Journal of the American Society of Brewing Chemists

The Science of Beer

ISSN: 0361-0470 (Print) 1943-7854 (Online) Journal homepage: http://www.tandfonline.com/loi/ujbc20

Enzymic Hydrolysis of Brewers' Spent Grain

N. Prentice & J. M. Refsguard

To cite this article: N. Prentice & J. M. Refsguard (1978) Enzymic Hydrolysis of Brewers' Spent
Grain, Journal of the American Society of Brewing Chemists, 36:4, 196-200, DOI: 10.1094/
ASBCJ-36-0196

To link to this article: https://doi.org/10.1094/ASBCJ-36-0196

Published online: 06 Feb 2018.

Submit your article to this journal

Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=ujbc20
Enzymic Hydrolysis of Brewers' Spent Grain 1/2
N. PRENTICE and J. M. REFSGUARD, 3 U.S. Department of Agriculture, Barley and Malt Laboratory, Madison, WI
53705
ABSTRACT
Enzyme hydrolytic products of brewers' spent grain (SG) have a
representative composition of: 9.6% total lipid,4.3%ash, 31% protein, 9%
c e l l u l o s e , 12% s t a r c h and /3-glucans, 19% p e n t o s a n s
(xylose:arabinose: 12:7), and 16% lignin. Treatment of commercial dried,
milled SG (CSG) with a crude cellulase preparation hydrolyzed all of the
starch and /?-glucans, about two-thirds of the cellulose and one-half of the
pentosans into the constituent monosaccharides. The cellulase did not
effectively hydrolyze the cellulose and pentosans in pilot wet, unmilled
SG (PSG). In about 6 hr, a protease hydrolyzed up to 87% of the protein in
CSG and PSG to soluble products, 85% of which had a molecular weight
less than 3500. After treatment of the CSG with crude cellulase plus
protease, about 53% was hydrolyzed to form soluble nutrients.
Key words: Brewery by-product, Soluble products, Utilization
ne of the voluminous residuals of brewing is the "spent grain"
O (SG) that remains after mashing. Nutritionally, the grain is far
from spent since the protein level may be 30% or greater (20).
Because SG contains large amounts of fibrous materials, it is
marketed primarily as ruminant feed. Recently, attention has
focused on the importance of fiber in diets (21), and bread is
produced (17) in which protein and fiber are increased by
replacement of about 15% of the wheat flour by finely milled SG.
This report describes the gross chemical composition of SG and
the susceptibility of carbohydrate and protein components to
enzymic hydrolysis.
EXPERIMENTAL
Samples
Dried Commercial Spent Grain (CSG). CSG from a malt-corn
mash was obtained from a brewery. The CSG, containing about 2%
trub and dried to about 7% moisture at about 45° C, was milled in a
Udy cyclone mill (Udy Analyzer Co., Boulder, CO). Hop extract
was used in wort preparation.
Wet Pilot Spent Grain (PSG). The wet SG from the pilot plant
mashing procedure described by Burkhart, Dickson and Hunt (6)
was frozen until use. PSG contained no trub.
Enzyme Activities
All activities were determined in the presence of excess substrate
and of enzyme concentrations with activity expressed as a linear
function of product yield. The procedures are outlined in Table I.
For carbohydrases a unit of activity was defined as the production
of 1 Mg glucose equivalent per hour as measured by reducing power
(12). For cellulase this unit of activity with filter paper substrate (8)
was the filter paper unit (FPU), and when the substrate was SG, the
unit was the spent grain unit (SOU). A unit of protease activity was
'Cooperative investigations, Barley and Malt Laboratory and College of
Agricultural and Life Sciences, University of Wisconsin, Madison 53705.
Mention of a trademark or proprietary product does not constitute a guarantee or
warranty of the product by the U.S. Department of Agriculture and does notimply its
approval to the exclusion of other products that also may be suitable.
The Barley and Malt Laboratory is supported in part by a grant from the Malting
Barley Improvement Association.
Presented at the 44th Annual Meeting, Toronto, May 1978.
'Research Chemist and Physical Science Aide, respectively.
