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Effect of proteinase and glucanase on RVA

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Effect of proteinase and glucanase on RVA Powered By Docstoc
					J. Food. Sci. Agric 1999 (Submitted)
Effects of Protein and -Glucans on Pasting and Thermal Properties of Oats
                                     1               1                        2                       1
                          Meixue Zhou , Kevin Robards , Malcolm Glennie-Holmes , and Stuart Helliwell

1
    School of Science and Technology, Charles Sturt University, PO Box 588, Wagga Wagga, NSW 2678, Australia
2
    NSW Agriculture, Agricultural Research Institute, Wagga Wagga, NSW 2650, Australia

                                                         ABSTRACT
Specific enzymes were used to decompose protein and -glucans in wholemeal oats prior to measuring pasting and thermal
properties of the meal, using rapid viscoanalysis and differential scanning calorimetry. -glucans were responsible for a large
proportion of these properties of the meals while the contribution of protein was much less. An extra transition process was
observed using DSC tests on wholemeal oats compared to isolated starch. This transition was not associated with -glucan or
protein. Pasting and thermal properties were correlated with each other and with compositional data. Processing effects on
thermal properties were also noted.

Key words: DSC; oat; glucan; protein; pasting.

                                                                   because of poor colour and poor flavour, and was
Introduction                                                       described by trained sensory panellists as having stronger
                                                                   starchy, weaker watery flavours as well as an unpleasant
Pasting and thermal properties of starch are the key factors
                                                                   adhesive mouthfeel (Zhou et al. 1999c).
responsible for the texture of cereal-based processed
products. Most previous studies of pasting properties of
                                                                       Table 1 Composition of oat samples.
cereals have concentrated on isolated starch because of the
problems of separating the effects due to other
components. However, unlike that from most cereals, oat            Component      Content (g kg-1)
starch cannot easily be separated from other components                           Cultivar
of the grain and the process of isolation may change the                          Mortlock      Yarran
properties of the starch. More importantly, from the               Moisture       1381         1431
consumer's point of view the pasting and thermal                   Protein        1886         1116
properties of the whole oat product are critical to its            Starch         5077         5761
acceptance. Thus, for studies on oats, it is more important        -glucan       471          461
to determine the pasting and thermal properties of the             Lipid          8 01         1011
whole meal. Of the non-starch components, lipid, -glucan
and protein will most influence these properties of the            Series II: Eight varieties of oats (Bimbil, Carrolup, Cooba,
whole meal. The effect of lipid on pasting properties of           Echidna, Euro, Mortlock, Pallinup & Yarran) were
oats has been examined (Zhou et al. 1999c). -glucans              grown in Condobolin. Futher samples of Mortlock and
form viscous gums (Autio et al. 1987) and contribute               Yarran were grown in Cowra and Parkes in NSW. These
significantly to water retention and processing behaviour.         samples were used to examine the effect of oat variety (and
The high content of gum, especially -glucans, in wet-             to a lesser extent, the effect of growing site) on the
milled oat bran had a marked effect on the viscosity of            relationship between RVA and DSC data and the effect of
heat- and -amylase-treated bran slurries (Jaskari et al           processing on DSC.
1995). Slurry viscosity was correlated with -glucan
concentration in oat flours and treatment of slurries with         The oat samples were dehulled with a laboratory scale oat
-glucanase lowered viscosity significantly (Zhang and             dehulling machine. Dehulled samples were ground with a
Moore 1997). There has been little work on the effect of           Falling Number Grinder fitted with a 0.8 mm screen.
protein on pasting and thermal properties of oat meal.             Moisture contents were determined by Near Infrared
                                                                   Reflectance Spectroscopy (NIR) using a Technicon 450R
This study examined the effect of -glucan and protein on          instrument based on calibrations provided by the Drought
the pasting and thermal properties of oat meal as measured         Evaluation Unit of NSW Agriculture. Starch was
by Rapid Viscoanalysis and Differential Scanning                   determined by a total starch kit and -glucans were
Calorimetry (DSC). The effect of processing on the                 determined by a total -glucan kit both supplied by Mega-
thermal properties of oat meal was also examined.                  Zyme (Deltagen, 31 Wadhurst Drive, Boronia 3155, Vic.
                                                                   Australia) and used according to the manufacturer's
Materials and methods                                              instructions. Protein contents of the two commercial
                                                                   samples were determined by Dumas combustion using a
                                                                   Lecor CNS 2000 instrument (improved AOAC method
Groat samples                                                      7.016 with modern technology). Lipid contents of the
Series I: Commercially grown samples of two Australian             commercial samples were determined by extraction using
cultivars, Yarran and Mortlock, were used in this study.           petroleum ether (bp 40-60, PE) for 16 h in Goldfisch
Table 1 characterises these two samples. These                     (plain-body) apparatus (AOCS official method Ai 3-75 -
commercial samples came from the same bulk lots as were            modified). Protein and lipid contents of the other samples
used in previous studies (Zhou et al. 1999a, 1999b,                were determined by NIR.
1999c). For food use purposes, Mortlock is preferred by
consumers, while Yarran is not acceptable to consumers




