Food Flavor and Safety - American Chemical Society

Chapter 15

Use of Enzyme-Active Soya Flour in Making White Bread

Downloaded by UNIV OF ROCHESTER on November 4, 2014 | http://pubs.acs.org Publication Date: May 11, 1993 | doi: 10.1021/bk-1993-0528.ch015

J. P. Roozen, P. A. Luning, and S. M. van Ruth Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, Netherlands

Native soya flour contains at least 3 lipoxygenase isoenzymes, which improve dough characteristics by peroxidizing unsaturated fatty acids followed by oxidation of proteins (rheology) and carotenoids (bleaching). The fatty acid peroxides formed can also degrade into volatile compounds interfering with the flavor of white bread. Dynamic headspace samples of this bread made with and without soya flour were analysed by gas chromatography (and mass spectrometry). Addition of soya flour increased the concentrations of several secondary lipid oxidation products. Storage of bread improvers containing enzyme active soya flour caused more loss of lipoxygenase activity in a paste than in a powder type of improver. As a consequence their bleaching action and formation of volatile lipid oxidation products were diminished to the same extent.

Haas and Bohn patented in 1934 (7) the use of enzyme active soya flour as a dough bleaching agent. The bleaching activity of soya bean lipoxygenase L2 is much higher than that of wheat lipoxygenase (2). Lipoxygenase catalyzes polyunsaturated fatty acid oxidation to yield hydroperoxides with intermediate free radicals able to cooxidize lipophilic pigments (5), fat soluble vitamins, and thiol groups of proteins. Lipoxygenase is entangled in the gluten fraction, when it is added to a flour-water suspension (4). To date the aroma of bread consists of a total of 296 volatile compounds (5), which can originate from different stages of bread making (Table I). The

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In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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Use of Enzyme-Active Soya Flour in White Bread 193

volatile compounds of wheat kernels normally increase on milling and storage of flours (maturation). New flavor compounds are probably formed by the disturbed metabolism of the grain after grinding. Maturation improves the performance of the flour in bread making, which is probably caused by oxidative changes in the flour components (6). These changes are propagated by the activity of lipase because it acts as a supplier of substrate for lipoxygenase. During dough making and development yeast fermentation induces enzyme activity responsible for additional flavor compounds or precursors (Table I). These compounds relate strongly to the ingredients used for the dough mixture, e.g. type of bread improver. The aroma of the finished bread depends to a large extent on the way of baking. In general the amine-carbonyl reactions play a dominant role in flavor formation in the crust, while the crumb retains mainly fermentation volatile compounds (7). For estimating the contribution of volatile compounds to bread aroma Rothe and coworkers (8) defined "aroma value" as the ratio of the concentration of some volatile compounds to the taste threshold value of the aroma. This concept was further developed by Weurman and coworkers (9) by introducing "odor value", in which aroma solutions were replaced by synthetic mixtures of volatile compounds in water. These mixtures showed the complexity of the volatile fractions of wheat bread, because none of them resembled the aroma of bread. Recently two variations of GC-sniffing were presented (10-11), in which the aroma extract is stepwise diluted with a solvent until no odor is perceived for each volatile compound separately in the G C effluent. The dilution factors obtained indicate the potency of a compound as a contributor to the total aroma. Bread improvers are utilized for enhancing the processing capabilities of raw materials. The bread made should have desirable physical and chemical properties and kept for some time during storage. The improvers are made up to fulfil specific dough and bread characteristics like texture, whiteness and aroma. The aroma of bread can be modified by oxidative degradation products of polyunsaturated fatty acids, and by physical absorbtion (selective) of flavor compounds in the matrix (e.g. starch). However, volatile products from hydroperoxide breakdown are considered beneficial in German bread (12) and detrimental in French bread (13) depending on the opinions of the consumers involved. In many countries additives are forbidden in bread, except aromas in, for instance, fruit-flavored bread. Natural additives like enzyme active soya flour get only little attention for application in bread improvers because: the active substances like enzymes are not fully understood and are present in variable amounts; beany and rancid flavors can negatively affect the aroma of bread; the risk of unknown volatile compounds formed during bread making, e.g. by enzymic reactions.


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Experimental Bread Sample Preparation. The recipes for making white bread consisted of wheat flour (regular, 1 kg), water (520 ml), baker's yeast (25 g), improver mix (24 g), salt (20 g) and soya flour (0 or 30 g). For studying the shelf-life of soya containing improver mixtures (20 C , 80 % rH) an improver paste with 0.5 % and an improver powder with 2.5 % soya flour were used in the recipe without further soya flour addition. The dough was mixed for about 8 min in a spiral mixer (Kemper) at 26 C and 80 % rH. Four loaves were formed, allowed for two fermentation periods (40 resp. 70 min) at 34 C and 80 % rH, and then baked (30 min at 225 C ) in a rotary oven (Siemens). After one hour of cooling about 90 g slices were packed in baker's paper and aluminum foil, and afterwards kept in cold storage for max fourteen days until sampling. e