This article is in the public domain and not copyrightable. It may be freely
reprinted with customary crediting of the source. The American Society of
Brewing Chemists, Inc., 1978.
196
the production of 1 mg of amino nitrogen per hour as measured by
Nesslerization (11).
Control of Microbial Growth
In all experiments at 50° C, microbial growth was not evident for
at least 6 hr. For longer reaction times microbial growth was
controlled either with 0.02% NaN 3 in the reaction mixtures or by
the use of autoclaved SG suspensions and enzyme solutions that
had been filtered through a sterile 0.54 membrane filter (Millipore
Corp., Bedford, MA 01730).
Enzyme Sources
Cellulase. The cellulase used was Onazuka SS from Trichoderma
viride (Yakult Biochemicals Co., Ltd., Tokyo) (14). Preliminary
work with several commercial cellulases showed that Onazuka SS
was the most active on SG under our experimental conditions.
Cellulase activity: 175 FPU/mg, 11 X 103 FPU/mg N; 300
SGU/mg, 19 X 103 SGU/mg N
Laminaranase activity: 180 U/mg, 11 X 103 U/mg N
Amyloglucosidase activity: 2.66 U/mg, 166 U/mg N
Xylanase activity: 400 U/mg, 25 X 103 U/mg N
Cellobiase activity: 272 U/mg, 1.7 X 103 U/mg N
Amyloglucosidase. The p r e p a r a t i o n used was
"Amyloglucosidase, Grade II" from the Sigma Chemical Co., St.
Louis, MO 63178.
Amyloglucosidase activity: 364 U/mg, 10.3 X 103 U/mg N
Laminaranase activity: 250 U/mg, 7.1 X 103 U/mg N
Xylanase activity: 290 U/mg N
0-Glucanase activity: 55 X 103 U/mg, 1.5 X 10" U/mg N
Cellulase activity: 0 FPU
Protease. The protease used was derived from Streptomyces
griseus (Type VI, Sigma Chemical Co., St. Louis). Other proteases
were less effective under our experimental conditions. (Activity: 7
U/mg, 82 U / m g N )
Chemical Analyses
Nitrogen, Crude Fiber, Ash, Lipids, Moisture and Lignin. SG
samples were analyzed for nitrogen, crude fiber, ash and moisture
by standard methods (1). Enzyme nitrogen was determined by
Johnson's method (11). Nonpolar lipids were extracted from the
SG with diethyl ether, and the SG residue was extracted with water-
saturated H-butanol. After removal of the water and n-butanol, the
residue was extracted with diethyl ether. This ether-soluble
material represented the polar lipids. Lignin was determined by the
standard method of the Association of Official Analytical Chemists
(2). Briefly, 5 g of SG was refluxed with 100 ml IN H2SO4
containing 2 g of cetyl trimethyl ammonium bromide for 1 hr. The
solid residue from this treatment is called "acid-detergent fiber."
Cellulosic materials in the dried acid-detergent fiber were extracted
by treatment at 20° C with 72% H2SO4 for 3 hr. The washed and
dried residue (crude lignin and ash) was weighed and ash was
determined by ignition. The loss in weight during ignition was
calculated as crude lignin.
Amino Acids. Samples were hydrolyzed for 22 hrat 1W°Cin6N
HC1 in tubes that had been flushed with nitrogen, evacuated and
sealed. Analyses were made with a Beckman Model 121 amino acid
analyzer (16).
Carbohydrates
Acid Hydrolyses. Starch, /3-glucans and pentosans were
hydrolyzed by refluxing 14 g of milled SG in 500 ml of IN H2SO4
for 4 hr. Cellulose was hydrolyzed by the method of Saeman,
No. 4 ASBC JOURNAL
Moore and Millett (18). Aliquotes of the hydrolysates were
neutralized with BaCOs and centrifuged. Supernatant solutions
were assayed for individual sugars by the method of Brobst, Scobell
and Steele (5), and starch plus /3-glucans was calculated from the
glucose obtained. This value was corrected for residual maltose in
the SG, which was removed by aqueous extraction of a separate
portion of the SG and assayed similarly.
Enzymic Hydrolyses. Glucose produced from starch by
amyloglucosidase was determined by the glucose oxidase-
peroxidase method (9). Sugars released from SG by cellulase and
amyloglucosidase were assayed directly (5).
Enzymic Treatments
Appropriate substrate and enzyme blanks were done with each
experiment and results were adjusted for these values.
