Rice Protein Aggregation During the Flaking Process

Journal of Cereal Science 28 (1998) 187–195 Article No. jc980199

Rice Protein Aggregation During the Flaking Process R. Mujoo∗, A. Chandrashekar† and S. Zakiuddin Ali∗ ∗Department of Grain Science and Technology †Department of Food Microbiology, Central Food Technological Research Institute, Mysore 570 013, India Received 8 September 1997

ABSTRACT The effect of roasting of soaked paddy rice and its subsequent flaking during production of rice flakes on solubility of rice proteins in various solvents was investigated. The solvents were so chosen to provide an understanding of the type of protein–protein interactions occurring during processing. There was an increase in protein solubility upon roasting of soaked paddy in fine hot sand (175–250°C) – a prelude to the flaking process – however, after subsequent flaking solubility decreased. Gel filtration studies indicated that aggregates with molecular weights greater than 4×107 kDa formed during flaking as a result of disulphide bonding. The aggregates were composed of 68, 31–34 and 24 kDa proteins. Of these, the 31–34 kDa proteins were identified as ‘putative prolamins’. These proteins require detergent for their initial solubilisation. The prolamins of 13–16 kDa were not involved in the formation of aggregates. Their solubility decreased after flaking. The higher molecular weight proteins involved in aggregate formation were susceptible to proteolysis, whereas the 13 kDa prolamin was resistant.  1998 Academic Press

Keywords: rice, protein, flaking, solubility, prolamins.

INTRODUCTION Rice is a very widely used staple cereal and is eaten either directly after cooking in boiling water or after processing to various products such as expanded, popped and flaked rice. In the Indian subcontinent flaked rice is usually produced by roasting fully soaked paddy in hot fine sand and then flaking in a unit called an edge-runner flaker1 or in a heavy duty cereal roller-flaker; a process developed by CFTRI, India2. It has been shown by many workers3–5, using the Osborne method of protein fractionation, with slight modification thereof, that proteins of wheat,

 : w.b.=wet basis; CFTRI=Central Food Technological Research Institute; B.S.S.=British standard sieve; ME=mercaptoethanol; SDS-PAGE= sodium dodecyl sulphate; 2-polyacrylamide gel electrophoresis; Tris=tris (hydroxy methyl) methylamine; PMSF=phenylmethyl sulphonyl fluoride. Corresponding author: S. Zakiuddin Ali. 0733–5210/98/050187+09 $30.00/0

sorghum and maize undergo change during processing. Most of these changes have been attributed to the formation of disulphide bonded aggregates between various proteins4–8. One possible consequence of the aggregation of proteins would be reduction in the protein digestibility as reported in sorghum9. This study was undertaken to investigate the changes in the solubility, aggregation and digestibility of protein in rice grain as a result of cooking, roasting and flaking. The solvents used for dissolution of proteins were so chosen that an understanding of the role of hydrogen and disulphide bonds in protein interactions during processing would be obtained. The enzyme trypsin was used for digestion to identify those proteins that may be resistant to proteolysis after processing. EXPERIMENTAL A long grain, high amylose (28·6%) paddy cultivar IR-64, which is currently being used by cottage  1998 Academic Press

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industry in India for producing flaked rice, was obtained from a local market. Milled rice grain average dimensions were length 6·7 mm, breadth 2·1 mm and thickness 1·7 mm.

