Volume 8
Issue 4
Biotechnology
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Available Online: http://www.ejpau.media.pl/volume8/issue4/art-76.html
FUNGAL PHYTASES IN WHOLEMEAL BREADMAKING III: NUTRITIONAL EFFECTS OF DIFFERENT PHYTASES IN RYE BREADS
Krzysztof Żyła1, Magdalena Mika1, Halina Gambu¶2, Anna Nowotny2, Beata Szymczyk3
1 Department of Food Biotechnology,
University of Agriculture in Cracow, Poland
2 Department of Carbohydrates Technology,
Agricultural University of Cracow, Poland
3 National Research Institute of Animal Production
Department of Animal Nutrition and Feed Science in Aleksandrowice, Poland
Nutrient digestibility in rye breads baked with the addition of microbial 3- and 6-phytases A at 5000 FTU/kg, and their combinations with phytase B at 30 AcPU/g, were evaluated in vitro and in growing rats. In comparison to rye flour, phytate content in the control bread, bread baked with 3-phytase A, and bread baked with 6-phytase A, was reduced by 91, 91, and 98 %, respectively. 3-Phytase A enhanced phosphorus retention and metabolic balances in rats and exerted detrimental effects on metabolic parameters of calcium. The effect was stimulated by a subsequent addition of phytase B. The opposite was true for 6-phytase A that repressed phosphorus and preserved calcium metabolic balances in rats. As compared to rats fed control breads, improved HDL/TC ratios were found in plasma of animals that consumed breads prepared with 3- or 6- phytase A, or with 6-phytase A and phytase B. Rats fed breads supplemented with 6-phytase A and phytase B had lowered contents of total lipids, triacyloglicerols, and total cholesterol in sera. Microbial phytases, and particularly 6-phytase A and phytase B, open new perspectives in applications of biocatalysts in baking.
Key words: rye bread, phytase A, phytase B, phytate, rat, calcium metabolism, serum cholesterol.
INTRODUCTION
Rye bread is known to possess the ability to decrease serum total and LDL cholesterol in humans [11], to beneficially affect the postprandial insulin response [8] and to decrease the concentrations of compounds that are putative colon cancer risk markers in middle-aged individuals [4]. In previous studies [24] we reported that enzymatically modified rye breads produced with the addition of fungal 6-phytase A and phytase B offered important advantages of enhanced texture in addition to increased loaf volumes. It was found also that in contrast to rye breads supplemented with a standard 3-phytase A, breads baked with 6-phytase A had fat content lowered by more than fifty percent. A less pronounced decrease in fat concentration of the wholemeal rye bread was observed also in breads supplemented with 0.2 % of pure myo-inositol. The purpose of the current investigation was to evaluate the extent of phytate breakdown in breads supplemented with different phytases and to learn changes in in vitro nutrient digestibility and in phosphorus and calcium metabolic balances in rats. Yet another objective of the study was to find possible changes in lipid profile of rats fed enzymatically modified rye breads.
MATERIALS AND METHODS
Materials
Wholemeal rye breads were baked using materials, enzymes and methods described previously [24]. Test kits for determination of lipids in sera were purchased from Olympus Diagnostica GmbH, Hamburg, Germany.
Determination of myo-inositol phosphates
Inositol phosphates (IP6-4) were determined by a modified Lehrfeld procedure [10] using ion-pair C18 reverse phase HPLC with refractive index detection as detailed previously [25].
In vitro determination of nutrients digestibility
An in vitro procedure designed to simulate digestion in the human intestinal tract was applied as described previously [25].