Cellulase. SG (10 g) was suspended in 100 ml 0.01M acetate
buffer, pH 4.8,containing2X 10 ,4X 10 4 ,9X 10 4 ,18X 10 4 and36X
10" FPU of cellulase. Suspensions were agitated (150 rpm) at 50° C
for three, six and nine days. After incubation, enzyme activity was
destroyed by 5 min at 100° C, and sugars in the filtrate were assayed.
Amyloglucosidase. Duplicate 10-g portions (dry basis) of CSG
and one portion each of 3.6 X 104 U and 7.3 X 104 U of amylo-
glucosidase were agitated at 150 rpm at the optimum temperature
of 50° C (3) in 250 ml of 0.01M acetate buffer, pH 4.3, for
four days. The suspensions were heated for 5 min at 100° C and
filtered, and the sugars in the filtrate were determined. PSG was
treated similarly except that 5.5 X 104 U and 73 X 104 U of enzyme
were used.
Protease. SG(10g)and 175—700 U of enzyme were suspended in
350 ml of water and agitated in a rotary shaker at 50°C for three
days. The pH was maintained at 7.5 by periodically adding as much
as 4 ml 0.27V NaOH solution. The mixture was centrifuged at 25,000
X g, and the solid portion was dried overnight at 105° C, then milled
in a Udy cyclone mill. Solid and corresponding liquid fractions
were assayed for nitrogen at one, two and three days.
In some experiments, 100 g of SG was incubated with 350,1,050,
and 1,750 U of protease and corresponding amounts of water for
periods to 24 hr. Periodically 10 ml of supernatant was removed for
determination of dissolved nitrogenous constituents.
Dialysis of Protein Hydrolysates
Subsamples of each supernatant were dialyzed exhaustively
against distilled water with Spectraphor membrane tubing, 3,500
molecular weight cutoff (Spectrum Medical Industries, Inc., Los
Angeles 60916), and with Visking membrane tubing, 10,000
molecular weight cutoff (Union Carbide Co., New York 10017).
197
Both dialyzable and nondialyzable portions were analyzed for
nitrogen.
Carbon Treatment of Protein Hydrolysates
The 1,500 ml supernatant was treated with 15 g of Darco G-60.
Absorbance of the solution before and after treatment was
determined at 500 nm. Nitrogen content was determined before and
after treatment.
Experimental Replication
All experiments, including the amino acid analysis, were done in
duplicate or triplicate. Deviations of individual results within 3% of
the corresponding means were accepted.
RESULTS AND DISCUSSION
Chemical Analyses
The compositions of CSG and PSG were similar, as shown by
chemical analysis (Table II). The variations in lipid and starch plus
/J-glucan may reflect differences in mashing conditions. The PSG
was prepared from a 70% malt/30% corn mixture, but the ratio in
the CSG preparation is not known. A difference in these
proportions may affect the level of starch plus /J-glucan residue.
The high level of dietary fiber supports the potential for the use of
SG in bread formulation (17).
Enzymic Treatment of CSG
Carbohydrates. Treatment of CSG with 36 X 104 FPU of
cellulase for three to nine days produced the hydrolytic products
listed in Table III. The yield of glucose was 18% after six days'
incubation. Yields of xylose and arabinose were about 6% and 4%,
respectively, and represent about half the total pentosans. The
lower levels of enzyme were not effective, even after nine days'
incubation. After nine days' incubation at the highest level of
enzyme treatment, an addition of 9 X 104 FPU of cellulase did not
measurably increase hydrolysis.
Because no low-molecular-weight hydrolytic products of
cellulose other than glucose were present, the enzyme preparation
must have cellotriase, cellotetraase and other similar enzymes in
addition to the demonstrated 272 U/ mg of cellobiase. Similarly, the
pentosanases of the enzyme preparation were responsible for
complete hydrolysis of about one-half of the pentosans to xylose
and arabinose.
Considering the 12% starch plus /J-glucan and 9% cellulose
(Table II), the 18% yield of glucose (Table III) is probably derived
from the starch, /3-glucan, and about two-thirds of the cellulose.