e

β


e

Sampling and Analysis. A frozen slice of bread was cut in pieces and stacked in an enlarged sample flask of an aroma isolation apparatus according to MacLeod and Ames (14). Volatile compounds were trapped on Tenax T A and afterwards thermally desorbed and cold trap injected in a Carlo Erba GC 6000 vega equipped with a Supelcowax 10 capillary column (60 m χ 0.25 mm i.d.) and a flame ionisation detector. Similar G C conditions were used for GC-MS identification of volatile compounds by dr. M.A. Posthumus (Dept. Organic Chemistry, V G MM7070F mass spectrometer at 70 eV EI, 75). Color evaluation was carried out with a panel of 10 assessors, who judged coded slices of white bread in a Kramer ranking test design (76). A spectrophotometric assay procedure was used to measure the lipoxygenase isoenzyme activities of fresh and stored soya containing bread improver pastes and powders (77).

Results and Discussion The number of volatile compounds collected from white bread by the dynamic headspace method are presented per class in Table Π. The values give only a partial impression, because they depend strongly on the conditions used for isolation and detection of volatile compounds. Overall this method seems to release 1/3 of the total number of compounds published for white bread (5). So, the data obtained can only be used for studying differences compared to control samples. Table III shows that l-octen-3-ol and 2-heptenal were only detected in soya containing bread and that the latter has significant larger peak areas of 1pentanol, 1-hexanol, l-penten-3-ol, hexanal and 2- heptanone compared to the control (p < 0.05). These differences could be caused either by addition of volatile compounds present in soya flour or by its lipoxygenase activity. The main volatile compound found in soya flour was limonene (75), of which the peak areas were similar in the soya flour containing bread and its control sample. Moreover minor volatile compounds of soya flour, like 1-pentanol, 1hexanol and hexanal, increased steadily in soya samples, as can be seen in Table


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Table I. Kind of volatile compounds formed in different stages of bread making


Grain

Dough making

Baking

(disturbed) metabolism of grain

Enzymes/yeast metabolism

Amine-carbonyl reactions

alcohols aldehydes ketones hydrocarbons

alcohols acetoïn organic acids

alkyl pyrazines pyrroles aldehydes ketones

Flour

esters lactones heterocyclic compounds

Table II. Number of volatile compounds detected in white bread samples with the dynamic headspace method Class of compound Acids Alcohols Aldehydes Bases Esters Furans Hydrocarbons Ketones Sulfur Compounds

Number found 6 13 11 6 9 5 2 10 2

Proportion* -3 -7 -3 -1 .5 -3 -2 -3 -1

•Proportion to the total number of these compounds presented by Maarse and Visscher (5)



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III. These results indicate that the composition of volatile compounds of soya containing white bread is hardly influenced by the volatile compounds of the soya flour itself and that lipoxygenase activity plays a major role. It is interesting to note that the two most potent detrimental volatile compounds of autoxidized white bread (2-nonenal and 2,4-decadienal; 18) were not detected in our soya containing white bread samples. But, these samples had additionally 2-heptenal and l-octen-3-ol, which belong to the flavor significant volatile compounds of oxidized soya bean oil (19). Apparently, soya lipoxygenase peroxidized available soya oil constituents more easily than wheat lipids. The enzymes of legume flour have been shown to peroxidize unsaturated fatty acids in all major lipids occurring in wheat dough (20). It has been noticed, that these enzymes were responsible for a "green" and bitter off-flavor of bread (21). In todays practice of making bread enzyme active soya flour will not be used as an additional ingredient, but as a component of a bread improver mixture. In that case the storage stability of soya lipoxygenase in bread improvers is an important factor for their bleaching action and formation of volatile lipid oxidation products. The latter effect is demonstrated in Table IV. Bread improver paste products declined fully during 5 months of storage, while powder products decrease only about 50 %. In average the difference between paste and powder is 32 % and significant at ρ < 0.01 in the Student-t test. The bleaching capacity of the soya flour added to bread improver paste and powder is shown in Figure 1. The sums of ranks of the slices of control bread are much higher ( = less white) than of the slices of the soya added ones. The difference in soya flour content of the improvers is also reflected here: sum of ranks for whiteness is significant lower (= more white) for bread with fresh and stored bread improver powder (p < 0.05). The effect of storage of the soya containing bread improvers gives a not significant increase in the sum of ranks. So, within the studied shelf-life of 5 months, these bread improvers can be considered as satisfactory for the end-users. The bleaching of plant pigments responsible for greyness of bread crumb, is related to lipoxygenase isoenzyme activities of native soya flour. These activities were measured in the soya containing bread improvers (Figure 2). The activities of the enzymes decreased much more in the paste than in the powder during storage. The higher activities of L2 and L3 isoenzymes of the powder are likely to be responsible for its larger bleaching capacity presented in Figure 1. Weber and coworkers (23) found that the isoenzymes mentioned are able to cooxidize caroteno'fds and that isoenzyme L I exhibits slightly carotene oxidation. The activity of isoenzyme L I decreased much more in the paste than in the powder improver (Figure 2). This might explain the differences in yield of volatile compounds in Table IV, assuming that these compounds originated mainly from lipids peroxidized by isoenzyme L I . As discussed, addition of enzyme active soya flour changes the composition of volatile compounds of white bread. In its practical application as a bread improver component, the soya lipoxygenase isoenzymes are sufficient stable for 5 months to meet the bleaching requirements.