0
Preparation of Rolled Oats                                      Results and discussion
Samples of each variety (10 kg) were dehulled and then
stored at 8C for the two days which had to elapse before       RVA
processing could proceed. Samples (3.5 kg) were                 Rapid viscograms are normally carried out using freshly
processed following normal commercial practice using            prepared slurries in distilled water. The use of buffer to
small scale steaming, kilning and rolling machines in           replace distilled water had no effect on the viscograms.
Uncle Tobys Research & Development Centre                       However, to ensure the complete degradation of the protein
(Wahgunyah, Victoria, Australia). The conditions were:          or -glucan, it was necessary to allow 1 hr of reaction time
steaming for 9 min, kilning at 100C for 45 min and 65C        prior to the test. The viscograms were substantially
for 15 min, cooling to room temperature, resteaming for 5       changed by this pretreatment (Figure 1). The reduction in
min and rolling to a thickness of 55 m.                        viscosity following soaking can be attributed either to
                                                                physical changes due to hydration or the action of intrinsic
Enzymes                                                         enzymes. Consistent with this latter interpretation,
                                                                viscograms run in the presence of silver nitrate (0.1
Proteinase K (EC 3.4.21.14) was supplied by Sigma Chemical      mMoles/g meal, a concentration sufficient to inactivate
Co. (St. Louis, MO USA). The -glucanase ((1,3)-, (1,4)--D-    amylases, proteases and glucanases, Glennie-Holmes,
glucanase, EC 3.2.1.73) was taken from a total -glucan kit     1995) were indistinguishable from those of freshly
manufactured by Mega-Zyme (Deltagen, Boronia, Vic.              prepared slurries.
Australia).
                                                                Following soaking in buffer for 1 hr, most RVA
                                                                measurements were affected by variety. The peak
RVA measurements                                                viscosities of Morlock and Yarran were significantly
Ground groat meal (4.0g, at 15% moisture content) was           decreased (35% and 11%, respectively). There was a slight
slurried in phospate buffer (20 mM, pH 6.5, 25 mL). The         increase in the trough of Mortlock (6%) while that of
pasting properties of the slurry were determined with a         Yarran was decreased significantly (14%). Final viscosity
Rapid Visco-analyser (RVA, Newport Scientific,                  and setback of Mortlock were increased 42% and 64%,
Warriewood, NSW, Australia) using a previously                  respectively, whereas those of Yarran decreased 29% and
developed profile (Zhou et al 1999a) with a stirring speed      40%. The two characteristics not varietally affected were
of 960 rpm for 10 sec and 115 rpm for the remainder of          time to peak viscosity (15% longer) and pasting
the test and with the temperature programmed to rise from       temperature (9% and 4% higher for Morlock and Yarran,
40C to 90C in 3 min, to hold for 6.5 min, to cool to 40C     respectively). These results indicate that Morlock
in 4.5 min and to hold for 5 mins. All measurements were        contained a lesser amount of intrinsic enzymes in the
as previously described (Zhou et al 1998).                      resting grain than Yarran. The detection of enzymatic
                                                                activity agrees with reports of -glucanase in
To study the effect of proteinase and -glucanase on the        ungerminated oat seeds (Wood et al. 1978). Doehlert et al.
viscogram, "blank" determinations were carried out by           (1997) also suggested that the loss of apparent viscosity in
mixing phosphate buffer with meal in an RVA can and the         raw and roasted oat meal slurries after an extended
can immersed in a 40C water bath for 1 hour before             incubation period was partly due to the action of -
running the RVA. Similarly, tests with either proteinase or     glucanase.
-glucanase in buffer were placed in the 40C water bath
for 1 hr before viscoanalysis. The times and quantities of      Effect of added       proteinase   and    -glucanase on
enzyme were chosen to ensure degradation of all the             viscograms
protein or -glucans in the meal. All measurements were         Viscograms of both meals were changed significantly
performed in duplicate.                                         (Figure 2 and Table 2) by the addition of -glucanase.
                                                                Although the -glucan content of these two varieties
DSC measurements                                                differed only slightly (Table 1), the addition of -
DSC was carried out by means of a Mettler Toledo DSC            glucanase caused differential responses. Added -
820 Differential Thermal Analyser. Meals (4.0 mg) were          glucanase had a greater effect on Mortlock with peak
suspended in an aqueous solution (15 µL; phosphate              viscosity, trough and final viscosity being decreased by
buffer, proteinase in buffer, or -glucanase in buffer) in an   36%, 14% and 34%, respectively, whereas the reduction in
aluminium pan (medium pressure) or a stainless steel pan        these measurements for Yarran was 12%, 11% and 6%,
(high pressure). To ensure complete enzyme activity, the        respectively. The addition of -glucanase decreased the
meal slurries were put in a 40C oven for one hour and          setback of Mortlock meals significantly (43%) while there
then moved to ambient temperature until testing                 was no significant effect on the setback of Yarran (2%
commenced. The temperature of the DSC was started at            higher). -glucanase also caused significant increases in
25C, raised to 115C (the maximum temperature before           time to peak viscosity and pasting temperature of Mortlock
the sealing of the aluminium pans fails) at the rate of         (4% and 23%, respectively). In contrast, time to peak
10C/min and then cooled to 25C at the rate of 50C/min.       viscosity and pasting temperature of Yarran were
                                                                unaffected by additions of -glucanase.
Statistical analysis
                                                                Additions of proteinase caused significant but minor
All analyses were performed at least in duplicate and           changes to some of the RVA parameters of both varieties
results recorded as the mean and standard deviation. The        and the effects were relatively small compared to -
data were analysed by single factor or multifactor analysis     glucanase. Peak viscosity and trough of both varieties
of variance following Snedecor and Cochran (1967).              were decreased by about 15% with added proteinase.
                                                                While final viscosity of both meals was little affected by
                                                                the addition of proteinase, the setback of both varieties




