Journal of Food Engineering 63 (2004) 177–184 www.elsevier.com/locate/jfoodeng
Effect of fermented/germinated cowpea flour addition on the rheological and baking properties of wheat flour Elin Hallen a, S glu ß enol I_ bano
b,*
, Paul Ainsworth
c
a
c
Department of Food Science, Swedish University of Agriculture Science, 75007 Uppsala, Sweden b Department of Food Engineering, Gaziantep University, 27310 Gaziantep, Turkey Department of Food and Consumer Technology, Manchester Metropolitan University, Old Hall Lane, M14 6HR Manchester, UK Received 19 April 2003; accepted 21 July 2003
Abstract There is a growing interest in fortifying wheat flour with high lysine material, such as cowpea flour, to improve the essential amino acid balance of baked food products. The use of cowpeas as a food source has not been utilised fully, especially in developed countries. In this research, wheat flour in a standard bread formulation was partially replaced with cowpea flour, germinated cowpea flour and fermented cowpea flour at levels of 5%, 10%, 15% and 20% (wt/wt). Composite flours were analysed for ash, protein, gluten contents and a-amylase activity as well as colour, farinograph and extensograph characteristics. Bread baked from composite flours was analysed for loaf volume and weight, texture, crumb-grain structure and colour. Increasing levels of cowpea flour in the blends resulted in changed flour characteristics such as ash and protein contents and colour. It also changed farinograph and extensograph characteristics, mainly by increased water absorption. Incorporation of cowpea flour exerted a certain volume depressing effect on the bread and gave a compact structure at higher substitution levels. Overall acceptable results were obtained based on characteristics of control bread. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Cowpea flour; Fermentation; Germination; Dough properties; Rheological and baking properties
1. Introduction Cowpea (Vigna unguiculata or V. sinensis), also known as black-eyed pea, is a leguminous crop of many tropical and subtropical areas and is an important grain legume in developing countries. It contains 24–26% crude protein and is rich in glutamic acid, aspartic acid and lysine, but low in sulphur amino acids. Cowpeas are low in fat and contain no cholesterol. The lipid content ranges from 0.7% to 3.5% and unsaturated fatty acids constitute more than two thirds of the total fatty acids (Prinyawiwatkul, McWatters, Beuchat, & Phillips, 1996). Considerable interest has been generated in fortifying wheat flour with high protein, high lysine material to increase the protein content and improve the essential amino acid balance of baked products, especially bread. The high lysine content (486 mg/g nitrogen) makes *
Corresponding author. Tel.: +90-342-3601200; fax: +90-3423601100. E-mail address:
[email protected] (S ß . I_ banoglu). 0260-8774/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0260-8774(03)00298-X
cowpeas an excellent enhancer of protein quality when combined with cereal grain proteins, which are low in lysine but rich in sulphur amino acids (Prinyawiwatkul et al., 1996). Also, the low crude fat content of cowpeas removes the need for a defattening step at flour production (McWatters, 1990). Despite its high protein content, the use of cowpeas as a food source has not been utilised to its full potential, particularly in the industrialised countries, mainly due to preparatory difficulties (Sharma, Bajwa, & Nagi, 1999). The constraint has also been the presence of indigestible oligosaccharides in cowpeas (Sosulski, Elkowiez, & Reichert, 1989), particularly raffinose and stachyose. They can be hydrolysed by intestinal anaerobic micro-organisms to produce flatulence or intestinal gas (Prinyawiwatkul et al., 1996). In addition to these, anti-nutritional factors such as trypsin and chemotrypsin inhibitors, responsible for reducing the digestibility of protein by inhibiting protease activity, and haemagglutinins, have been detected (Liener, 1979). Research has shown that germination (Sathe, Deshpande, Reddy, Gell, & Salunkhe, 1983) and fermentation (Zamora &
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Nomenclature 10C 10F 10G 15C 15F 15G 20C 20F 20G 5C
wheat flour with 10% untreated control cowpea flour wheat flour with 10% fermented cowpea flour wheat flour with 10% germinated cowpea flour wheat flour with 15% untreated control cowpea flour wheat flour with 15% fermented cowpea flour wheat flour with 15% germinated cowpea flour wheat flour with 20% untreated control cowpea flour wheat flour with 20% fermented cowpea flour wheat flour with 20% germinated cowpea flour wheat flour with 5% untreated control cowpea flour