Preparation of flakes The paddy was processed to prepare rice flakes as per the traditional method using edge-runner (ER) unit and the modern roller–flaker (RF) flaking unit. Edge-runner1 flakes were prepared by soaking the paddy overnight to a moisture content of about 30% followed by roasting manually in batches of 1 to 2 kg in hot fine sand at 250°C. The roasted paddy, separated from the sand by sieving, with a moisture content of about 18 to 20%, was immediately fed into an edge-runner unit rotating at 300 rev/min. The unit consists of a perforated, round, flat thick sieve (with a raised edge). An idle roller is mounted at the edge of the sieve and the roasted paddy is flaked during passage between the edge and the roller. The dried husk and part of the bran layers are removed through the rotating sieve and the rice grains are flattened by repeated pressing as they pass between the idle roller and the edge of the sieve. The flaking was carried out for 35 and 60 s to obtain thick (1·3 mm) and thin (0·7 mm) flakes respectively. Roller flaking2 involves an instantaneous, single step pressing of the rice grain. The soaked paddy was roasted in a continuous mechanical roaster with sand at 175°C. The roasted paddy (moisture content 20–22%) was dehusked, debranned, tempered and then flaked by passing through a heavy duty roller–flaker consisting of two parallel steel rolls (diameter 50 cm, length 60 cm exerting pressure of 40 t m−1) mounted close to each other, and rotating in opposite directions. The clearance was adjusted to obtain thick (1·0 mm) and thin (0·3 mm) flakes. Samples were collected during successive stages of processing of paddy to flakes. This included raw paddy, roasted paddy, thick and thin flakes from the edge-runner and roller–flaker flaking processes. All samples were dried to about 12–13% moisture (w.b.), ground first in a Bu¨hler Laboratory Grain Mill (type MLI 204, Bu¨hler-Miag, Italy) and then in a Fritsch Pulverisette-14 (Fritsch Gmbh, Germany) to pass a 100 mesh British Standard Sieve (B.S.S) screen. All the samples were equilibrated to 12–13% moisture content (w.b.). The

samples used to determine changes in protein during processing were raw rice flour (PR), rice flour cooked in water at 96°C for 20 min (CPR), roasted polished rice (RPR), thick flakes from the edge-runner (TkF-ER) and roller–flaker (TkF-RF) and thin flakes from the edge-runner (TnF-ER) and roller–flaker (TnF-RF). Protein extraction Twenty milligrams of each flour defatted by Soxhlet extraction using 60–80°C petroleum ether was extracted using the following solvents: (a) 2% sodium dodecyl sulphate (SDS); (b) 2% SDS/5% 2-mercaptoethanol (2-ME); (c) 2% SDS/6 urea; and (d) 2% SDS/5% 2-ME/6 urea. Each sample was extracted twice and the extracts pooled. Prolamins were extracted from 100 mg flour with 70% 2-propanol, with or without 5% 2-ME. The residue was re-extracted twice in the same volume of solvent, the supernatants pooled and evaporated at 40°C. Prolamins were also recovered from protein extracts of 100 mg or raw rice flour with 2% SDS/ 6  urea/5% 2-ME. The extract was centrifuged for 20 min at 6000 g treated with equal volume of 2-propanol/5% 2-ME to obtain a 50%(v/v) 2propanol/2·5% 2-ME solution. The supernatant obtained after centrifugation was extensively dialysed against 50% 2-propanol for 10 h to remove the original solvent. The dialysed solution was evaporated at 40°C and the residue extracted with 70% 2-propanol/5% 2-ME prior to separation by SDS-PAGE. Estimation of nitrogen content Protein in the extracts was quantified from optical density values at 280 nm. Polished rice flour (2 g) was extracted with 20 mL SDS/ME. Optical density and Kjeldhal nitrogen values were determined for each sample. The relationship between 280 nm absorption at different extract concentrations to the nitrogen content was essentially linear. The nitrogen content of all the other extracts was therefore obtained from their optical density at 280 nm. Electrophoresis of protein extracts Proteins extracted by the various solvent systems from the different samples were subjected to so-

Changes in rice proteins on processing

Table I

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Content of nitrogen (% of total nitrogen) extracted with different solvents from raw, roasted and flaked rice

