- Email: [email protected]
Proteolysis of Alcohol-Treated Soybean Meal Proteins by Bacteroides ruminicola, Bacteroides amylophilus, Pepsin, Trypsin, and in the Rumen of Steers
Proteolysis o f A l c o h o l - T r e a t e d S o y b e a n M e a l Proteins b y
Bacteroides amylophilus,
Bacteroides ruminicola,
Pepsin, T r y p s i n , and in t h e R u m e n of Steers G. L. LYNCH, P. J. van der AAR, 1 L. L. BERGER, G. C. FAHEY, JR., and N. R. MERCHEN Department of Animal Sciences
University of Illinois Urbana 61801 ABSTRACT
untreated soybean meal or soybean meal treated with 70% ethanol at 80°C indicated that no detectable soluble glycinin or conglycinin escaped ruminal degradation.
Sodium dodecyl sulfate-gel electrophoresis and cation exchange chromatography were used to examine degradation of treated and untreated soybean meal protein fractions by Bacteroides amylopbilus H181, Bacteroides ruminicola Bx4, pepsin, trypsin, and intraruminally. Soybean meal treatments consisted of 30% vol/vol isopropanol, 40% propanol, or 50% ethanol at 22°C or 70% ethanol at 80°C. Water-soluble protein fractions were applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient of .03 to .27 and then .27 to 1.0 M. The four protein fractions with the highest absorbance at 276 nm were dialyzed against distilled water prior to being subjected to enzymatic hydrolysis. Soybean meal treated with 40% propanol had the greatest reduction in absorbance of all effluents at 275 nm, followed by soybean meal treated with 50% ethanol or 30% isopropanol. Comparison of electrophoretie patterns over time showed that B. amylopbilus H181, degraded protein subunits more rapidly than B. ruminicola B14. Protein subunits with the highest molecular weights were the most rapidly degraded by B. arnylopbilus H181, B. ruminicola B14, pepsin, and trypsin. Hydroxylapatite chromatography of omasal fluid from steers supplemented with
INTRODUCTION
Soybean meal (SBM) is a common protein source in ruminant diets and is rapidly degraded by ruminal microorganisms. Ruminal digestion of protein is less efficient than postruminal digestion (4) because some of the degraded protein N is lost from the rumen as ammonia. Ammonia is converted into urea in the liver and much of it is subsequently excreted in the urine. Various treatments have been examined for their ability to improve resistance of SBM protein to ruminal microbial proteases. Formaldehyde (19) and heat (9) treatments reduced degradation of SBM protein by ruminal microbes. However, excessive treatment resulted in depressed postruminal availability as measured by chick growth bioassay (20, 22). Soybean meal treatments involving alcohols, with and without addition of heat, depressed ruminal protein degradation (13, 24) but did not affect the nutritive value for chicks (12, 25). Development of a commercial process to extract soy flakes with aqueous isopropanol (1) generated interest in the effect of these solutions on susceptibility of soy protein to enzymatic degradation. In vitro fermentation of different feed proteins with Bacteroides amylopbilus indicated wide variation in the degradation rates of different soluble proteins, and the relationship between degradation rate of soluble and insoluble proteins is inconsistent (14). Reduced N disappearance in situ was observed Received July 1, 1987. when SBM was treated with aqueous alcohols Accepted March 14, 1988. (5, 12, 24); however, hydrolysis of alcohol1CLO -- Instituut Voor de Veevoeding "De Scho- treated SBM protein by Aspergillis sojae octhorst", Meerkoeten Weg 26, 8218 NA Lelystad, curred faster than that of untreated SBM (8). The Netherlands. 1988 J Dairy Sci 71:2416-2427
2416
PROTEOLYSIS OF ALCOHOL-TREATED SOYBEAN MEAL PROTEINS Effect of alcohol treatments on proteolysis of SBM proteins by ruminal microbial and mammalian proteases and on in vivo degradation of specific SBM proteins has not been reported before. The purpose of this study was to determine effects o f various alcohol treatments on different SBM protein fractions and to measure proteolysis of these fractions by microbial proteases, pepsin, trypsin, and within the rumen. MATERIALS AND METHODS Soybean Meal Treatment
Hexane-extracted, dehulled SBM was treated with various aqueous alcohol solutions at two temperatures. Treatments with 30% vol/vol isopropanol, 40% vol/vol propanol, or 50% vol/vol ethanol were conducted at 22°C. These concentrations were chosen because earlier work (24) indicated that they resulted in the greatest depression in ruminal degradation. One hundred gram SBM were soaked in 250 ml of alcohol solution for 30 min. After soaking, SBM was filtered through Whatman No. 41 filter paper and air-dried. The heat processed treatment involved adding 22.7 kg SBM to 76.8 L of 70% vol/vol ethanol heated to 80°C. The mixture was stirred for 30 min, after which the liquid was drained and the SBM dried at 77°C for 12 h. Soluble proteins were isolated from untreated SBM treated at 22°C with 30% isopropanol, 40% propanol, or 50% ethanol from SBM treated with 70% ethanol at 80°C, or from omasal fluid from steers fed untreated SBM, SBM treated with 70% ethanol at 80°C, or ureacasein. Forty-gram SBM samples were suspended in 200 ml distilled water and agitated for 1 h in a 40°C water bath prior to centrifugation for 20 min. The supernatant was exhaustively dialyzed against .03 M potassium phosphate buffer containing .1% 0~-mercaptoethanol, followed by gel filtration over a Sephadex G-25 column (Pharmacia Fine Chemicals, Inc. Milwaukee, WI). An amount of extract containing 200 mg protein (10) was applied to a hydroxylapatite column (2 × 30 cm). The column was eluted with a discontinuous phosphate gradient of .03 to .27 M then .27 to 1.0 M (6) using an Ultragrad (LKB-Produkter AB, S-16125 Bromma, Swed.) gradient mixer. Proteins eluting from the column were detected