Fields, 1979) of cowpeas can not only improve nutritional quality by increasing protein content but also reduce these undesirable factors. Fermentation of cereals improves amino acid composition and vitamin content, increases protein and starch availabilities and lowers levels of anti-nutrients such as trypsin inhibitor (Chavan & Kadam, 1989). Changes in the proximate composition of germinated seeds are expected effects of germination (Ologhogbo & Fetuga, 1986) and the nutritional benefit of legumes can be improved before incorporation into legume supplemented products by utilising this (Fernandez & Berry, 1989). Germination induces an increase in free limiting amino acids and available vitamins with modified functional properties of seed components. Germination has been shown to decrease anti-nutritional factors like those mentioned above and also increase the fat, protein and crude fibre contents (Uwaegbute, Iroegbu, & Eke, 2000). The maximum nutritional benefits can be achieved by complementing cereals with cowpeas at the ratio of 45:15 (cereal:cowpea, wt/wt), which yield amino acid scores closer to the FAO/WHO/UNU standard (Prinyawiwatkul et al., 1996). However, nonglutenous protein adjuncts exert a volume depressing effect on bread when used at the relatively high levels necessary to accomplish the desired amount of fortification. They also change the absorption, mixing tolerance and other physical properties of doughs. Only small amounts of flour or isolates have been found to give acceptable volume in the end products. Fermented or unfermented cowpea flour can be used in breads, baby/weaning foods, chips, and extruded snacks. Cowpea and peanut flour have been reported to
5F 5G a b BU C F G HFN L N RH SD VC VL VR VS
wheat flour with 5% fermented cowpea flour wheat flour with 5% germinated cowpea flour hunter red–green hunter yellow–blue Brabender unit untreated control cowpea flour fermented cowpea flour germinated cowpea flour Hagberg falling number hunter lightness Newtons relative humidity standard deviation volume of container loaf volume volume of rapeseeds specific volume
successfully replace up to 20% wheat flour in cookies (McWatters, 1978) and doughnuts (McWatters, 1982a, 1982b), at least 43% in muffins and up to 20% in bread (Mustafa, Al-Wessali, Al-Basha, & Al-Amir, 1986). The purpose of this research is to partially replace wheat flour in a standard bread formulation with germinated, fermented and unfermented (control) cowpea flour to improve protein quality and quantity without affecting loaf volume and overall acceptability.
2. Materials and methods 2.1. Cowpea flour production Cowpeas were subjected to natural lactic acid fermentation and germination followed by grinding to a fine flour. Flour from untreated cowpeas was produced as control to compare any changed properties that might occur due to the two pre-treatments. Wheat flour was partially substituted with these flours at levels of 5%, 10%, 15% and 20% by weight. Properties of the resulting composite flours were determined, as well as properties of the bread produced from composite flours. 2.1.1. Control cowpea flour (C) Cowpeas were washed and then dried on perforated trays at 50 °C for 24 h. They were ground to fine flour through a 1 mm mesh screen using a hammer mill. 2.1.2. Germinated cowpea flour (G) Cowpeas were washed in cold running water and soaked in tap water for 8 h at room temperature. The
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hydrated seeds were spread on trays lined with previously sterilised moist muslin sheets and covered with aluminium foil. Germination went on for three days in an incubator at 25 °C. They were dried at 50 °C for three days, after which formed roots and testa were rubbed off. Dried, germinated seeds were ground through a 1 mm mesh screen. 2.1.3. Fermented cowpea flour (F) Unfermented, washed and dried cowpeas (as in Section 2.1.2) were ground through a 1 mm mesh screen. The flour was then mixed with water (1:4, wt/wt) to form a slurry followed by and addition of 5% sugar by weight of flour. The slurry was left to ferment in trays at 25 °C for four days until the pH of the slurry reached 5.50. The fermented slurry was dried at 50 °C and then ground through a 1 mm mesh screen to produce fermented cowpea flour. 