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was increased significantly (9% and 30%, respectively for      prepared. The statistically significant correlations are now
Morlock and Yarran). The time to peak viscosity of the         discussed. Groat protein content was negatively correlated
Yarran meal was decreased significantly by adding              with trough (-0.82**). -glucan content was significantly
proteinase, whereas that of Morlock was unchanged. The         correlated with breakdown (0.82**), final viscosity (-
proteinase had no significant effect on the pasting            0.69*) and time to peak viscosity (-0.60*). The Tp1 of the
temperature of either meal.                                    meals was positively correlated with protein (0.68*) and -
                                                               glucan (0.78**) content and negatively correlated with
-glucans contribute most of the viscosity of soluble          lipid content (-0.73**). -glucan had a close correlation
extracts of oats (Autio et al 1987, Bhatty 1992), thus it is   with both H2 (-0.92**) and Tp2 (-0.62*).
not surprising that decomposition of -glucans had a much
greater effect on meal pasting properties than                 The pasting properties showed close correlation with
decomposition of protein. As the two varieties contained       thermal properties. Peak viscosity was positively
the same amount of -glucan as substrate but different         correlated with H1 (r = 0.71**); Trough was negatively
levels of intrinsic glucanases, the pretreatment stage         correlated with Tp1 (r = -0.74**); Breakdown was
permitted greater reduction in viscosity in the Yarran thus    positively correlated with Tp1 (r = 0.80**) and negatively
reducing the opportunity for added enzymes to express full     with H2 (r = -0.69*) and Tp2 (r = -0.70*); Final viscosity
activity. When the results of the standard (freshly mixed,     was negatively correlated with Tp1 (r = -0.67*) and
without soaking) procedure and the -glucanase treated         positively with H2 (r = 0.73**) and Tp2 (r = 0.66*);
test are compared, both varieties showed an identical          Setback was correlated with Tp2 (r = 0.68*); Time to peak
(43%) reduction in viscosity. This overall reduction           viscosity was negatively correlated with Tp 1 (r = -0.67*)
indicated that the -glucan had been completely destroyed.     and pasting temperature was positively correlated to H2 (r
                                                               = 0.61*) and Tp2 (r = 0.78**). These results suggest that as
DSC                                                            the second transition has been identified as the result of the
The data obtained by DSC using wholemeals were the             breakdown of the amylose:lipid complex (Hartunian Sowa
enthalpy and temperature corresponding to endothermic          and White 1992), the pasting temperature measured by
transitions occurring during heating (Figure 3, Table 3).      RVA was similarly asigned to this process.
Three transition processes were detected in Morlock and
Yarran, the first corresponding to the loss of starch          Effect of processing on DSC
crystallinity (Paton, 1987), the second to the transition of   Our previous study showed that processing of oats
the amylose-lipid complex (Hartunian Sowa and White            changed the pasting properties of the meals significantly
1992; Zhou et al 1999b) and the third to interactions          (Zhou et al 1999c), as also shown by Doehlert et al.
between other components of the meal and starch (Zhou et       (1997), and Zhang et al. (1997). Processing also had a
al 1999b). The loss of starch crystallinity has been defined   significant effect on thermal properties of the meals (Table
as the point of gelatinisation and thus H1 (Table 3) is       4).