2417
by monitoring column effluent at 276 nm with a Unicord S monitor (LKB-Produkter AB). The four largest fractions from untreated SBM and SBM treated with 30% isopropanol, 40% propanol, and 50% ethanol at 22°C were extensively dialyzed against distilled water, frozen, and stored. Soluble proteins in omasal fluid from steers fed untreated SBM, 70% ethanol-treated SBM at 80°C, or urea-casein were prepared by compositing samples b y treatment, followed by centrifugation at 35,000 × g, with the supernatant dialyzed against .03 M phosphate buffer containing .1% 0~-mercaptoethanol. Proteins were separated as above by applying 24 mg protein to the hydroxylapatite column. Omasal fluid was collected as described previously (13). Amino Acid Analysis
Fractions isolated from untreated SBM were hydrolyzed with 6N He1 for 22 h at 110°C prior to analysis for amino acid composition with a Beckman 119CL amino acid analyzer (Beckman Instruments, Inc., Palo Alto, CA). Release of ~-amino groups was determined as described by (17). Digestion rates of various protein fractions were expressed as micrograms glycine equivalents released per milligram enzyme protein - 1 • minute - 1 • In Vitro Bacterial Digestion
The bacteria, Bacteroides amylophilus strain H181 and Bacteroides ruminocola strain B14, were grown and maintained on media described by Schaefer et al. (21). Eighteen hours after transfer to fresh media, bacteria were centrifuged at 8000 × g. The pellet containing the bacteria was suspended in 1 ml .05 M phosphate buffer (pH 7.6). Bacterial cells were disrupted using a French press cell (20,000 psi) and suspended in 25 ml .05 M phosphate buffer (pH 7.6). Three milliliters of water-soluble protein extract were incubated with 1 ml bacterial suspension. After 1, 3.5, and 6 h of digestion, 1-ml samples were immediately analyzed for free (~-amino groups. Rates of protein hydrolysis for the protein fractions were determined b y linear regression. In Vitro Trypsin and Pepsin Digestion
Three milliliters of water-soluble protein extract from untreated SBM and 30% isoJournal of Dairy Science Vol. 71, No. 9, 1988
2418
LYNCH ET AL.
propanot, 40% propanol, and 50% ethanoltreated SBM were incubated with .10 mg trypsin in 1 ml .05 M phosphate buffer (pH 7.6) at 25°C. After 1, 4, and 7 min, 1-ml samples were taken and digestion was stopped by adding .1 ml of 1 N HC1. Prior to analysis for free a-amino groups, .1 ml 1 N NaOH was added to the tubes. Rates o f protein hydrolysis were determined by linear regression. To evaluate susceptibility to pepsin, 3 ml of water-soluble protein extract fractions were incubated with .4 mg pepsin in 1 m l . 1 N HC1 at 30°C. After 1, 9, and 17 min, 1-ml samples were taken, neutralized with .2 N NaC1 and analyzed for free a-amino groups. Rates of protein hydrolysis were determined by linear regression. Electrophoresis
Composition of protein fractions isolated from untreated SBM extract was examined using SDS P A G E (26). Also, effects of hydrolysis by various proteases on electrophoretic patterns of SBM protein over time were studied. Hydrolysis conditions were similar to those described previously. The molecular weight of subunits was determined with a standard protein solution consisting of lysozyme (Mr 14,300), trypsinogen (M r 24,000), ovalbumin (M r 45,000), bovine plasma albumin (M r 66,000), and phosphorylase a (M r 92,500). Pepsin and trypsin digestions were stopped after 0, 1, 2, 4, 10, 30, and 60 min. Bacterial digestions were stopped after 0, 4, 8, 12, and 24 h of digestion. Ruminal Protein Degradation
Three mature Hereford steers (average 508 kg) and three mature Simmental steers (average 594 kg) fitted with ruminal cannulae were used to measure soluble SBM protein outflow from the rumen. Treatments consisted of three N supplements: untreated SBM, SBM treated with 70% ethanol at 80°C, and a urea-caseincorn mix. Nitrogen supplements were added to a basal diet (Table 1) to achieve a 14.4% CP final diet, with approximately 70% of the dietary CP provided by N supplements. Steers were fed daily at 0800 and 2000 h and were tethered in solid floor stalls in a temperaturecontrolled (18.3°C) room under continuous lighting. Journal of Dairy Science Vol. 71, No. 9, 1988
Each experimental period was 14 d in length. Days 1 through 10 were for diet adaptation with d 11 through 14 used for collection of omasal fluid samples. Cobalt EDTA (23) was continuously infused into the rumen to serve as omasal fluid flow marker. A p p r o x i m a t e l y 75 ml of omasal fluid were collected five times daily; collection times were randomized so that omasal samples were collected every 70 min of a 24-h period. Omasal samples were collected by inserting a polyethylene tube (.95 cm i. d., 1.59 cm o. d.) into the omasum through the reticulo-omasal orifice. A plastic strainer was attached to the omasal end of the tube and held approximately 12 cm into the omasum by 2 x 10 cm rubber flanges positioned on each side of the reticulo-omasal orifice. Fluid samples were collected by attaching the opposite end of the tube to a filter flask and applying negative pressure with a vacuum pump. Omasal samples were composited, centrifuged at 14,000 x g, and frozen at - 2 0 ° C prior to protein isolation. Statistical Analysis
Data on rate of digestion were analyzed b y analysis of variance. When significant differences were observed, treatment means were compared by the F test-protected, least significant difference method (3).