2.2. Tests on composite flours 2.2.1. Rheological analysis Measurements of water absorption, dough development time, dough stability, and degree of softening were made by using a farinograph (model 8 101, Brabender OHG, Duisburg, Germany) according to AACC 54-21 method (Approved methods of the American association of cereal chemists, 2000). Dough extensibility and maximum resistance to a extension were determined using an extensometer (model 8 600, Brabender OHG, Duisburg, Germany) using AACC method 54-10 (Approved methods of the American association of cereal chemists, 2000). Water absorption is the amount of water that the flour can absorb until the dough consistency reaches 500 BU. Dough development time is the time required for the curve to reach its maximum height (i.e. 500 BU). Dough stability is the time needed before the dough consistency starts to decline from 500 BU line. Dough weakening is the reduction in the dough consistency from 500 BU line after 5 min. The farinograph records how far the dough stretches before breaking. This distance (in cm) on the chart is termed extensibility and is a measure of dough elasticity. The maximum force reached as the dough stretches is known as the dough resistance. 2.2.2. Gluten content Ten gram of flour was mixed to a stiff dough with 5 ml water. The dough was placed in the washing chamber of the gluten washer and run for 15 min. The gluten ball was gently kneaded under a tap with coldrunning water to wash away any remaining starch or coarse particles. Surplus water was removed. The gluten ball was cut into twelve pieces, placed in a tin and dried in the drying oven for 30 min at 155 °C. After cooling in a desiccator, weight of dry gluten was recorded. Tests were done in duplicates and presented as a mean value (Approved
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methods of the American association of cereal chemists, 2000). 2.2.3. a-Amylase activity The a-amylase activity was determined by measuring the HFN (AACC method 56-81B) (Approved methods of the American association of cereal chemists, 2000). A water bath was brought to the boil and kept boiling briskly. Twenty-five millilitre of room tempered, distilled water was placed in a tube and 7.0 g of flour added. To obtain a uniform suspension, a stopper was fitted and the tube shaken vigorously up and down 25 times. The tube with a stirrer was placed in the boiling water bath and the timer started immediately. After 5 s, the suspension was stirred by hand at a rate of two stirs per second for a total of 60 s. At exactly 60 s, the stirrer was released and the microswitch turned into position. When the stirrer had dropped by its own weight to the point where the microswitch was activated, the timer stopped and the falling number could be recorded. Results were presented as a mean value of three runs. The HFN gives an indication of amylase activity in the flour such that the longer it takes for the stirrer to drop on its own weight to the point where the switch is activated the lower the amylase activity. 2.2.4. Ash content Ash content was analysed according to AOAC standard method (Official methods of analysis, 1995). Results were presented as a mean value of triplicates for each composite flour. 2.2.5. Colour Flour colour was analysed using a Hunter Colour Lab colorimeter (model D 25-2, Hunter Associates Laboratory Inc., Reston Virginia, USA). In the Hunter-Lab colorimeter, the colour of a sample is denoted by the three dimensions L, a and b. The L value gives a measure of the lightness of the product colour from 100 for perfect white to zero for black, as the eye would evaluate it. The redness/greenness and yellowness/blueness are denoted by the a and b values respectively. 2.2.6. Crude protein content The protein content was analysed according to AOAC method 955.04 (Official methods of analysis, 1995). A factor of 6.25 was used to calculate crude protein content. Tests were done in triplicates. 2.3. Bread making and bread properties 2.3.1. Bread making Bread was made using a Hobart mixer according to a activated dough development technique with 1000 g flour, 20 g yeast, 18 g salt and water determined by
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farinograph to give a dough consistency of 500 BU. The dough temperature was 30 °C. Flour and salt were sieved together into the mixing bowl. Water and yeast were mixed and added to the flour with the mixer running at speed 1. After mixing for one minute, the speed was changed to no. 2 for 10 min. The dough was divided into three 460 g pieces, rounded up and left covered for 10 min. They were moulded and placed in greased tins and then placed in a prover, 42 °C/ 80%RH, for about 50–60 min until the dough piece was level with the top of the tin. The bread was baked in an oven at 230 °C for 25 min. For each flour blend three batches of dough were made, each baked to three loaves of bread.