Sample

2-propanol

2-propanol +ME

SDS

SDS+urea

SDS+ME

SDS+ME +urea

SEM∗

PR CPR RPR TkF-ER TkF-RF TnF-ER TnF-RF SEM†

1·93aA 6·51fA 5·47eA 4·73dA 3·56cA 3·36bcA 3·01cA ±0·004

9·24dB 9·16dB 9·32dB 8·62cB 8·44bcB 8·28bB 8·03aB ±0·003

20·0bC 14·6aC 28·6cC 16·8aC 16·7aC 17·8abC 16·8aC ±0·005

23·2bC 14·6aC 32·6cC 21·1bD 21·0bD 17·8aC 22·0bD ±0·009

44·3bD 34·5aD 67·0dD 48·5bE 53·6cE 65·1dD 66·2dE ±0·014

52·6bE 32·8aD 67·0cD 62·4cF 62·9cF 64·5cD 64·6cE ±0·026

±0·013 ±0·006 ±0·025 ±0·009 ±0·012 ±0·005 ±0·011

PR=raw rice; CPR=cooked polished rice; RPR=roasted polished rice; TkF-ER=thick edge runner flakes; TkF-RF= thick roller flakes; TnF-ER=thin edge runner flakes; TnF-RF=thin roller flakes. All values are mean of triplicate extractions. Values of same column or row followed by different letters differ significantly (PΖ0·05) according to Duncan’s New Multiple Range test. Difference between treatments (columns) for each solvent are indicated by small letters. Differences between solvents (rows) for each treatment are indicated by capital letters. ∗ Standard error of mean between solvents for each treatment. † Standard error of mean between treatments for each solvent.

dium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli10 with slight modifications. The extracted supernatants were mixed with an equal volume of SDS-sample buffer (0·125  Tris-HCl, pH 6·8, 4% w/v SDS/5% w/v 2-ME) and heated at 96°C for 3 min before separating on electrophoresis. Gels were stained for protein using Coomassie Brilliant Blue R-250 (0·05%) in methanol–acetic acid– water (25:10:65 v/v/v) and de-stained in the above solution without the dye. Proteins used as molecular weight standards were lysozyme (14 300), carbonic anhydrase (29 000), ovalbumin (43 000), bovine serum albumin (68 000) and phosphorylase b (97 400) obtained from Bangalore Genei, India. Gel filtration SDS/urea extracts from rice flour, cooked rice flour and thin flakes from the edge-runner and roller–flaking process, were fractionated by ascending column chromatography (Pharmacia column, 1·6×70 cm) using Sepharose CL-2B gel (Pharmacia, Sweden) at a flow rate of 15 mL/h using 0·1% SDS as eluent. The void (Vo) and the total volume (Vt) of the gel column was determined using isolated waxy starch and glucose respectively. Molecular weight assignments of the void volume peak and the fractionated peaks was based on the standard curve provided by the manufacturer for

the gel with respect to the kav values for globular proteins. Fractions were collected in 3 mL aliquots and analysed spectrophotometrically at 280 nm. The optical density of each aliquot was expressed as a percentage of the total sum of optical densities of all the aliquots collected. The peak fractions were dialysed extensively against double distilled water and freeze dried. The dried samples were dissolved in SDS/urea/2-ME and subjected to SDS-PAGE. Digestibility Flours from different samples (50 mg) were incubated with trypsin (EC 3.4.21.4, Sigma Chemical Company U.S.A.) at two concentrations, 1590 and 12 720 units, in Tris buffer (20 mM, pH 7·2) at 37 °C for 60 min. Enzyme action was stopped by adding phenyl methyl sulphonyl fluoride (Sigma Chemical Co, U.S.A.), 1 mM in 2-propanol. Protein was extracted from the residue obtained after centrifugation of the digest with SDS/urea/2-ME and subjected to SDS-PAGE as described earlier. Statistical analysis The data were subjected to analysis of variance conforming to completely randomised design and the means of triplicate extractions were separated using Duncan’s New Multiple Range test11.

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S

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Figure 2 SDS-PAGE profiles of proteins from raw rice extracted with (a) 2-propanol/2-ME; (b) SDS/2-ME/urea fraction re-extracted with 2-propanol/ME.