In vivo experiment on rats
The experiment was conducted following the Guidelines for Animal Care and Treatment set by the Council of European Communities and was approved by the local Animal Ethics Commission. Growing male albino Wistar rats, at 6 wk of age, weighing 100 ± 1.5 g were housed in stainless steel cages, maintained at 25°C, with 12 h light: dark cycle and unlimited access to feed and water. After adaptation period, the animals were randomly divided into six dietary treatments of five animals and fed bread powders as the sole dietary component. During the second week of feeding a total collection of faeces and urine was performed and body weights and food intakes were recorded. Performance parameters were recorded also for the second experimental period from week 3 to 5. Biological indices, and namely, apparent digestibility coefficient (ADC) for calcium and phosphorus, calcium and phosphorus retention (balance), and percent retention per absorption (% R/A) were calculated as described in details by Nestares with co-workers [16]. Additionally, we calculated balance/intake ratio (bal/int) for both phosphorus and calcium. At the completion of the experiment rats were deprived of food overnight, anesthetized intraperitoneally with sodium tiophenal (Biochemie, Viena, 25 mg/100 g body weight) and killed by withdrawing blood from heart. Blood samples were centrifuged (4000 g, 10 min) and the serum samples were analysed using commercially available test kits for total cholesterol (TC), HDL-cholesterol (HDL), and triacyloglicerols (TG). The concentration of total lipids in serum was determined enzymatically using standard kits (Cormay) on a Beckman DU640 spectrophotometer.
Statistical analyses
Data were subjected to one-way or two-way analysis of variance using Statgraphics Plus for Windows 5.1 statistical package. Mean differences were determined using Fisher’s least significant difference test. Statistical significance was accepted at p < 0.05.
RESULTS AND DISCUSSION
Effects of two forms of phytase A, alone, and in combination with phytase B, on phytate hydrolysis and on in vitro digestibility of nutrients in wholemeal rye breads
Less than 10 % of initial phytate (IP6) content in rye flour was found in breads made without added enzymes (Table 1). Assuming negligible role played by the 3-phytase of yeast in dephosphorylation of rye phytates during breadmaking, endogenous rye phytate-degrading enzymes (phytase and acid phosphatases) must have performed initial phases of phytate hydrolysis in control breads. Much higher extent of dephosphorylation (91 %) attained in rye breads as compared to wheat breads [25] may be explained by a two-fold higher endogenous phytases A, and a three-fold higher endogenous acid phosphatases (phytase B) activity in rye than in wheat [22, 3, 5]. In breads prepared with 6-phytase A, additional, substantial reduction in phytate was observed, whereas 3-phytase A did not change the content of IP6 in experimental breads. The addition of phytase B along with 3-phytase A reduced phytate concentration by about one third and simultaneous application of 6-phytase A and phytase B caused complete conversion of phytate into lower phosphate esters of myo-inositol. In comparison to their original content in rye flour, inositol pentaphosphates (IP5) concentrations in rye breads made without exogenous enzymes were higher, whereas in breads prepared with different kinds of phytase were similar, or higher. Centeno with co-workers observed complete conversion of inositol pentaphosphates into lower esters of myo-inositol during germination of rye grains [3]. Myo-inositol phosphate esters accumulated by the action of microbial 6-phytase A were probably similar to intermediates produced by the phytase of rye, whereas microbial 3-phytase A surely produced different kind of inositol phosphates. Breads made with microbial 6-phytase A and the others prepared with 3-phytase A must have differed therefore, in the composition of myo-inositol phosphates. Among products released by the action of 6-phytases, myo-inositol 1,2,3,6- and 1,2,3,4- tetrakis(phosphates), as well as myo-inositol 1,2,3-tris(phosphates) have been reported in several studies [15, 17], although Konietzny and Greiner [9] provided contradictory data. These substances with the preserved 1,2,3 array of phosphate moieties on the myo-inositol ring are known to possess powerful antioxidant function [18], to enhance calcium absorption as well as to act as second messengers of the signal transduction systems [20]. From nutritional standpoint important question arises whether the metabolically active intermediates of phytate degradation are used up by growing yeast population or are retained in baked breads. Research to clarify the issue is in progress.