TABLE I
Procedures for Enzyme Assays

Enzyme Assay" Substrate Buffer pH Time Volume of Reaction

(hr) Mixture (ml)

0-1,4-Glucan glucohydrolase 150 mg filter paper 0.1M acetate 4.8 4 2.2

(cellulase) 3.2.1.4 100 mg SG" 0.01M acetate 4.8 4 11.0
<*-l,4-Glucan glucohydrolase 12.5 mg soluble 0.01 M acetate 4.8 0.25 1.5
(amyloglucosidase) starch
/3-I,3(4)-Glucan glucanohydrolase 6.75 mg laminaran 0.0 \M acetate 4.7 4 2.0
(laminaranase) 3.2.1.6
/3-1,4-Xylan xylanohydrolase 6.75 mg xylan 0.0 \M acetate 4.7 4 2.0
(xylanase) 3.2.1.8
/3-D-Glucoside glucohydrolase 34 mg cellobiose 0.1 Af acetate 4.6 1 4.2
(cellobiase) 3.2.1.21
/3-1,4-Glucan glucanohydrolase 25 mg barley 0.02A/ acetate 5.0 1 2.5
(/3-glucanase, gumase) 3.2.1.4 /3-glucan
Peptidyl peptide 250 mg SO" O.IM phosphate 8.0 16 10.0
hydrolase (protease) 3.4.4
"Enzyme assay and nomenclature of the International Union of Biochemistry (10).
"SG = Spent grain.
198 HYDROLYSIS OF SPENT GRAIN
Chemical analysis of the residue (after digestion with cellulase)
revealed a cellulose residue that represented about 2% of the CSG.
This is further supported by the quantities of glucose released by
treatment of CSG with amyloglucosidase. Both levels of treatment
afforded a 13% yield of glucose, which is comparable to 12% yield
from the starch and /3-glucans by acid hydrolysis (Table II). Since
essentially all the glucose released by this amyloglucosidase derives
from starch and /3-glucans, the amyloglucosidase in the cellulase
preparation probably converted the starch completely to glucose
also. Pure amyloglucosidase does not hydrolyze amylopectin
completely unless traces of a-amylase are present. Unless highly
purified, fungal amyloglucosidase preparations contain a-amylase,
and the crude preparations convert starch completely to glucose
(22).
After the protein of the SG was removed by treatment with
protease and the solid residue was treated with cellulase, yields of
sugars from carbohydrase activity were essentially the same as
those obtained without protease treatment. Similarly, prior
extraction of lipid had no effect.
After removal of starch and /8-glucans by amyloglucosidase,
incubation of CSG with two successive 4.4 X 104 FPU of cellulase
produced from the cellulose a quantity of glucose representing 4%
of the original weight of the CSG (Table IV). Some of the pentosans
also were hydrolyzed, as indicated by release of 2.8% xylose and
TABLE II
Composition of Spent Grains"
Constituent PSG" CSG'
(% Dry Basis)
Vol. 36
1.2% arabinose based on the CSG weight. Thus, the total yield of
glucose from treatment of CSG first with amyloglucosidase (13%)
and then with cellulase (4%) approached the 18% yield from long
incubation with the cellulase preparation alone (Table III).
Removal of starch, gums and pentosans by refluxing in IN
HiSOt before treatment with cellulase produced results similar to
prior treatment with amyloglucosidase (Table IV). Again, 4.4% of
the original weight of the SG appeared as glucose, which represents
about one-half of the total cellulose.
Proteases. Table V shows the proteolysis of CSG during a three-
day period. The upper limit of hydrolysis (80—87%) was
approached after one day when 10 g of CSG and at least 350 U (50
mg) of enzyme were used. From an economic aspect, this indicates
that appreciable quantities of expensive enzyme and lengthy
incubations may be needed to attain near complete protein
hydrolysis. Some benefit seemed to be gained by larger scale
incubations. When 100 g of CSG was incubated with 1,750 U (250
mg) of protease, this level of hydrolysis could be attained in about 6
hr.
In an experiment in which 80% of the CSG protein was
solubilized (Table VI), the soluble protein fraction was not
significantly enriched in any of the essential amino acids relative to
the original CSG. About 80% of the amino acids, except cystine,
arginine and glutamic acid, appeared in the soluble fraction. Since
amide nitrogen appeared to be high in the solubilized protein as
indicated by the high level of ammonia from the soluble protein, the
glutamic and aspartic acid amides may have been proportionately
higher in the soluble than in the insoluble protein. In addition, some
amide nitrogen in the insoluble protein may have been hydrolyzed
by amidases in the protease preparation and thus appeared as
ammonia in the soluble fraction.