3. Results and discussion 3.1. Results on composite flours Fig. 1 shows the ash content of the flours studied. The white wheat flour used in this study had an ash content of 0.55% (dry basis). Values for the composite flours ranged from 1.0% to 1.4% and the more wheat flour that was substituted for cowpea flour in the mixes, the higher was the ash content. Beans were not dehulled or decorticated before milling; therefore, as expected, a higher content of ash in the more substituted flours was obtained. The differently treated flours were not notably
2.3.2. Loaf volume and weight Loaf volume was measured using rapeseed replacement method after 15 min from oven (K€ oksel, Sivri, € zboy, Basßman, & Karacan, 2000). The loaf was put in O a metallic container with known volume (VC ). The container was topped up with rapeseed, the loaf removed and the volume of the rapeseed noted (VR ). Loaf volume (VL ) could then be calculated and recorded according to VL ðmlÞ ¼ VC VR After cooling 1 h, the same loaves used for measuring volume, were weighed on digital scales, W (g). Specific volume (VS ) of bread was calculated as VS ðml=gÞ ¼
VL W
Fig. 1. Ash content of composite flours (%, dry basis).
2.3.3. Crumb-grain structure Crumb-grain structure was evaluated visually and judged according to the Dallman scale 1–8 with higher Dallman scale numbers indicating smaller pores and more dense structure in bread (K€ oksel et al., 2000). 2.3.4. Colour of crumb and crust The colour of bread crumb and crust was measured using Hunter Colour Lab in the same way as for the flour (Section 2.2.5). 2.3.5. Texture of crumb Ninety minute after coming out of the oven, crumb firmness was analysed with a texture analyser (Stable Micro Systems Ltd, Godalming, UK) following AACC method 74-09 (Approved methods of the American association of cereal chemists, 2000). A return distance of 30 mm, 5 g trigger force and a 35 mm diameter cylindered aluminium probe was used. Three 2.5 cm thick slices were measured from one loaf out of each batch. Force in Newton was measured at 6.25 mm and a mean value was calculated for each loaf and then a mean of these to get a value for each blend.
Fig. 2. Crude protein content of composite flours (%, as is).
E. Hallen et al. / Journal of Food Engineering 63 (2004) 177–184
Fig. 3. Wet weight of gluten from 10 g composite flour samples.
different within each percentage group (p > 0:05), except for the 10F and 15F flours who were diverging somewhat with a higher ash content than those for 10C/10G and 15C/15G, respectively. As can be seen in Fig. 2, the protein content of the F flour mixes was considerably higher than those of C and G (p > 0:05). The protein level of the germinated flour was somewhat higher than that of the untreated control flour, but not nearly to the same extent. Fig. 3 shows the wet weight of gluten and decreasing gluten content can be seen the higher the level of cowpea flour in the samples. This effect is a self-evident result from the substitution of wheat flour and also gluten with bean flour, diluting the amount of gluten in the composite flours. Flour and dough characteristics analysed with Brabender Farinograph and Simon Extensometer are presented in Table 1. A correlation between the flour water absorption and increasing levels of cowpea flour can be seen. The water absorption capacity of flour depends on and is increased by lower moisture content, higher bran content, higher protein content, higher pentosan levels, more damaged starch and higher enzymatic activity. By looking at the protein levels represented in Fig. 2 and the rheological characteristics in Table 1, it can be noted that flour water absorption increases the higher the protein
181
content of the flour. In a study by (Deshpande, Rangnekar, Sathe, & Salunkhe, 1983), it was shown that wheat–bean composite flour blends had greater water absorption capacities than wheat flour. The addition of bean flour to wheat flour is expected to increase the protein content of the blends, since legumes generally contain more proteins than the cereals. The greater water absorptions of composite flours, therefore, could be an additive effect. Approximately 70–90% of dry bean proteins are water soluble, whereas gluten, the major fractions constituting approximately 80–90% of total wheat flour proteins, are water insoluble. The higher water absorption of the composites could hereby be explained by the high water absorption of the beans (K€ oksel et al., 2000). Proteases hydrolyse peptide linkages which are between amino acids. This effect induces a partial destruction of protein network. It increases the dough viscosity and decreases the mixing time. The decreased mixing time and stability indicate weakening of dough strength. Although this can be hard to read out from the data in Table 1 (dough stability), it was definitely notable at the time of the experiment when handling the different dough. The cowpea flours gave weaker dough with increased dough weakening at higher substitution levels, although more extensible in the case of C and especially G flour (Fig. 4). For the germinated flour, 20G resulted in poor extensibility compared to 5G/10G/ 15G and extremely sticky dough for all substitution levels. Samples with higher incorporation of cowpea flour demonstrated lower dough resistance (Fig. 5), most significantly so in the case of G, which exhibited very low values for all substitution levels and almost no resistance at all at 20G. The 5C showed the highest resistance, with a quite steep slope down to the much lower values for 15C/20C. Results for the F flours showed an opposite trend compared to the other two treatments, with a higher dough resistance and an
Table 1 Rheological properties of doughs from composite floursa Flour blend
Flour water absorption (%)
Dough development (min)
Dough stability (min)
Dough weakening (BU)
Dough extensibility (cm)
Dough resistance (EU)
5C 10C 15C 20C 5G 10G 15G 20G 5F 10F 15F 20F
62.4 64.1 65.6 66.5 65.0 65.8 66.1 66.5 67.0 69.0 69.7 70.2
2.0 2.5 4.5 4.0 1.5 2.0 2.0 1.5 2.0 2.0 1.5 2.0
1.5 2.0 2.0 1.5 4.5 3.0 3.0 4.0 3.0 4.0 3.5 3.5
120 130 90 100 100 120 140 – 130 110 140 140
15.1 16.0 18.0 16.8 19.5 21.5 21.3 15.6 17.5 17.4 16.7 15.2
355 266 132 143 194 84 63 29 228 216 241 259
a
Mean values of triplicates, SD 6 ±5%.
