Extraction of Protein and Solids From Wheat Bran

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J Sci. Food Agric. 1985 36 5-10

Extraction of Protein and Solids from Wheat BranPeter J. Roberts, David H. Simmondsa, Michael Woottonand Colin W. WrigleySchool o Food Technology, University of New South Wales, Kensington, New South Wales 2033 andCSIRO, Wheat Research Unit, North Ryde, New South Wales 2113, Australia(Manuscript received I May 1984)

The influence of pH , time, temperature, cellulase treatment and washing condi-tions on the recovery of solids and protein from Australian wheat bran wasinvestigated. Highest yields (83 of available protein, 72 of total solids) wereachieved by extracting at pH 12 and washing with water at pH 7. However,acceptable yields could be obtained under near neutral conditions by extracting atpH 6.5 for 16h followed by three washes at pH7 . Under these conditions 72 ofthe protein and 55 of the total solids were extracted and greater stability of thevitamins thiamin, riboflavin and pyridoxine was expected. The use of cellulase in anattempt to disrupt the aleurone cell walls did not improve the recoveries of eitherprotein or solids.Keywords : Wheat bran; protein extraction; thiamin; riboflavin; pyridoxine.

1. IntroductionInexpensive sources of protein from plants and microorganisms have been extensively investi-gated to supplement and even replace more expensive animal protein in the diet of the world'sincreasing populat ion. Various fractions from the milling of cereals, particularly wheat bran andgerm, offer potential in this regard. Protein concentrates have been prepared from rice branusing a wet alkaline process,' while the preparation and properties of a corn germ isolate havealso been reported.* Protein has been extracted from wheat bran, with recoveries increasing from28 at pH 6.5 to 84 at pH 12.5.3 Wet alkaline processes have also been used to produceprotein concentrates from various wheat milling product^.^.^

It is apparent from this work that wet extraction under strongly alkaline conditions (pH 12.5)offers substantial advantages in terms of protein yield. However, vitamins such as thiamin,riboflavin and pyridoxine which are known to be concentrated in wheat bran, have poor stabilityat high pH6 and could be rapidly destroyed under these extraction conditions. Strongly alkalineconditions may also lead to changes in the functional properties of the recovered protein,especially if the bran is contaminated with endosperm-derived material containing gliadin andglutenin proteins from the storage fraction of the grain.

The fact that up to 20 of the total protein present in the grain is contained in the bran layers,particularly in the germ and aleurone, suggests that a close examination of extraction proceduresapplicable to these millstreams is warranted. The present work was therefore undertaken toinvestigate the isolation of protein from wheat bran under milder conditionsof pH, in an attemptto obtain acceptable protein recoveries while reducing the destruction of any vitaminssimultaneously extracted. The protein-depleted bran so produced would also be useful in theformulation of high-fibre based products.

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2. Mater ia ls and methods2.1. B r a nA f inely divided comm ercial bran f rom m ixed Austral ian wheat var ie t ies was obtain ed from C.S.McIntyre Flour Mills , Newcastle , New South Wales. This mater ia l contained protein (14.8%,calculated as Nx6.25) , mois ture (12 .1%) , fa t (3 .4%) , ash (4 .6%) and starch (5.9%).2.2. Extraction proceduresFor all extract ions, deionised water (150ml) was added to bran (15g) in a Virt is homogeniserflask. Th e p H was immediately adju sted to th e desired level using HC1 or N aO H ( 1 ~ ) . h esuspension was the n hom ogenised using a Virtis 45 homogenise r (T he Virt is Co mpany G ardner ,New York) for 4x1 min per iods with 1 min intervals between each. The products f rom fourhomogenisat ions were combined for each subsequent steeping regime. Following homogenisa-t ion , the p H was readjus ted t o th e des i red leve l us ing 1~ HC1 or N a O H as requi red .Extraction of the homogenised suspension was carried out by heating in a water bathmaintained at the desired temperature with continuous mechanical s t i r r ing for the specif iedtimes. Th e p H was mon itored dur ing the extract ion procedure and , wh ere necessary, wasreadju sted. Varia t ions w ere less than 0.2 p H units .At the end of the extract ion per iod the samples were f i l tered under vacuum through cheesecloth and the residue was resuspended in the wash solution. The suspension was st ir red for 10min at approximately 25C before filtering as described above. A residue to solvent ratio of 1:10was used and pH was adju sted using IMN aO H o r HC1 as requi red . This procedure was repea tedup to th ree t ime s. Washings were com bined with the or iginal extract an d stored at 3C pr ior toanalysis which was ca rried o ut within 24 h. The bran residue was dried in a vacuum oven at 150Cto constant weight .