Lipid TABLE IV
Nonpolar 6.0 8.6 Hydrolysis of Starch-Depleted
Polar 0.7 1.0 Commercial Spent Grains with Cellulase"
Ash 4.3 4.3
Protein (N X 6.25) 36.0 31.0
13.0
Sugars (% of SGb)
Crude fiber 15.0
Nitrogen free extract 42.0 44.0 Method Glucose Xylose Arabinose
Cellulose 11.0 9.0
Starch and /3-glucansd 6.7 12.0 Amyloglucosidase
Pentosan e 21.0 19.0 1st application of cellulase' 3.0 2.8 1.2
Lignin 19.0 16.0 2nd application of cellulase 1.0 0.9 0.2
Dietary fiber' 46.0 43.0 Refluxed with IN H 2 SO 4 d 4.4 0.6
"Values are means of triplicate determinations. "10 g (dry wt) CSG was depleted of starch before cellulase
b
PSG = Pilot wet, unmilled spent grain. application.
C
CSG = Commercial dried, milled spent grain. h
Values are means of four replications.
d
Corrected for residual maltose. "Each application: 4.4 X 104 FPU, five days' incubation, 50°C.
c
Xylose:arabinose = 14:7 for PSG, 12:7 for CSG. d
Reflux time = 4 hr.
'Dietary fiber = 100 - (Lipid + ash + protein + starch and
/3-glucans).

TABLE V
Proteolysis of Commercial Spent Grain°'b
TABLE III
Hydrolysis of Spent Grains (10 g Dry Basis)
with Cellulase (36 X 104 FPU) Enzyme Incubation Protein Protein
Level Time Hydrolyzed Solubilized
(Days) (%) (g)
Incubation Sugars (% of SGb)
SG" (Days) Glucose Arabinose Xylose 175 1 69 1.4
2 78 1.2
CSGC 3 12 3.0 3.1 3 81 1.4
6 18 4.0 6.1
9 18 4.1 6.4 350 1 82 2.3
2 84 2.3
d
PSG 3 4.7 2.5 3.5 3 83 2.4
6 5.0 2.5 3.7
9 4.0 2.8 3.4 700 1 78 2.3
"SG = Spent grain. 2 82 2.4
3 87 2.5
^Values are means of duplicates.
'CSG = Commercial dried, milled spent grain. "10 g of commercial spent grain (3.1 g of protein) dry basis.
d b
PSG = Pilot wet, unmilled spent grain. Values are means of triplicates.
No. 4 ASBC JOURNAL 199
TABLE VI
Distribution of Commercial Spent Grain Protein Nitrogen after Proteolysis2