termed the enthalpy of gelatinisation and Tp1 the
temperature of gelatinisation. Most DSC studies on oats        The H1 of the processed meals was reduced in every
have been based on isolated starch and values reported for     cultivar, on average by 1.4 J g-1. Tp1 was altered on
the gelatinisation and amylose:lipid complex transition        average by –1.0C (Table 4) with a range between the
enthalpies were about 9 J g-1 and 3 J g-1 respectively         varieties of +0.1 to -2.5C. Therefore, processing would
(Hartunian-Sowa and White 1992, Paton 1987). The use of        reduce the energy input required to gelatinise the product,
wholemeals in present study acounts for the third trasition    but individual varieties had a characteristic response to
and also for the reduced enthalpies (see Table 3).             processing which was also affected by growing site. For
                                                               example, as shown in Table 4, Morlock grown at Cowra
There were no significant differences of enthalpy between      had H1 reduced by 2.8 J g-1 whereas the H1 of the same
varieties for H1 and H2, whereas H3 differed in             cultivar grown at Condobolin was reduced by only 0.4 J g-
varieties with Mortlock having higher enthalpy. Mortlock       1
                                                                 . The non-zero value of the enthalpy of gelatinisation of
had significantly higher Tp1 and Tp3. There was no             processed oats, H1, which occurred in all cultivars
difference in Tp2 between varieites.                           indicates that complete gelatinisation had not occurred
                                                               during processing despite the fact that grains are steamed
Effect of added proteinase and -glucanase on DSC              twice before rolling. This was supported by the moisture
The two varieties responded similarly in peak temperature      content of the grain, which did not change significantly
to the addition of -glucanase (Figure 3, Table 3). When       during processing (Zhou et al 1999b). Nevertheless, there
the -glucans were broken down, Tp1 was increased by           was a significant decrease in the transition enthalpy, H1,
8C, Tp2 by 5 - 6C and Tp3 by 3 - 5C. These data are         following processing, indicating some disorganisation of
consistent with those obtained with the RVA (Table 2).         the crystalline structure (Table 4).
With the exception of H3 of Mortlock and H2 of Yarran,
the enthalpies of the two varieties were little affected by    The H2 was also reduced by processing in every cultivar
the addition of -glucanase. The changes following the         (Table 4) with an average reduction of 0.4 J g-1. Of
addition of proteinase were smaller that those following       particular interests was the similartity in H2 values of
the addition of -glucanase.                                   Morlock and Yarran both before and after processing.
                                                               Since this enthalpy change is associated with amylose:lipid
                                                               complex, it suggests that the complex is similar in both
Correlations between RVA and DSC data
                                                               varieties despite the large difference in oil content (Table
                                                               1). Therefore, it is the proportion of amylose capable of
A correlation matrix (17 x 17, not presented) relating         forming a complex with lipid which is limiting in both
compositional (protein, -glucan & lipid), RVA and DSC         cultivars. Tp2 was increased, on average, by 2.0C after
measurements on the 12 oat samples (series II) was             processing, and therefore the amylose:lipid complex was