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cowpea flour gave progressively darker flours, as indicated by the falling L-values. The statistical analysis of the data in Table 2 showed that the changes in a and b values were not significant at the 5% level. 3.2. Bread making and bread properties
Fig. 4. HFN of composite flours.
increase rather than decrease at 10/15 levels of substitution. The weakening of dough due to the addition of bean flour to wheat flour is in agreement with earlier observations of bean flours. Possible reasons for this weakening of dough due to the addition of bean flour might include an effective decrease in wheat gluten content (dilution effect), competition between dry bean proteins and wheat flour proteins for water and possible proteolytic activity in the dry bean flours. It is also known that bean proteins alone do not have food dough forming properties and that the supplemental proteins disrupt the well defined protein–starch complex in wheat flour bread suggesting a weakening of dough. The proteolytic activity in bean flours has been documented, and since flours had not been subject to treatments to inactivate this, it could very well be a possible explanation for dough weakening. HFNs for the different composite flours are given in Fig. 4. In bread, too much amylase activity will cause wet, sticky breadcrumbs with large voids in the loaf, and too little causes dry, crumbly breadcrumbs and high loaf density. The germinated flours all had considerably lower HFN and hence a higher a-amylase activity than the both two other cowpea flours and the wheat flour. Hunter Lab colour of wheat flour and control cowpea flour can be seen in Table 2. A higher substitution for
Table 3 Weight and volume of loaves made from 460 g dough piecesa
Table 2 Colour of floursa
Wheat flour 5C 10C 15C 20C 5G 10G 15G 20G 5F 10F 15F 20F a
All flour blends produced nice, dome-shaped bread. The cowpea flour resulted in more sticky dough compared to ordinary wheat flour dough, which made it somewhat more difficult to handle at baking. This was especially true for the G flour mixes, which produced very sticky and weak dough, as also evident from earlier farinograph/extensograph analysis. Incorporation of cowpea flour in the dough had a certain negative effect on loaf volume and also the specific volume of bread, as presented in Table 3. This is due to the reduction in the wheat structure forming proteins (Akobundu, Ubbaonu, & Ndupuh, 1988) and a lower ability of the dough to enclose air (see also earlier discussion). In Table 3 it can be seen that while C breads stay on more or less the same specific volume over the substitution range (around 3.8 ml/g) and F differ 10% between 5F/10F and 15F/20F (from 4.1 to 3.7 ml/g), the G bread specific volume is decreased by 23%; from 4.0 ml/g for 5G to 3.1 ml/g for 20G. Incorporation of cowpea flour resulted in more compact bread with a more dense structure. The crumbgrain structure was evaluated visually and numbered according to the Dallman scale as presented in Table 4. Although the G flour had the strongest volume depressing effect on bread, F flour produced bread with smallest pores. Texture analysis showed that the higher the level of cowpea flour in the bread, the harder was the texture (Fig. 5). All flours showed the same increase in force resistance, the 5% level being no different from an allwheat bread at around 1.8 N, but a more than a 60%
L
a
b
92.6 92.4 91.5 90.5 90.3 89.8 88.1 87.0 86.3 89.2 87.3 86.0 84.2
)4.4 )5.0 )4.4 )4.0 )4.3 )3.8 )4.3 )4.5 )4.5 )3.7 )3.8 )4.2 )4.2
8.8 9.5 9.4 9.1 9.4 9.3 10.1 10.3 10.7 9.0 9.1 9.6 9.9
Mean values of triplicates, SD 6 ±5%.