RESULTS Protein solubility S

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Quantitative Data on the quantity of the nitrogen extracted from different samples using various solvents are presented in Table I. More nitrogen was extracted by 2-propanol from cooked and roasted-polished rice than from raw and flaked rices. Addition of 2-ME to 2-propanol increased the amount of nitrogen extracted. However, the quantity of proteins extracted between samples was not very different except for a decreased extractibility from thin flakes. SDS extracted more protein than 2propanol 2-ME, with least nitrogen being extracted from cooked rice and the highest from roasted polished rice. Addition of 2-ME to SDS increased protein solubility by two to three times over that of SDS alone. Addition of urea to the SDS further increased the solubility. The highest quantity of protein was extracted from samples with SDS/urea/ME. More protein was extracted from flakes than from raw and cooked rice. About 30% of rice protein still remained unextracted by

Figure 1 SDS-PAGE profiles extracted from rice preparations with (a) SDS; (b) SDS/2-ME; and (c) DS/2-ME/urea. Lanes a–g represent: raw rice; cooked polished rice; roasted polished rice; thick edge-runner flakes; thick roller–flaker flakes; thin edgerunner flakes; and thin roller–flaker flakes respectively. The lane S on the left side of each figure shows molecular weight markers: lysozyme (14 300); carbonic anhydrase (29 000); ovalbumin (43 000); bovine serum albumon (68 000) and phosphorylase b (97 400).

Changes in rice proteins on processing

191

16 3 14

12

2

% OD (280 nm)

10

8

6 1 4

2

0 10

20 Vo

30 Fraction number

40

50 Vt

Figure 3 Gel filtration profiles of rice flour proteins from raw rice (-); cooked polished rice (--); thin roller–flakes (···) and thin edge-runner flakes (-·-·-) extracted with SDS/urea.

the SDS/urea/ME solvent and the use of an alkaline medium may enable more protein to be extracted. Qualitative The SDS-PAGE profile of proteins extracted with SDS, SDS/2-ME and SDS/2-ME/urea are presented in Figure 1 (a–c) respectively. A triplet at 31–34 kDa, and a 20 kDa protein were present as intense bands, and faint bands in the range 60–55 kDa and at 29, 24 and 11 kDa were observed in raw rice proteins extracted with SDS [Fig. 1(a)]. Protein bands in SDS extracts from cooked rice flour were faint due to decreased extractibility of proteins after cooking. A triplet band with molecular weight of 39–42 kDa, and intense bands at 65 kDa and 20 kDa were characteristic of roasted polished rice. The bands at 31–34 and 20 kDa were less intense in roasted polished rice than in raw rice. After flaking there was an overall reduction in the intensity and number of bands. A

greater reduction was observed in the intensity of protein bands extracted from the edge-runner than from the roller–flaker flakes. Fainter bands were seen for extracts from thin flakes than from thick flakes. The intensity of 14 kDa band in all flakes was similar to that from raw rice. In addition a band at 11 kDa was visible in these samples. When flours were extracted with SDS/2-ME and SDS/2-ME/urea new bands appeared in the high molecular weight region (90, 85 and 80 kDa) and the protein bands at 34, 32, 31, 24 and 20 kDa were more intense than when extracted with SDS [Fig. 1 (b,c)]. Additional bands at 39–42 kDa were seen only in roasted polished rice, and the 24 and 17 kDa bands were darker. Proteins extracted by these two solvents from flaked rice were similar to those from raw rice flour. The profile of proteins was similar to those extracted by SDS/2-ME, though more protein was extracted by SDS/2ME/urea. The solubility of the protein of flaked samples in SDS/2-ME or SDS/2-ME/urea was less from thinner than from thicker flakes.

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16 3 14

2

12

% OD (280 nm)

10

8

6 1 4

2

0 10

20 Vo

Figure 4

30

40

50 Vt

Fraction number

Gel filtration profiles of thin edge-runner flakes extracted with SDS (-); SDS/urea (-·-); SDS/urea/2-ME (···).