2
stabilised by processing.                                                                      Iowa State University Press. Ames, Iowa, USA.
                                                                                          Wood P J, Siddiqui I R, Paton D 1978 Extraction of high
                            References                                                         viscosity gums from oats. Cereal Chem 55 1038-
                                                                                               1049.
Autio K, Myllymäki O, Mälkki Y 1987 Flow properties of
                                                                                          Zhang D, Doehlert D C, Moore W R 1997 Factors
      solutions of oat -glucans. J Food Sci 52 1364-
                                                                                               affecting viscosity of slurries of oat groat flours.
      1366.
                                                                                               Cereal Chem 74 722-726.
Bhatty R S 1992 Total and extractable -glucan contents
                                                                                          Zhou M X, Glennie-Holmes M, Robards K, Helliwell S
      of oats and their relationship to viscosity. J Cereal
                                                                                               1999a RVA protocol for determining the pasting
      Sci 15 185-192.                                                                          properties of oats. J Cereal Sci (Submitted).
Doehlert D C, Zhang D, Moore W R 1997 Influence of                                        Zhou M X, Glennie-Holmes M, Robards K, Helliwell S
      heat pretreatments of oat grain on the viscosity of                                      1999b. Effects of processing and short-term storage
      flour slurries. J Sci Food Agric 74 125-131.                                             on the pasting characteristics of slurries made from
Glennie-Holmes M 1995 Studies on barley and malt with                                          raw and rolled oats. Food Aust (In press).
      the RVA. (1) The effect of variation in physical and                                Zhou M X, Roberts G L, Robards K, Glennie-Holmes, M,
      chemical parameters. J Inst Brew 101 11-18.                                              Helliwell S 1998 Effects of sowing date, nitrogen
Hartunian-Sowa M, White P J 1992 Characterisation of                                           application, and sowing rate on oat quality. Aust J
      starch isolated from oat groats with different amount                                    Agric Res 49 845-51.
      of lipid. Cereal Chem 69 521-527.                                                   Zhou M X, Robards K, Glennie-Holmes M, Helliwell S
Jaskari J, Henriksson K, Nieminen A, Suortti T, Salovaara                                      1999c Effects of oat lipids on groat flour pasting
      H, Poutanen K 1995 Effect of hydrothermal and                                            properties. J Sci Food Agric 79:(In press).
      enzymic treatments on the viscous behaviour of dry-
      and wet-milled oat brans. Cereal Chem 72 625-631.
Paton D 1987 Differential scanning calorimetry of oat
      starch pastes. Cereal Chem 64 394-399.
Snedecor G W, Cochran W G 1967 Statistical method. The