The influence of cellulase in extractability of protein and solids by cell wall degradation wasinvest igated using the enzy me Cellulase 200 00 (Pf izer Pty Ltd. W est Ry de, New Sou th Wales) a ta level of 1% in the or iginal extract ion m edium . Th e extract ion was a llowed to proceed for 16 ha t the tempera ture and pH optima for this enzyme (60 C, pH 4.5).2.3. Analysis of extract ion an d b ran residuesProtein c on ten t was dete rm ine d directly on t he ex tracts following digestion for 2 h at 370C in aTechnicon BD-20140 block digester. Analysis of the digests employed a salicylateinitroprussideprocedure using a Technicon A utoan alyser system (Sampler model 11, P u m p M o d e l I , ManifoldNo . 116-D531-01, Colo r imete r I).The moisture content of the bran residues and the solids content of the extracts weredetermined by drying to constant weight in a vacuum oven according to standard methods ofanalysis (Off ic ia l Methods of Analysis of t he AOAC 12 th e dn) . F o r sc a nn ing e l e c t r onmicroscopy, bran and extracted bran were placed on to a luminium stubs using double sidedadhesive tape without fur ther treatment.

Table 1. Effect of extraction time and cellulase treatment on the yield of protein and solids fromwheat bran

Extractiontime(h)

08

162432

Total protein extracted Total solids extractedcellulase cellulase~

57 5765 6974 6557 6067 69

cellulase + cellulasea)51 4858 5153 5253 5251 51


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Extraction of protein an d solids from w heat bran

90r8

I401 2 3 40 5 6 7 8 90

Extraction temperature ( C)Figure 1 . Effect of temperature on extraction of protein 0 ) and solids 0 ) at pH 6.5 for 16h followed by three washes atpH 7.

3 Results and discussionTh e yields of protein an d solids by extracting for various times at 60C and p H 4.5, ollowed bythree washes a t pH 7, with and without 1 % added cellulase , are shown in Table 1.I t wil l be seen th at the addit ion of cellulase offered n o adv antages in yield of protein or solidseven though ext rac tion was ca rr ied o ut a t the op t imum p H of the enzyme. I t is a lso apparen t thatextraction fo r 8-16 h achieved op tim um yields of both protein and solids. Extraction fo r 16 h wasused in subsequent ex per iments because of the convenience of overnight extraction although 8 his more a t tract ive commercial ly. Three washing steps, a procedure adopted as standardthroughout th is s tudy, proved to be ad equa te for maximum solids and protein recovery.Th e effect of tem per atu re on protein and solids recovery was studied by extract ing at p H 6.5for 1 6h a t va rious tempera tures be tween 10 and 90C. The yields o btaine d are shown in Figure 1.It will be seen that although the highest yields of protein and solids were obtained at 60 and70C respectively, l it t le var ia t ion was encou ntered over the wh ole tem peratu re range. A stand ardextract ion temperature of 60C was therefore adopted for the remainder of the s tudy. At th istemperature , protein recovery was a t a maximum. However , for a commercial operat ion theenergy savings of extract ing at a mb ient temp erature would probably mo re tha n offset the sl ightlylower protein and solid yields obtain ed.The inf luence of pH of both extract ing and washing media was a lso invest igated. Bran wasextracted to 60C for 1 6 h using extract ing solutions of p H 2 , 4.5, .5 and 12. Recovery of proteinand solids was de te rmined for each ext rac tion p H , fo llowing three washes a t each p H 2 , 7 and12). Th e result s a re sum mar ised in Table 2.

Table 2. Effect of extraction and washing pH on recovery of protein andsolids from wheat branTotal protein extractedafter washing (5 ) Total protein extractedafter washing (9 )~~xtractionP H p H 2 p H 7 p H 1 2 p H 2 p H 7 u H 12

2.0 66 66 73 51 51 544.5 61 66 74 51 50 596.5 64 72 73 51 55 4912.0 74 83 83 61 72 60


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Table 3. Particle size distribution of bran

P. J. Roberts e l al.

Mesh sizeBran retained

( )2.0 mm1.4 mm1.0 mm425 pm300 pmPan

60 P m

3.56 . 1

26.026.013.310.413.5

Figure 2. Scanning electronmicrographs of bran before (a)and after (b) extraction at pH6.5, ambient temperature for16 h followed by three washesat pH 7.