Individual Amino
Amino Acid N Acid N in
In Spent Grain In Solid Residue In Supernatant Supernatant
(g/100 g N) (20 g N) (80 g N) (%)
Lysine 4.0 1.0 4.3 81
Histidine 4.0 0.9 2.9 76
Ammonia 12.4 2.2 15.3 87
Arginine 8.9 1.5 4.2 74
Aspartic acid 4.1 0.9 3.3 79
Threonine 2.5 0.6 2.4 80

Serine 3.4 0.8 3.0 79

Glutamic acid 13.5 2.3 12.8 85
Proline 9.8 1.7 6.9 80
1/2 Cystine 1.1 0.2 0.6 75
Glycine 4.0 1.0 3.1 76
Alanine 5.3 1.2 5.2 81
Valine 3.8 0.9 3.2 78
Methionine 1.2 0.2 1.0 83
Isoleucine 2.6 0.6 2.3 79
Leucine 6.7 1.3 6.0 82
Tyrosine 1.6 0.3 1.3 81
Phenylalanine 3.0 0.7 2.5 78

Recovery, Kjeldahl N basis, ' 92 92 100

"100 g of spent grain (dry basis) hydrolyzed with 1,750 U of protease for 5 hr at 50° C; > of the Kjeldahl N solubilized. Values are
means of duplicates.