                    Table 2 Changes of RVA measurements with enzyme treatments

Variety        Treatment                      PV               T              BD              FV          SB             TTPV            PT
                                              (RVU)            (RVU)          (RVU)           (RVU)       (RVU)          (min)           (C)
               Buffer + 0 hr                  461a             205b           256a            532b        327c           6.48c           66.9c
Mortlock       Buffer + 1 hr                  411b             219a           192b            756a        537b           7.43b           73.0b
               -glucanase + 1 hr             261c             189c           72c             496c        307c           7.72a           90.1a
               Proteinase + 1 hr              375b             182c           193b            765a        583a           7.33b           72.7b
               Buffer + 0 hr                  410a             282a           128a            669a        387a           7.39b           86.5b
Yarran         Buffer + 1 hr                  265b             243b           22c             474c        232c           8.57a           89.7a
               -glucanase + 1 hr             232c             217c           15d             445d        228c           8.31a           90.0a
               Proteinase + 1 hr              239c             186d           53b             488b        302b           7.58b           90.0a
PV: Peak viscosity; T: Trough; BD: Breakdown; FV: Final viscosity; SB: Setback; TTPV: Time to peak viscosity; PT: Pasting temperature.

                         Table 3 Changes of DSC measurements with enzyme treatments

Variety           Treatment                          H1              Tp1              H2             Tp2              H3              Tp3
                                                     (J g-1)          (C)             (J g-1)         (C)             (J g-1)          (C)
                  Buffer + Ambient                   5.7a             50.3a            1.2a            81.0a            1.3b             104.1a
Mortlock          Buffer + 40C                      6.2a             51.5b            1.2a            81.8a            1.2b             104.5a
                  -glucanase + 40C                 5.9a             59.5c            1.3a            87.5b            0.7a             109.2b
                  Proteinase + 40C                  5.8a             52.1b            1.3a            81.7a            1.4b             105.6a
                  Buffer + Ambient                   5.1a             46.4a            1.2a            81.3a            0.5a             104.7a
Yarran            Buffer + 40C                      5.8b             48.6b            1.2a            82.8b            0.7a             107.1b
                  -glucanase + 40C                 6.1b             56.7c            2.0c            87.1c            0.4a             109.8c
                  Proteinase + 40C                  5.7b             49.2b            1.6b            82.3ab           0.5a             105.5a
H1, H2 and H3 are the enthalpies of first, second and third transition process, respectively; Tp1, Tp2 and Tp3 are the peak temperatures
of first, second and third transition process, respectively.

                         Table 4 Thermal properties of twelve samples before and after processing

                    Before Processing                                                             After Processing
                    H1       Tp1        H2          Tp2         H3          Tp3            H1        Tp1         H2          Tp2        H3       Tp3
                    (J g-1)   (C)       (J g-1)      (C)        (J g-1)      (C)           (J g-1)    (C)        (J g-1)      (C)       (J g-1)   (C)
Bimbil              5.0       50.5       1.0          81.0        0.4          104.1          3.8        49.0        0.8          83.1       1.1       105.7
Carrolup            4.9       50.4       1.1          81.7        1.2          104.5          4.1        50.3        0.4          83.8       0.8       103.7
Cooba               4.9       50.5       1.2          81.9        0.8          103.9          3.1        49.4        0.8          83.0       0.4       106.4
Echidna             5.3       50.9       1.0          81.9        1.0          103.7          3.2        49.8        0.6          82.4       1.1       102.9
Euro                5.2       51.2       1.1          81.6        2.0          104.0          4.6        48.7        0.6          84.4       /         /
Mortlock            5.0       51.7       1.2          81.6        1.6          101.8          4.6        51.2        0.6          84.5       1.1       105.4
Pallinup            4.7       52.4       0.9          81.8        0.9          101.9          3.1        50.5        0.8          84.5       1.1       104.1
Yarran              4.7       49.0       1.2          82.8        1.6          103.6          3.4        48.2        0.0          84.0       /         /




3
Cowra-Mortlock     6.2       49.1      1.1        81.2      0.8       104.5    3.4       49.2       0.7        82.0       0.8      104.3
Cowra-Yarran       5.8       46.8      1.5        83.2      0.6       104.3    3.6       45.9       0.6        84.5       0.8      103.1
Parkes-Mortlock    5.9       48.8      1.3        81.4      0.7       103.1    4.7       47.8       0.8        84.8       0.9      104.2
Parkes-Yarran      4.1       45.7      1.2        82.0      1.0       102.9    2.7       45.7       0.8        85.0       1.0      104.9
Average            5.1       49.8      1.1        81.8      1.0       103.5    3.7       48.8       0.7        83.8       0.9      104.5


Captions for Figures
Figure 1 RVA viscograms of Morlock and Yarran before (         ) and after (       ) soaking in buffer for 1 hr.
Figure 2 Changes in RVA viscograms of Morlock and Yarran before (               ) and after the addition of either -glucanase (           ) or proteinase (
                     ).
Figure 3 Effect of enzymatic treatments on thermal properties.




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