Introduction
Proteins are the important components of wheat (
Triticum aestivum L.) that contributes to its end-use quality (
Weegels et al., 1996). Variations in protein content and composition markedly modify flour quality, especially for bread baking (
Weegels et al., 1996;
Laflandra et al., ;
Branlard et al., 2001). Wheat proteins are traditionally separated on the basis of their solubility as albumins, globulins, gliadins, and glutenins (
Shewry et al., 1995). Within 10-15 days after anthesis (DAA), the first two fractions reflect only metabolic and structural proteins, and then, they accumulate in the developing starchy endosperm (Gupta et al., 1991;
Singh et al., 1991;
Gupta et al., 1993). The gliadin and glutenin fractions consist of storage proteins accumulated during the grain-filling period and are used as nitrogen sources during seed germination (
Shewry and Halford, 2002).
A fifth fraction, classified as lipoproteins, can be isolated (
Marion et al., 1994). The research on lipoproteins in wheat flour began quite early (
Blochet et al., 1991). Some lipoproteins are involved in the formation of cell membranes and hydrophobic layers, while some of them play an important role in the transport of fatty acids or their CoA derivatives, such as the lipid transfer protein (LTP) thionins (
Douliez et al., 2000). In addition, nonmembrane amphiphil proteins such as puroindolines have a large effect on grain hardness and dough rheological properties (
Dubreil et al., 1998;
Giroux and Morris, 1998). “Puroindolines from wheat flour are basic proteins that contain five disulfide bridges and a unique tryptophan-rich domain that is involved in lipid recognition and could contribute to the formation and stability of dough foams” (
Simeone and Lafiandra, 2003).
Albumins and globulins accumulate from anthesis to approximately 20 DAA and then remain at an almost constant level. Storage proteins accumulate from approximately 6 DAA to the end of grain filling (
Gupta et al., 1996;
Stone and Nicolas, 1996). Gliadin accumulation occurs earlier in the grain-filling period than glutenin accumulation (
Panozzo et al., 2001;
Deng et al., 2004). Recently, it was observed that amphiphils accumulated during the early stages of grain development and so did the albumins and globulins, although their rate of accumulation did not decrease as much as that of the albumins globulins (
Triboi et al., 2003). This pattern of accumulation is in good agreement with their supposed functions in the formation of cell membranes and walls and also as a protein associated with lipid storage in the endosperm (
Douliez et al., 2000). Amphiphils can, in part, provide for the intra-and-extra cellular transport of hydrophobic molecules during grain filling. Lipid-protein interactions also play an essential role in lipid functionality during cereal processing. Generally, the functionality of proteins has been researched by reconstitution studies, but the kinetics of lipoproteins accumulation and the way that they affect the dough mixing quality has rarely been reported. Thus, the objectives of this research were to study the lipoproteins’ formation and accumulation and further to analyze their relationships with dough mixing quality by using six cultivars known differing in flour quality characteristics.
Methods
Materials
Six winter wheat cultivars having different bread-making qualities were used. They were classified into three groups according to their basic characteristics, that is, Gaocheng8901 (Gao8901) and PH3259 (strong gluten), Youmai2 (YM2) and Youmai3 (YM3) (medium strength gluten), and 97-4 and 101 (weak gluten) (Table 1).
Experimental design
The six cultivars were grown at the experimental fields at Tai’an Research Farm of Shandong Agricultural University, in 2002-2003. The randomized plot area was 12 m2, with three replicates. Total nitrogen (0.89±0.05 g·kg-1), available phosphorus (45.37±0.02 mg·kg-1), and available potassium (80.35±0.01 mg·kg-1) were contained in the soil from depths of 0 to 20 cm.
Plant sampling and protein extraction
At anthesis, single heads (about 100) with a uniform size were marked. Kernel samples were taken at intervals of 5 days after anthesis until harvest. Grains harvested from about 100 whole heads were treated by liquid nitrogen. The individual grains freeze dried and were then milled using Perten 3100 mill (Perten instruments, AB. Sweden).
The extraction method was similar to those described by
Marion et al. (1994) and
Triboi et al. (2000), but some modifications were made such that the lipid bound proteins, which are designated lipoproteins, were extracted from 0.300 g of whole meal. During each extraction step, the samples were continuously stirred and shaken in an oscillating water incubator. Soluble and insoluble fractions were separated by centrifugation at 8000×g for 20 min at the extraction temperature. First, the albumins and globulins were removed by extracting at 20°C with 7 mL of 0.05 mol·L
-1 NaCl, 0.05 mol·L
-1 sodium phosphate buffer at pH 7.8, and then centrifuged. The supernatant was discarded. Lipoproteins were extracted at 20°C from the pellet using 7 mL of 2% (v/v) TritonX-114, 0.1 mol·L
-1 NaCl, and 0.05 mol·L
-1 sodium phosphate buffer at pH 7.8. The supernatant was kept refrigerated at 4°C for further study.
Protein content determination
The supernatant solution containing lipoproteins was measured for protein content by the auto-Kjeldahl instrument using the procedures, as described in method AACC 46-11 (
AACC International, 1983).
Physical-chemical analysis
Mature grains were milled to flour using Brabender Quadrumat Senior mill (Brabender OHG. Duisburg, Germany) (flour extraction rate=70%). Physical dough tests were performed using Brabender Farinograph (Brabender OHG. Duisburg, Germany) with a 50-g mixing bowl, following the AACC 54-21 (
AACC International, 1983) method. The mixing properties of the flour were also tested on Mixograph (Lincoln, Nebraska, U.S.A.) with a 10-g sample bowl according to the method AACC 54-40 (
AACC International, 1983).