Table 1. In vitro nutrient digestibilities and myo-inositol hexa- and pentaphosphates content in wholemeal rye breads made with different forms of phytases |
|
Bread with added ingredient |
ANOVA |
|||||
none |
3-A |
6-A |
3A+B |
6A+B |
p |
SEM |
|
IP6 (%) |
0.103 |
0.100 |
0.025 |
0.030 |
n.d. |
||
IP5 (%) |
0.145 |
0.072 |
0.041 |
0.023 |
0.056 |
||
IP4 (%) |
0.092 |
n.d. |
n.d |
n.d. |
0.074 |
||
P dial (mg/g) |
1.138a |
1.313b |
1.359b |
1.527c |
1.596d |
0.0154 |
0.0001 |
Ca dial (mg/g) |
0.379b |
0.354a |
0.384b |
0.354a |
0.361a |
0.0059 |
0.0035 |
Protein dial (mg/g) |
80.80a |
81.95a |
83.03a |
88.04b |
89.72b |
1.068 |
0.0001 |
Sugars dial (mg/g) |
355.6a |
360.5ab |
349.12a |
378.3b |
378.8b |
6.366 |
0.0267 |
Amounts of IP6 and IP5 in the wholemeal rye flour were 1.104%, and 0.037%, respectively Values are means of five determinations a-e – Means within rows with no common superscript letter differ significantly at p<0.05 n.d. – not detected |
Dephosphorylation of breads was clearly reflected in increased concentrations of phosphorus released from breads during in vitro digestions. Breads prepared with 6-phytase A had the contents of dialyzable calcium similar to breads produced without enzymes whereas in all other kinds of breads the concentration of dialyzable calcium was significantly reduced. The application of phytases A enhanced concentrations of dialyzable protein and reducing sugars and simultaneous application of phytases A and B additionally increased amounts of these compounds freed from rye breads by the in vitro digestion method.
Effects of wholemeal rye breads baked with two forms of phytase A, phytase B, and myo-inositol on growth performance and on phosphorus and calcium metabolic balance in growing rats
There were significant effects of experimental breads on rats’ growth performance (Table 2). Animals fed the control breads or breads made with 3-phytase A, alone or in combination with phytase B, had feed intakes superior to those fed breads prepared with 6-phytase A or with myo-inositol. The feed intakes were not reflected, however, in daily weight gains, final body weights and feed efficiencies of the animals tested, since in rats consuming breads made with 6-phytase A these parameters did not differ from those observed in rats fed breads prepared without additives or with 3-phytase A. The addition of phytase B on top of 3-phytase A did not significantly change growth performance of rats, but phytase B added along with 6-phytase A additionally reduced certain performance parameters in rats. The lowest values of performance parameters were observed in rats fed bread made with the addition of myo-inositol. Rats that consumed rye breads enriched with 6-phytase A had relatively low feed intakes, but they also had a superior feed efficiency, and, in consequence, gained normal body weights. The “high density” of nutrients in breads made with 6-phytase A cannot be related to any of the digestion parameters measured in vitro.
Table 2. Growth performance of rats fed experimental rye breads made with different phytases or myo-inositol |
Parameter |
Bread with added ingredient |
ANOVA |
||||||
none |
3-A |
6-A |
3-A+B |
6-A+B |
inositol |
SEM |
p |
|
Feed intake (g/rat/day) |
14.58b |
14.62b |
14.03a |
14.52b |
14.17a |
14.12a |
0.0829 |
0.0001 |
Weight gain |
1.495c |
1.533c |
1.505c |
1.467bc |
1.333ab |
1.276a |
0.0503 |
0.0044 |
Final body weight (g) |
148.2bc |
149.5c |
148.3bc |
147.2bc |
143.0ab |
141.0a |
1.875 |
0.0178 |
Feed efficiency |
9.81ab |
10.12ab |
9.35a |
9.89ab |
10.70bc |
11.14c |
0.3167 |
0.0052 |