Wheat flour 5C 10C 15C 20C 5G 10G 15G 20G 5F 10F 15F 20F a
Weight (g)
Volume (ml)
Specific volume (ml/g)
416.1 433.1 427.9 415.7 415.6 419.0 416.4 418.5 423.5 417.6 412.0 415.1 419.7
1560 1657 1597 1627 1490 1667 1587 1470 1330 1710 1680 1547 1540
3.7 3.8 3.7 3.9 3.6 4.0 3.8 3.5 3.1 4.1 4.1 3.7 3.7
Mean values of triplicates, SD 6 ±5%.
E. Hallen et al. / Journal of Food Engineering 63 (2004) 177–184
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Table 4 Crumb-grain structure according to the Dallman scalea
Dallman number
Wheat flour
5C
10C
15C
20C
5G
10G
15G
20G
5F
10F
15F
20F
6
5
7
6
6
5
5
6
7
8
8
8
8
Higher number indicates smaller pores and more dense structure. a Mean values of triplicates, SD 6 ±5%.
3.5
Force (N)
3.0
Control
2.5
Germinated
2.0
Fermented
mainly lysine, and lead to amino acid sugar reaction products (polymerised protein and brown pigments). This reaction may compromise the nutritional value of foods through the blocking and destruction of essential amino acids such as lysine and other essential nutrients (Hurrell, 1990).
1.5 1.0 0
5
10
15
20
4. Conclusions
25
Cowpea flour (%) Fig. 5. Texture analysis of bread made using composite flours with varying percentages of cowpea. Force measured at 6.25 mm.
increase could be noted at the 20% substitution levels, with values closer to 3 N. Highest value was found for 20G, 3.18 N, which also was in accordance to other results for crumb-grain structure and specific volume of bread. The colour of crust and colour of crumb (Table 5) got progressively darker as the bread contained higher levels of cowpea flour. Also the crumb got a darker, brownish colour from the cowpea flour. This darkening of the cowpea containing bread might have been attributed to an increased Maillard reaction taking place during baking of the loaves, due to the high lysine content of cowpeas. In the Maillard reaction reducing carbohydrates react with free amino acid side chains of proteins,
Adding dry bean flours to wheat flour does affect most dough properties as measured by farinograph (p > 0:05). These effects seem to increase as the level of bean flour in the blends is increased. Increased water absorption of wheat–bean composite flours may provide more water for starch gelatinisation in the doughs during baking and may prevent stretching and tearing of gluten strands. Results show that substituting wheat flour for cowpea flour up to a level of 20% produces bread with characteristics similar to control bread (p > 0:05). One problem if producing bread with cowpea composite flours would probably be that you get quite sticky dough which might be difficult to handle. The use of different handling procedures may be need to overcome this problem. A sticky dough indicates that the water absorbtion rate is low. A longer holding time (after mixing) before moulding may overcome the problem.
Table 5 Colour of crust and crumb of breadsa Crust
Wheat flour 5C 10C 15C 20C 5G 10G 15G 20G 5F 10F 15F 20F a
Crumb
L
a
b
L
a
b
34.4 36.9 33.8 32.1 28.8 31.3 30.4 28.2 27.8 36.8 32.4 29.5 30.7
4.6 7.9 7.7 6.6 7.0 6.5 7.9 6.9 7.8 9.5 8.4 7.2 7.1
16.3 15.2 13.0 8.2 8.1 10.7 9.2 7.6 9.1 15.4 12.8 7.9 8.9
78.7 74.9 72.6 66.4 60.3 72.5 72.4 65.2 60.4 71.9 66.7 59.7 57.8
6.8 8.6 8.1 6.0 4.4 8.0 8.2 6.6 8.0 8.8 7.5 3.9 4.0
16.2 17.1 16.3 15.2 15.8 17.0 17.7 16.5 17.1 16.9 15.7 13.5 13.5
Mean values of triplicates, SD 6 ±5%.
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