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Extraction of protein and solids from wheat bran 9

It is apparent from these data that alkaline extraction conditions give the highest recoveries ofboth protein and solids. This is consistent with earlier finding^ ^ Ho wev er , th e yields o btaine d inthis study at less alkaline pH values are significantly higher than those obtained in the previouswork. ' This may be du e both to the s ta te of subdivision of the different bran sam ples used in thetwo ser ies of experiments and to dif ferences in the extract ion t imes used. A third importantvar iable is the am ou nt of end ospe rm mater ia l adher ing to the in ner margin of the a l euron e cells .I t can b e seen from sieving analysis (Table 3) that almost two-thirds of the fine bran particleswere less than 1mm in size , smaller than normal and coarse bran streams. Normal commercialsamples of the la t te r pass over 1mm mesh. I t was found tha t ext ract ion a t 60C, p H 6 .5 for 16hfollowed by 3 washes a t p H 7 w ithout homogenisat ion resulted in only minor changes t o yields ofprotein and solids ( less than 2%) compared to exper iments in which homogenisat ion was used.Yields in th e presen t study unde r less a lkaline condit ions (72% of protein a t pH 6.5) should becompared with previous results (28% of prote in a t p H 6.5).3 This improvement is a t tr ibuted toth e f iner bran size and longer extract ion t imes used.Examination of the scanning electron micrograph in Figure 2a shows the presence of starchye ndospe r m on the unextracted bran. Based on the level of 5.9 starch found in the bran, thismay be est im ated as approximately 7.5% of the unextracted mater ia l . This adher ing endos permmaterial contains relatively high proportions of storage protein, the concentration of whichdecreases f rom the sub-aleurone to the central por t ions of the endosperm. Treatment withaque ous solutions a t ne ar neutral ity, extracts less of the end ospe rm storage protein th an wouldalkaline solutions. Chan ges in th e appearan ce of t he b r a n as a result of extract ion can b e seen bycomparing the scanning electron micrographs in Figure 2. Washing has removed all adheringstarchy material together with the contents of most of the a l euron e cel ls . The m ain are a in bo thelectron micrographs (Figure 2a and 2b) shows the inner surfaces of the bran while the outersurface can be seen in the lower lef t por t ion. Prel iminary studies have shown the presence offa int bands representing endosperm protein in the e lectrophoretic and isoelectr ic focusingpatterns of the bran extracts , a long w ith bands characterist ic of the bran protein .W hen ext rac t ion was under taken us ing non-alka line condit ions , t r ea tment a t p H 6 .5 ( theunadjus ted p H of bran) , fo llowed by three washes a t p H 7 (unadjus ted pH of washed bran) gavethe best yields of bo th p rotein and solids. I t is therefore likely th at on an industr ia l scale , tapwater could be used as a conv enien t , che ap and effective solvent for both extract ion and washing.

4. ConclusionsThis study has established conditions for the effective recovery of protein and solids from wheatand solids f rom w heat bran using neutral extract ing solutions. Th e results indicate that extract ionwith t ap wa ter a t p H 6 .5 for 16 h a t amb ient temp eratures , fol lowed by three washes with tapwater wil l recover abou t 72% of the tota l protein an d 55 of th e solids com par ed with 83 and72% respectively und er alkaline conditions (p H 12). T he improv ement in yield at non-alkalinep H levels com par ed to previous research is a t tr ibut ed to the f iner bran size and longer extractiontimes used in this s tudy. Digest ion of the cell walls with cellulase offers no advantages forcommercial use. The results open up the possibility of carrying out fur ther work on the drying,composition and utilisation of th e extra cted mater ia l and the insoluble high-f ibre residue.

References1.2.3.

Connor, M . A . ; Saunders, K. M . ; Kohler, G . 0 Rice bran protein concentrates obtained by wet alkaline extraction.Cereal Chem. 1076, 55 488-496.Nielsen, H. C. ; Inglett, G. E.; Wall J . S.; Donaldson, G. L. Corn germ protein isolate: preliminary studies onpreparation and properties. Cereal Chem. 1973, 50 435-443.Fellers, D. A ; Sinkey, V . ; Shepherd, A. D.; Pence, J W . Solubilisation and recovery of protein f rom wheat millfeeds.Cereal Chem. 1966, 43, 1-13.


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Lindsay, G. W . ; Saunders, R. M.; Kohler, G. 0 Protein concentrates from wheat shorts, rice bran and soy flour byextraction with cheese wheys. J Food Sci. 1977, 42 1365-1369.Saunders, R . M. ; C o n n o r, M. A . ; Edwards, R . H . ; Kohler , G . 0 Preparation of protein concentrates from wheatshorts by a wet alkaline process. Cereal Chem. 1975, 52 93-101.Marks, J . A Guide o the Viturnin. Medical and Technical Publishing Co . Lt d, England, 1975.Cooke. W. M . ; Simpson, W . E. Determination of amm onium in Kjeldah l digests of crop s by an automated procedure.J Sci. Food Agric. 1971, 22 9-10Kent, N . L . Subaleu rone endosperm cells of high protein content. Cerenl Chem. 1966. 43 585-601.Hinton, J . J . C. The distribution of vitamin B I and nitrogen in the wheat grain. Proc. R . Sci. (London) 1947, B134418-429.Morris, V . H . ; Alexander, T. L.; Pascoe. E . D. Studies of the composition of the wheat kernel. 111. Distribution of ashand protein in central and peripheral zones of whole kernels. Cereal Chem. 1946, 23 54@547.

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Extraction of Protein and Solids From Wheat Bran