Rats were fed powdered experimental breads for 5 weeks a-c – Means within rows with no common superscript letter differ significantly at p<0.05 |
Apparent digestibility coefficients (ADC) and metabolic balances of phosphorus and calcium in rats paralleled intakes of these elements from experimental feeds (Table 3). As compared to rats consuming the control breads, the addition of 3-phytase A, or 6-phytase A and phytase B, or myo-inositol resulted in significant decreases in phosphorus intakes of rats. To the contrary, rats fed breads prepared with 3-phytase A and phytase B received more phosphorus than animals fed the control diets. Similar relationships between the type of phytase used in bread preparation and metabolic responses in rats consuming the breads were observed for ADC and metabolic balance of phosphorus. Changes in phosphorus retention to absorption ratios and in balance to intake coefficients suggest that breads made with 3-phytase A enhanced, while those prepared with 6-phytase A exerted detrimental effect on phosphorus metabolism in rats. For calcium metabolism, however, the opposite was true. In comparison to rats consuming breads with no enzyme added, the animals fed breads prepared with 3-phytase A had decreased values of both metabolic parameters, but those fed 6-phytase A had calcium retention to absorption ratios and balance to intake coefficients similar to rats fed the control bread. Phytase B, and myo-inositol exerted detrimental effects on all of measured parameters of phosphorus and calcium metabolism in rats. Phosphorus and calcium metabolic balances in rats consuming experimental, enzymatically modified rye breads clearly show that while microbial 3-phytase A alone, and, to a higher extent, along with phytase B, improves phosphorus metabolic parameters, 6-phytase A (endogenous, or microbial and endogenous) enhances calcium retention to absorption and balance to intake ratios. Similar observations have recently been made in growing broilers fed corn-soybean feeds supplemented with different phytases [19]. Our data confirm that in breads made with the activity of 6-phytase A (endogenous, or microbial and endogenous) not distorted by other phytase activity, calcium digestibilities measured in vitro and in growing rats were the highest.
Table 3. Phosphorus and calcium metabolic balance in rats fed rye breads made with different phytases or myo-inositol |
Bread additive |
Phosphorus Calcium |
||||||||
Intake mg/day |
ADC mg/day |
Balance mg/day |
% R/A bal/int |
Intake mg/day |
ADC mg/day |
balance mg/day |
% R/A bal/int |
||
None |
26.9c |
19.66d |
6.18c |
31.47c 0.230c |
2.23d |
1.81e |
0.794d |
43.67e 0.355d |
|
3A |
24.2b |
17.38c |
7.29d |
41.98d 0.302d |
1.99ab |
1.58c |
0.636c |
40.28d 0.319c |
|
6A |
24.1b |
17.62c |
3.85b |
21.89b 0.159b |
2.03bc |
1.53bc |
0.688c |
44.69e 0.338d |
|
3A+B |
29.1d |
24.26e |
10.81e |
44.53e 0.372e |
2.10c |
1.70d |
0.530b |
31.17b 0.252b |
|
6A+B |
22.2a |
16.57b |
3.05a |
18.43a 0.137a |
1.91a |
1.46ab |
0.506b |
34.69c 0.264b |
|
inositol |
21.8a |
15.88a |
2.89a |
18.00a 0.133a |
1.90a |
1.45a |
0.398a |
27.71a 0.209a |
|
ANOVA |
|||||||||
SEM |
0.209 |
0.125 |
0.149 |
0.672 0.0059 |
0.0327 |
0.025 |
0.0222 |
0.993 0.0089 |
|
P |
0.0001 |
0.0001 |
0.001 |
0.0001 0.0001 |
0.0001 |
0.0001 |
0.0001 |
0.0001 0.0001 |
ADC – apparent digestibility coefficient; % R/A – percent retention per absorption; bal/int – balance per intake. Rats were fed powdered experimental breads for 5 weeks. a-e – Means within columns with no common superscript letter differ significantly at p<0.05 |
Effects of rye breads produced using the two forms of phytase A and phytase B on lipids profile in blood plasma of rats.
