synthesis ability. By using such long fermentation times phytate degradation was obtained in all strains and in all phytate-containing media (data not shown). The growth of LAB strains did not result in phytate degradation in any SM.
Strains D648, E998 and SKQ grew well in the SLM, with rapid growth during the first 15–20 h (Fig. 1). As seen in Fig. 2 and Table 4, the experiments using SLM resulted in dramatic phytate degradation, despite a high inorganic phosphorus content in SLM samples. However, this increased phytase activity in SLM was not found for the LAB strains. The variation in initial sample composition meant that the phytate and inorganic PO4 contents could not be compared between experiments, so each fermentation was evaluated separately. The quantification of microbial growth by colony count did not work well using pea flour samples with strains D192 and D642. However, the increased growth during fermentation was evident when other parameters (pH, PO4 and IP6) were monitored. The growth was also visually obvious due to the massive formation of spherical mycelia containing pellet colonies with approximate diameters ranging from 1 to 10 mm. No microbial growth or change in pH, PO4 content or phytate degradation was observed in the autoclaved control flasks, indicating that the autoclaving was efficient in eliminating the endogenous microbial flora and the endogenous pea phytase activity. The microbial growth caused a lowering of pH for all strains except D648 (Fig. 3). As seen in Fig. 4, the PO4 content increased during fermentation with strains E998, D642 and D648, suggesting that their phytase activities were higher than their PO4 demands. Saccharomyces cerevisiae SKQ and A. niger D192 consumed inorganic phosphorus and degraded phytate simultaneously during fermentation which may indicate high PO4 requirements or a tendency to store phosphorus. Yeasts, for example, are known to store large quantities of polyphosphate.
Table 4. The concentration of IP6 (μmol l−1) during semiliquid pea flour fermentation at 30°C using fungal strains Rhizopus oligosporus (D192) and Aspergillus ficuum (D642)
The addition of PO4 to SLMb did not cause any noticeable phytase repression. As seen in Table 5, both yeast strains YS18 and YS-pPHO5 dramatically reduced the IP6 content during fermentation. The strains showed similar pH and growth patterns, reaching about 108 cfu ml−1 in 24 h. This growth yield is lower than that for SKQ in the previous experiment and can be explained by the difference in strain characteristics rather than the addition of PO4. These fermentations did not cause any significant changes in PO4 levels due to the high initial content of PO4.
Table 5. The pH and concentration of IP6 (μmol l−1) during semiliquid pea flour fermentation with PO43− addition (10 mmol l−1) at 30°C
Phytate degradation was obtained for strains D192 and D648 with autoclaved SSM (Table 6). The phytate degradation in the reference sample suggests that the autoclaving did not completely inactivate the endogenous pea phytase.
Table 6. The concentration of IP6 (μmol IP6 g−1 pea flour) during fermentation of autoclaved semisolid pea flour at 30°C
This study has shown the importance of adequate methodology for assessing the microbial production and activity of phytase. When using SM, the appropriate content of phytate and elimination of phosphorus contamination may be necessary. When dealing with low phytase activity, direct IP6–IP3 quantification is a superior analysis method due to the possibility of detecting the formation of lower IPs. The indirect method of Howson and Davis (1983) and Shieh and Ware (1968) was found to be a poor indicator of microbial phytase activity. When using SM, phytase synthesis seems to be more easily repressed by inorganic phosphorus, in contrast to more complex media such as semiliquid pea flour. For some strains the fermentation had to continue for very long periods, such as 10 d, in order to obtain substantial phytate degradation in SM3. This may indicate a minor constitutive production of phytase that requires a long incubation time to degrade substantial amounts of phytate. By using several combinations of phytate and inorganic phosphorus content the final biomass yield and phytase repression could be compared. Single samples were used but the tested strains showed identical trends for biomass and IP6 degradation in the different media. Therefore, the results can be judged and compared as relative results. The highest phytate degradation was obtained using medium SM3b, containing both inorganic phosphorus and phytate, which may be interpreted as showing that the initial microbial growth is facilitated by access to inorganic phosphorus and the phytase is synthesized as the inorganic phosphorus content decreases. Medium 3b, with both PO4 and phytate, resulted in a lower biomass yield than medium 3d, with solely PO4, indicating that the use of IP6 may reduce final biomass yield.