Dialysis of the solubilized protein hydrolytic products through
casings with 10,000 and 3,500 molecular weight limits showed that
molecular weights were less than 10,000 for 92% of the products
and less than 3,500 for 82%. Decolorization with carbon before
dialysis removed 7% of the nitrogenous constituents but did not
alter the molecular weight distribution. Absorbance at 500 nm was
decreased from 0.62 to 0.25 (pale yellow).
Enzymic Treatment of PSG
Treatment of PSG with 36 X 10" FPU of cellulase for three to
nine days produced the hydrolytic products shown in Table III.
Comparison of Tables II and III indicates that PSG carbohydrates,
particularly the pentosans, are less susceptible than those in CSG to
hydrolysis by the cellulase preparation. Although total glucose
expected from the starch, /J-glucans and cellulose in PSG was about
18% (Table II), a maximum of 5% was obtained (Table III). The
expected yield of pentosans was 21% (Table II), but only 6% was
obtained (Table III). By contrast, the glucose expected from CSG
was 21% (Table II), and about 18% was obtained (Table III).
Similarly, 19% pentosans were expected from CSG (Table II) and
about 10% was obtained after enzyme hydrolysis (Table III). When
PSG was dried at 45° C and milled, its susceptibility to
carbohydrase was similar to that of CSG.
The starch and /3-glucan in PSG were somewhat less susceptible
to hydrolysis by amyloglucosidase than that in CSG. CSG and PSG
have 12% and 6.7% (dry basis) starch plus /3-glucan, respectively
(Table II). While all of this in CSG appeared to be hydrolyzed by
amyloglucosidase, application of 5.5 X 104 U and 73 X 104 U of
amyloglucosidase to PSG afforded 4 and 5.1%, respectively, as
glucase from starch plus /3-glucan.
The starchy endosperm cells in the distal tips of the barley kernel
are not exposed to hydrolytic enzyme action until late in
germination (4) and are likely to be poorly modified. Furthermore,
these distal tips are left largely intact by the relatively coarse milling
of malt for mashing. The fine milling of the CSG probably
permitted a more efficient substrate-enzyme interaction for
carbohydrate hydrolysis than was permitted by the unmilled PSG.
Protease hydrolyzed the protein of the PSG, despite the large
particle size, as readily as the protein of CSG.
The increasing world population and threat of food shortages
have stimulated efforts to produce edible proteins (15). Mashing is
designed to optimize carbohydrate hydrolysis, but the hydrolysis of
the barley reserve proteins occurs at a much slower rate (13). As a
result, these reserve proteins, especially the prolamine hordein, are
the main components of the SG. Of the barley protein groups
(albumin, globulin, prolamine and glutelin), on a percent of protein
basis, the prolamine fraction contains less of the essential amino
acids lysine, arginine, threonine, valine, methionine, leucine and
phenylalanine than each of the other three protein groups (7).
Nevertheless, because of the high protein level in SG, the essential
amino acids compare favorably with some foods such as whole
wheat flour (17), on a sample weight basis. The bland solution of
enzymically hydrolyzed carbohydrate and protein from a by-
product such as SG may provide potential uses for these nutrients.
Imaginative and efficient nutritional uses for by-products of
brewing should provide an answer to claims that the use of grain in
brewing deprives the world of needed food (19).
Acknowledgment
We wish to thank George Robbins and James Gilbertson for determining
amino acids.
Literature Cited
1. AMERICAN ASSOCIATION OF CEREAL CHEMISTS. Approved
methods of the AACC(7thed.).Methods46-12,32-15,08-01 and 44-
15; all approved April 1961. The Association: St. Paul, MN (1962).
2. ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS.
Official Methods of Analysis (12th ed.). Methods 7.057 and 7.058.
The Association: Washington, DC (1975).
3. BARKER, S. A., and FLEETWOOD, J. G. J. Chem. Soc., London, p.
4857 (1957).
4. BRIGGS, D. E. Planta. 108: 351 (1972).
5. BROBST, K. M., SCOBELL, H. D., and STEELE, E. M. Amer. Soc.
Brew. Chem., Proc. 1973, p. 43.
6. BURKHART, B. A., DICKSON, A. D., and HUNT, W. N. Amer. Soc.
Brew. Chem., Proc. 1953, p. 34.
7. FOLKES, B. F., and YEMM, E. W. Biochem. J. 62: 4 (1955).
8. GRIFFIN, H. L. Anal. Biochem. 56: 621 (1973).
200 HYDROLYSIS OF SPENT GRAIN
9. HAUGAWA, S., and SMOLENSKY, D. C. J. Agric. Food Chem. 18:
902(1970).
10. INTERNATIONAL UNION OF BIOCHEMISTRY. Enzyme
nomenclature. Elsevier Publishing Co.: New York (1965).
11. JOHNSON, M. J. J. Biol. Chem. 137: 575 (1941).
12. LUCHSINGER, W. W., and CORESKY, R. A. Anal. Biochem. 4: 346
(1962).
13. MIKOLA, J., PIETILA, K., and ENARI, T-M. J. Inst. Brew. 78: 388
(1972).
14. MOORE, W. E., EFFLAND, M. J., and MILLETT, M. A. /. Agric.
food Chem. 20: 1173(1972).
15. OKE, O. L. Tropical Sci. 15: 139 (1973).
16. POMERANZ, Y., and ROBBINS, G. S. Cereal Chem. 49: 560 (1972).
17. PRENTICE, N., and D'APPOLONIA, B. L. Cereal Chem. 54: 1084
Vol. 36
(1977).
18. SAEMAN, J. F., MOORE, W. E., and MILLETT, M. A. Methods in
carbohydrate chemistry. Vol. Ill, ch. 12. Academic Press: New York
(1963).
19. SEGEL, E. Brew. Dig. 51(3): 26 (1976).
20. TOWNSLEY, P. M. Tech. Quart. Master Brew. Assn. Amer. 11: 262
(1974).
21. TROWELL, H., and BURKETT, D. Refined carbohydrate foods and
disease. Some implications of dietary fiber. Ch. 21. Academic Press:
New York (1975).
22. WHELAN, W. J. Biochem. J. 122: 609 (1971).
[Received March 15, 1978]