Qualitative profile of prolamins Rice prolamins are usually reported to be of the size 13–16 kDa12. However, a 23 kDa prolamin has also been reported13. Recently Wallace et al.14 and Hamaker et al.15 have developed a method for the extraction of zeins and kafirins from maize and sorghum respectively. Proteins were extracted in borate buffer (pH 10) containing detergent (1% SDS) and reducing agent (2% 2-ME). Non-prolamins were precipitated by the addition of ethanol, leaving the ethanol soluble proteins in the solution. More prolamins were extracted by this procedure than by direct extraction of flour with aqueous ethanol and 2-ME. This modified solvent system was applied to rice. The SDS-PAGE of the prolamins extracted in this modified solvent system is presented in Figure 2. Only 13–16 kDa bands proteins were extracted with aqueous alcohol and 2-ME, whereas additional bands at 31–34 kDa and 24 kDa were observed when proteins were extracted by the modified procedure. It is there-

S

a

b

c

d

e

f

g

h

Figure 5 SDS-PAGE profiles of peak fractions after extraction with SDS/urea. Lanes a and b represent peaks 2 and 3 of raw rice; lanes c–e represent peaks 1, 2 and 3 of cooked polished rice and lanes f–h represent peaks 1, 2 and 3 of thin edge-runner flakes. Lane S again shows molecular weight markers.

Changes in rice proteins on processing

(a)

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fore, suggested that these proteins may also be ‘prolamins’. Gel filtration profile of proteins

S

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Raw rice, cooked polished rice, and thin flakes from edge-runner and roller-flaker proteins were extracted with 2% SDS/6 urea, separated on Sepharose CL-2B column and eluted with 0·1% SDS. The elution profiles showed three peaks, one at the void volume and two others at kav 0·68 and 0·86, referred to as peaks 1, 2 and 3 respectively (Fig. 3). The void volume peak (1) of raw rice had an apparent molecular weight of 4×107 kDa, and comprised about 2·5% of the total area under peaks. There was an increase in the size of void volume peak for protein extracted from cooked rice. The proportion of the void volume peak comprised about 15% of the total area under peaks in proteins extracted from the flaked rice. The size of this fraction was larger for proteins extracted from edge-runner flakes (15%) than from the roller–flaker flakes (10%). In another experiment, when thin flakes from the edge-runner process were extracted with SDS, SDS/urea and SDS/urea/2-ME prior to gel filtration, it was observed that inclusion of 2-ME to the extracting solvent, reduced the size of peak 1 (Fig. 4). The SDS-PAGE profile (Fig. 5) of peaks 2 and 3 from raw rice showed that all rice proteins were present. Protein bands at 68, 31–34 (triplet) and 24 kDa were observed in those samples that formed large aggregates in all samples and were similar in banding pattern to proteins of peak 2 and peak 3.

(b)

S (c)

Digestibility with trypsin The SDS-PAGE profile of rice proteins extracted from raw and processed rice after digestion with different trypsin concentrations showed that the proteins from processed rice flours were more Figure 6 SDS-PAGE profiles of proteins extracted from rice preparations with SDS/urea/2-ME after digestion with different concentrations of trypsin: (a) Control; (b) 1590; and (c) 12 720 units of trypsin. Lanes a–g represent: raw rice; cooked polished rice; roasted polished rice; thick edge-runner flakes; thick roller–flaker flakes; thin edge-runner flakes; and thin roller–flaker flakes respectively. Lane S again shows molecular weight markers. S

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susceptible to trypsin degradation than those from raw rice (Fig. 6). The 32 and 24 kDa ‘prolamin’ bands involved in aggregation, were resistant to proteolysis in raw rice at both enzyme concentrations. In roasted polished and flaked rice these proteins were resistant to trypsin at the lower enzyme concentration, but were completely degraded at higher enzyme concentrations.