Generally, animals consuming breads made with exogenous phytases had significantly reduced content of total cholesterol in blood plasma (Table 4). Rats fed rye breads prepared with the addition of myo-inositol had total cholesterol content in serum that did not differ from that observed in animals fed rye bread made without additives. In rats fed breads made with 3-phytase A alone, or in combination with phytase B, however, elevated levels of total lipids and triacyloglicerols were found. High levels of these compounds were observed also in sera of rats consuming breads made with myo-inositol. Animals fed rye bread prepared with 6-phytase A followed different pattern of lipid profile changes in blood serum as they had significantly lower contents of total lipids in sera than rats fed breads made with 3-phytase A. The highest values of HDL/total cholesterol ratio were observed in sera of rats fed breads prepared with 6-phytase A, 3-phytase A, or 3-phytase A and phytase B, whereas the lowest values, in rats fed control breads prepared with no additives. Rats fed breads made with 6-phytase A along with phytase B had plasma lipid profile particularly advantageous with low levels of total lipids, total cholesterol and triacyloglicerols as well as a high HDL/total cholesterol ratio. 3-Phytase A added to breads with or without phytase B exerted quite different effects on plasma lipid profile in rats.
Table 4. Effects of experimental whole rye breads made with different phytases or myo-inositol on lipids profile in blood serum of rats |
Lipid fraction |
Bread with added ingredient |
ANOVA |
||||||
none |
3-A |
6-A |
3-A+B |
6-A+B |
inositol |
SEM |
p |
|
TC (mg/dl) |
76.04d |
64.69ab |
58.12a |
67.54bc |
60.38a |
73.77cd |
2.352 |
0.0001 |
HDL (mg/ dl) |
40.95ac |
43.77bc |
36.62a |
38.92a |
40.02ab |
45.23c |
1.635 |
0.0121 |
(HDL/TC) |
0.54a |
0.68d |
0.63bd |
0.58ab |
0.66cd |
0.62bc |
0.023 |
0.0026 |
TG (mg/dl) |
30.04ab |
36.36b |
30.91ab |
36.18b |
25.63a |
35.46b |
2.357 |
0.0196 |
TL(mg/dl) |
156.83bc |
163.2c |
144.9ab |
161.2bc |
140.22a |
163.48c |
5.631 |
0.0248 |
Rats were fed powdered experimental breads for 5 weeks a-c – Means within rows with no common superscript letter differ significantly at p<0.05 |
Several nutritional factors like antioxidants [19], β-glucan [6], faba been seeds [13] phospholipid-protein complexes [14] oleic acid-rich oils [7] or dietary taurine [23] have been reported to improve lipids profile in the blood plasma of rats, but enzymatically generated myo-inositol phosphates have not been elucidated for that purpose. From possible mechanisms described in the literature that offer explanation of cholesterol lowering effects caused by different dietary components we would like to emphasize relations between the effects created by 6-phytase A and altered lipids content of rye breads [24]. Phospholipids to triacyloglicerols ratio modulated by the enzyme in yeast population growing during breadmaking [1, 2] could affect both the bile acid cholesterol cycle in rat liver, as well as change the composition of cell membranes due to changes in the signal transduction system of rat.
Although it is known that altered concentrations of plasma lipoprotein fractions are not the main arthrosclerosis risk factor [12] and rats are not regarded as a perfect model for studying the disease [21], additional dietary benefits resulting from enzymatic modifications of rye bread offer a relatively new and exciting area for research and technological modifications in baking. For proper enzymatic modifications of baking technology minimal activities of phytases causing the beneficial effects reported in this study that allow to produce whole rye bread with unique nutritional and functional properties, remain to be established in future research.
ACKNOWLEDGEMENTS
Authors would like to acknowledge financial support from the Polish Research Committee (grant Nb. AR 73/31/PBZ/021/P06/99).
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Krzysztof Żyła
Department of Food Biotechnology,
University of Agriculture in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
phone/fax: (+4812) 662 4794
email: kzyla@ar.krakow.pl
Magdalena Mika
Department of Food Biotechnology,
University of Agriculture in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Halina Gambu¶
Department of Carbohydrates Technology,
Agricultural University of Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Anna Nowotny
Department of Carbohydrates Technology,
Agricultural University of Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Beata Szymczyk
National Research Institute of Animal Production
Department of Animal Nutrition and Feed Science in Aleksandrowice, Poland
32-083 Balice, Poland
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