Data analysis
SPSS11.0 was used to compare the difference of amphiphil means and correlation analysis.
Results
Lipoproteins accumulation
The lipoproteins had begun to accumulate 5 days after anthesis (Table 2). Initially, similar changes in accumulation were found for different cultivars, and a peak appeared at about 15 DAA, but the accumulation pattern was different. For cultivars with strong gluten (Gao8901 and PH3259), the accumulation in lipoproteins content trended upward from 5 DAA to 15 DAA, dropped at 20 DAA, but then markedly resumed increasing from 20 DAA on. The amount of lipoproteins accumulated was about 0.9 μg per kernel per day in Gao8901. PH3259 also increased slightly up to 30 DAA.
Mean comparison tests were produced by considering the six cultivars having different qualities. Significant differences in lipoprotein content were found in different cultivars within the same days after anthesis (Table 2). These results indicated that the total accumulation of lipoproteins at different development stages could be related to the cultivars.
The accumulation of lipoproteins in YM2 and YM3 cultivars per kernel per day increased by 0.28‰ and 0.47‰, respectively, from 5 DAA to 15 DAA. Peaks appeared at 15 DAA, dropped quickly, and remained stable when approaching maturity.
In contrast with strong gluten cultivars, the rate and amount of accumulation of lipoprotein proteins for the weak gluten cultivars were markedly greater up to 15 DAA. Although the amount was very small at the beginning, the accumulation rate per day was about 72 μg. The accumulation pattern for 101 increased first to a plateau, maintained a period, then declined, and finally increased slightly at maturation, which is similar to that for albumin and globulin (
Deng et al., 2004).
Correlation between lipoproteins content and dough rheological parameters
Table 3 indicates that lipoproteins from different stages showed different correlations with Farinograph and Mixograph parameters. From 15 DAA to 20 DAA, a great number of coefficients appeared negative between lipoproteins and dough rheological characteristics except for MTI. While from 25 DAA to 30 DAA, lipoproteins are positively correlated with major Farinogram and Mixogram parameters. All these correlation coefficients did not appear to be statistically significant.
These results indicated that lipoproteins at different stages had different effects on dough mixing parameters and also suggested that there was an optimal lipoproteins time, which would avail to dough mixing characteristics.
Discussion
In the present study, lipoproteins were extracted at different development stages of wheat kernels prior to storage protein synthesis. The accumulation rates and totals of lipoproteins followed a similar initial trend in the same groups, with a peak occurring at 15 DAA. However, the total accumulation rates appeared to be different in cultivars. Perhaps this difference is caused by the genotype or the interactions between lipoproteins and other proteins. These need to be further studied.
It was observed that the formation of lipoproteins apparently began earlier than 5 DAA, as the albumins-globulins did, but the accumulation rates for lipoproteins did not decrease as much as that for the albumin and globulin fractions (
Deng et al., 2004). Perhaps this is because the latter are a relatively homogeneous group of proteins, while the lipoproteins appear diverse, containing many proteins such as lipid-bound proteins, lipid-transferable proteins, puroindolines, and so on. These proteins, perhaps, have different accumulations during grain development, which will affect the lipoproteins accumulation. Therefore, the lipoproteins can be separated into fractions by electrophoresis, and then each fraction will be researched, which will be a good method to study the accumulation of lipoproteins. This work is to be further studied by high-performance capillary electrophoresis (HPCE) in our laboratory. In addition, the accumulation trend of YM2 was very similar to that of YM3 because they share the same parents.
It is well known that the storage proteins, gliadins, and glutenins play a critical role in governing wheat flour properties, including baking quality (
Dachkevitch and Autran, 1989;
Gupta et al., 1993;
Popineau et al., 1994;
Ciaffi et al., 1996;
Gupta et al., 1996;
Triboi et al., 2000). Although lipoproteins contribute to the spreading and stability of lipid and lipid-protein films, the role of lipoproteins in dough mixing properties is not well known except for the work done with the puroindolines of mature kernels, as reported by
Dubreil et al. (1998). In the current study, the correlation coefficients between lipoproteins and major dough mixing properties appeared not to be significant. The lipoproteins, to some extent, appeared to not strongly affect the Farinograph and Mixograph properties. At present, the influencing mechanism has been unknown in wheat, and the components of lipoproteins have been individually unthinkable for the effect. Perhaps the amount of lipoproteins would influence their distribution between structural and storage proteins. Therefore, a larger number of samples with different quality properties, including grain hardness, should be used to further study the mechanism between lipoproteins and dough mixing properties. In addition, exchange and reconstitution studies between lipoproteins and base flours with different characteristics would be another good method to study this mechanism.
Conclusions
The study on the effects of using different types of wheat on lipoproteins formation and accumulation gave new insights into the accumulation of lipoproteins. Lipoproteins accumulated in the grain during the early stages, that is, the cell division stage. The initial accumulation trend of lipoproteins in the same type of cultivars was similar. However, the total accumulation amount of lipoproteins produced during grain development was significantly different among cultivars. Therefore, the type of cultivars might affect the rates and total accumulation of lipoproteins during grain filling. As for the cultivars studied, the relationships between lipoproteins and dominating dough mixing properties appeared not to be statistically significant.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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