DISCUSSION The results clearly indicate that disulphide bonds are formed during the cooking or processing of rice. The formation of these bonds is indicated by the increased solubility of protein in solvents containing 2-ME. These disulphide bonded proteins were shown to form large aggregates. Decrease in solubility of wheat, sorghum and maize and soy proteins on thermal processing has been reported earlier due to formation of disulphide cross linkages3–5,16–18. The present study indicates that disulphide bonded aggregates are formed during flaking rather than roasting. Thus, the mechanical energy input during flaking appears to initiate the formation of disulphide bonds. The possible conversion of mechanical energy to bonding energy has not however, received much attention. Proteins in the edge-runner flakes were aggregated to the highest degree. This might occur because the grains are fed into the flaker when they are very hot whereas in the case of rollerflaking they are fed into the flaker at relatively low temperatures (50–60°C). Furthermore, the grains are compressed instantaneous in the roller-flaker unit in one operation whereas in the edge-runner process repeated pressing is needed to obtain flakes. It is suggested that the amount of protein in the aggregates might be used to estimate the amount of mechanical energy expended on flaking of rice. Some of the proteins involved in the aggregation appear to be ‘prolamin’ like proteins. In sorghum and maize, proteins rich in cysteine and gammaprolamins have been implicated in disulphide bonding19. Such proteins have not hitherto been identified in rice. The work presented indicates that some prolamins in rice are bonded tightly to other proteins and require detergent for their initial solubilisation. The nature of these proteins needs further investigation. The digestibility of many proteins is increased

during processing as reported, for example, in the thermal treatment of wheat, maize, rice and barley20–22. In rice the 24 and 31–34 kDa ‘prolamin’ like proteins are more resistant than other proteins to trypsin treatment at low enzyme levels than at higher enzyme concentrations. A 10–13 kDa rice prolamin was found to be resistant to proteolysis at both enzyme concentrations as reported by other workers21,23. Acknowledgements Ms Rajni Mujoo acknowledges with thanks the University Grants Commission, New Delhi (India) for the award of a Research Fellowship. REFERENCES 1. Ananthachar, T.K., Narasimha, H.V., Shankara, R., Gopal, M.S. and Desikachar, H.S.R. Improvement of the traditional process for rice flakes. Journal of Food Science and Technology 19 (1982) 47–50. 2. Narasimha, H.V., Ananthachar, T.K., Shankara, R., Gopal, M.S. and Desikachar, H.S.R. Development of a continuous process for making rice flakes. Journal of Food Science and Technology 19 (1972) 233–235. 3. Dexter, J.E. and Matsuo, R.R. Changes in sphaghetti protein solubility during cooking. Cereal Chemistry 56 (1979) 394–398. 4. Vivas, N.E., Waniska, R.D. and Rooney, L.W. Effect of tortilla production on proteins in sorghum and maize. Cereal Chemistry 64 (1987) 384–389. 5. Ummadi, P., Chenoweth, W.L. and N.G., P.K.W. changes in solubility and distribution of semolina proteins due to extrusion processing. Cereal Chemistry 72 (1995) 564–567. 6. Chandrashekar, A. and Kirleis, A.W. Influence of protein on starch gelatinization in sorghum. Cereal Chemistry 65 (1988) 457–462. 7. Hamaker, B.R. and Griffin, V.K. Changing the viscoelastic properties of cooked rice through protein disruption. Cereal Chemistry 67 (1990) 261–264. 8. Hamaker, B.R. and Griffin, V.K. Effect of disulphide bond-containing protein on rice starch gelatinization and pasting. Cereal Chemistry 70 (1993) 377–380. 9. Hamaker, B.R., Kirleis, A.W., Mertz, E.T. and Axtell, J.D. Effect of cooking on the protein profiles and in vitro digestibility of sorghum and maize. Journal of Agriculture and Food Chemistry 34 (1986) 647–649. 10. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage 14. Nature 227 (1970) 680–685. 11. Steel, R.G.D. and Torrie, J.H. ‘Principles and procedures of statistics’, McGraw-Hill Book Co., New York (1980) pp 107–109. 12. Padhye, V.W. and Salunke, D.K. Extraction and characterisation of rice proteins. Cereal Chemistry 56 (1979) 389–393. 13. Mandac, B.E. and Juliano, B.O. Properties of prolamin

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