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Table III. Amounts of volatile compounds (ng/kg) detected in white bread by dynamic headspace analysis; CONTROL is without and SOYA is with 30 g soya flour addition (see recipe; 15)


Compound 1-Pentanol 1-Hexanol l-Penten-3-ol l-Octen-3-ol Hexanal 2-Heptenal 2-Heptanone

CONTROL

SOYA

6 ± 1.5 9 ±2 4 ± 1.5 nd 78 ± 14 nd 6 ± 1.4

14 ± 2 61 ± 11 26 ± 2 3 ± 0.6 173 ± 23 14 ± 1.9 10 ± 1.9

nd = not detected.

Table IV. Influence of 16-23 weeks of storage of soya containing bread improver PASTE and POWDER on differences (d) in relative decreases (%) of G C peak areas of selected volatile compounds of white bread (22) Compound 1-Butanol 1-Pentanol l-Penten-3-ol 1-Hexanol l-Octen-3-ol Hexanal Heptanal Octanal Nonanal Decanal

PASTE 53 112 106 100 87 25 25 100 100 90

POWDER

d*

-7 50 80 36 62 43 50 50 46 64

60 62 26 64 25 -18 -25 50 54 26

* mean of d is 32 % and Student-t: ρ < 0.01.



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Figure 1. Influence of 16-23 weeks of storage of soya containing bread improver paste and powder on visual whiteness of slices of bread. Kramer ranking test design (1 = most white sample, etc.); (C) = control, bread improver without soya flour has been used (based on data mentioned in text of 22).

Figure 2. Influence of 16-23 weeks of storage of soya containing bread improver paste and powder on lipoxygenase isoenzyme LI, L2 and L3 activity. * = no activity detected (based on data mentioned in text of 22)


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Literature Cited 1. 2.


3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Haas, L.W.; Bohn, R.M. Chem. Abstr. 1934, 28, 4137. Grosch, W.; Laskawy, G.; Kaiser, P. Z. Lebensm. Unters. Forsch. 1977, 165, 77-81. Nicolas, J. Ann. Technol. Agric. 1978, 27, 695-713. Graveland, A. Biochem. Biophys. Res. Commun. 1970, 41, 427-434. Maarse, H.; Visscher, C.A. Volatile Compounds in Food. Qualitative Data; TNO-CIVO Food Analysis Institute: Zeist, The Netherlands, 1987. Bellenger, P.; Godon, B. Ann. Technol. Agric. 1972, 21, 145-161. Schieberle, P.; Grosch, W. Z. Lebens. Unters. Forsch. 1991, 192, 130-135. Rothe, M.; Thomas, Β. Z. Lebensm. Unters. Forsch. 1963, 119, 302-310. Mulders, E.J.; Maarse, H.; Weurman, C. Z. Lebens. Unters. Forsch. 1972, 150, 68-74. Acree, T.E.; Barnard, J.; Cunningham, D.G. Food Chem. 1984, 14, 273-286. Schieberle, P.; Grosch, W. Z. Lebensm. Unters. Forsch. 1987, 185, 111-113. Kleinschmidt, A.W.; Higashiuchi, K.; Anderson, R.; Ferrari, C.G. Bakers Dig. 1963, 37(5), 44-47. Drapron, R.; Beaux, Y.; Cormier, M.; Geffroy, J.; Adrian, J. Ann. Technol. Agric. 1974, 23, 353-365. MacLeod, G.; Ames, J. Chem. Ind. 1986, 175-177. Luning, P.A.; Roozen, J.P.; Moëst, R.A.F.J.; Posthumus, M.A. Food Chem. 1991, 41, 81-91. O'Mahony, M. Sensory evaluation offood: statistical methods and procedures; Marcel Dekker Inc.: New York, NY, 1986. Engeseth, N.J.; Klein, B.P.; Warner, K. J. Food Sci. 1987, 52, 1015-1019. Grosch, W. In Autoxidation of unsaturated lipids; Chan, H.W.-S., Ed.;Academic Press: London, 1987; pp 95-139. Frankel, E.N. J. Sci Food Agric. 1991, 54, 495-511. Morrison, W.R.; Panpaprai, R. J. Sci. Food Agric. 1975, 26, 1225-1236. Drapron, R.; Beaux, Y. C.R.Acad. Sci., Ser. D 1969, 268, 2598-2601. Van Ruth, S.M.; Roozen, J.P.; Moëst, R.A.F.J. Lebensm. Wiss. Technol. 1992, 25, 1-5. Weber, F.; Laskawy G.; Grosch, W. Z. Lebensm. Unters. Forsch. 1974, 155, 142-150.

RECEIVED January 29, 1993


Food Flavor and Safety - American Chemical Society

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