The results with SLM clearly showed that access to inorganic phosphorus was not sufficient to repress phytase synthesis. This can be interpreted as either the repression by inorganic phosphorus not being as effective in this sample matrix or the synthesis of phytase being promoted by a component in the pea flour which is absent in the SM. Several reports have been published regarding the repression of phytase synthesis in micro-organisms by inorganic phosphorus, e.g. Shieh et al. (1969) and Shieh and Ware (1968). The yeast strains SKQ and YS18 produced phytase in SLM, in contrast to previous results (unpublished) where the PO4-induced repression of phytase synthesis in CBS medium was very pronounced. Not even the addition of PO4 in SLMb induced phytase repression. In contrast to previous data (unpublished) using the yeast medium YPD, the yeast strain YS-pPHO5 with constitutive phytase expression did not show the expected superior phytate degradation in SLMb compared with the wild type strain YS18. This behaviour may be explained by the possibility of the strains producing several phytase enzymes, with different expression regulation. Recent data have demonstrated the presence of multiple phytase enzymes in S. cerevisiae (unpublished).
Han et al. (1987) showed the importance of the moisture content, finding optimal phytase production at 25–35% moisture. However, the moisture contents in SLM and SM were about 95% or higher. Papagianni et al. (1999) explained the increased phytase activity after the addition of wheat bran in two types of media during A. niger fermentation by the idea that providing a slow-release organic phosphorus source could reverse phosphorus repression. However, this theory fails to explain the present results since the SLM is completely dissolved and homogeneous and a slow release of organic phosphorus is unlikely. The repression of phytase synthesis by inorganic phosphorus seems to be less significant with higher medium composition complexities. It is not known, however, what components in the complex media account for the reduced repression.
The phytate degradation was less complete when using semisolid pea flour because the SSM contained larger amounts of phytate. It could also be explained by slow microbial growth in the SSM. This emphasizes the importance of having sufficiently high water activity and medium homogeneity for proper growth during fermentation. The phytate degradation in the reference sample (Table 6) suggests that autoclaving did not completely inactivate the endogenous pea phytase. The results suggest semisolid pea flour to be an unsuitable substrate for the tested micro-organisms.
The application of micro-organisms in vegetable samples presented special difficulties. The exclusion of endogenous phytase sources is fundamental to ensure that the phytase activity originated from the micro-organisms. The accessibility of inorganic phosphorus in the samples may repress the microbial phytase production. Furthermore, almost complete phytate degradation is required to improve iron bioavailability (Simpson et al. 1981; Brune et al. 1992; Hurrell et al. 1992), which implies high microbial phytase production. Several previously published effects of phytate degradation during the fermentation of vegetable food can be explained by the activity of the endogenous phytase. The fermentation may increase the accessibility of phytase and phytate through proteolysis and starch hydrolysis by microbial enzymes. The pH change during fermentation is also important due to the effect on protein solubility and the activity of the endogenous enzymes.
The phytase of E. coli E998 has been studied previously by Greiner et al. (1993), who concluded that it was a periplasmic enzyme. The activity is reported to be optimal at 55°C and pH 4·5, which indicates that the phytase activity is not perfectly adapted to the optimal growth temperature of the host.
No satisfactory scientific evidence showing degradation of phytate by a wild type LAB has been presented with regard to phytase production by LAB. The tested LAB strains seem to require less phosphorus for growth than the other strains. Lactic acid bacteria are adapted to environments rich in nutrients and energy and, therefore, have dispensed with their biosynthetic capability (Axelsson 1998). Due to the rich environments where LAB exist, there may never have been an evolutionary selection of LAB with respect to phytate-degrading capacity. Thus, to date it is uncertain whether there are any wild type LAB with the ability to produce a phytate-degrading enzyme. Even if there are any LAB with a phytase-producing ability, the production of sufficient phytase enzyme for efficient phytate degradation in food is unlikely. The use of gene techniques for insertion of the necessary genes for phytase production into LAB is likely to be more successful than screening for naturally occurring phytase-producing LAB strains.
The authors thank Kristina Hasselblad and Nils-Gunnar Carlsson (Department of Food Science, Chalmers University of Technology, Göteborg, Sweden) for discussion and excellent technical assistance. Many thanks are also due to Arja Laitila, Erja Malinen, Eija Lemola, Kaisa Saloniemi and Pirjo Tähtinen (VTT, Espoo, Finland) for planning and performing experiments. Gerd Jonsson (SIK, The Swedish Institute for Food and Biotechnology, Göteburg, Sweden) is acknowledged for technical assistance. This work was supported by Swedish Council for Forestry and Agricultural Research, project no. 50.0473/98, and FAIR CT 95–0193 and is part of NUTRIPEA C1004-95.