RESULTS
Elution patterns resulting from separation of water-extractable proteins on the hydroxylapatite column from untreated SBM or from SBM treated with 30% isopropanol, 40% propanol, and 50% ethanol are presented in Figures 1, 2, and 3. Four major and two minor peaks were observed. The first fraction eluted with .03 M phosphate buffer, the second between .18 and .23 M phosphate, the third with .27 M phosphate, and the fourth between .4 and .7 M phosphate. Two minor peaks were observed between .08 and .12 M and .23 and .25 M phosphate. The relative absorbance at 276 nm of soluble protein fractions from SBM treated with 30% isopropanol is superimposed on the pattern found for untreated SBM in Figure 1. The isopropanol-treated sample contained more proteins that eluted at .03 M and between .18 and .23 M phosphate and less of the proteins
PROTEOLYSIS OF ALCOHOL-TREATEDSOYBEAN MEAL PROTEINS TABLE 1. Ingredient and chemical composition of basal diet (dry basis). Item
(%)
Ingredient composition Corncobs, ground Cornstarch grits Prairie hay, ground Dextrose Casein Urea Dicalcium phosphate Limestone Vitamin mix ~
45.0 30.14 12.5 11.0 .5 .4 .2 .16 .1
Chemical composition Dry matter Crude protein Acid detergent fiber
79.7
4.31 26.4
Contains 3,300,000 IU vitamin A, 330,000 IU vitamin D, and 22,000 IU vitamin E/kg.
1
2
2419
that eluted in fractions 3 and 4. The same observations were made for the samples from SBM treated with 40% propanol (Figure 2) and 50% ethanol (Figure 3). Reduction in peak 3 and 4 proteins was largest for the 40% propanol treatment. Fifty percent ethanol treatment resulted in a reduction in size of these peaks intermediate between isopropanol and propanol. Four fractions of the water-extractable proteins from untreated SBM were obtained as shown in Figure 1. The amino acid composition of these fractions is presented in Table 2. Fraction 1 contained high concentrations of threonine, alanine, and glycine and low concentrations of leucine relative to the crude SBM protein fraction applied to the column. Fraction 2 contained relatively lower concentrations of serine, leucine, tyrosine, and phenylalanine. Fraction 3 was the only one in which mea-
3
4
qD z
:E z
0 "o -I-
(D ',N
.,4
m
U.I
0
r-
..... i 100
300
EFFLUENT VOLUME
500
127o3
-I -¢
700
(ML)
Figure 1. Absorbance (276 nm) pattern of soybean meal proteins ( ) applied to an hydroxylapatite column and eluted with a discontinuous phosphate buffer (. . . . ) in comparison to the elution pattern for soybean meal treated with 30% isopropanol ( ). Journal of Dairy Science Vol. 71, No. 9, 1988
2420
LYNCH ET AL. 1
2
3
4
Z O
:E Z
i/:,,)l/},J / ,,,,'://
gO UJ
j
m O r-
-4 .<
jj~ jl ....
~'
.03
100
300
5~
EFFLUENT
VOLUME
700
(ML)
Figure 2. Absorbance (276 nm) pattern of soybean meal proteins ( - - ) applied to an hydroxylapatite column and eluted with a discontinuous phosphate buffer (. . . . ) in comparison to the elution pattern for soybean meal treated with 40% (vol/vol) propanol ( --).
1
2
3
4
l\
\ :E Z
III I
\
I
I
I
I
1.00
"u
O
A
gO N
I
I
/
UJ
/
/
"u -I-
/
-4 m O
/ ._/ 11
.27
Iij 100
-4
J
.03
300 EFFLUENT
500 VOLUME
700
(ML)
Figure 3. Absorbance (276 nm) pattern of soybean meal proteins ( ) applied to an hydroxylapatite column and eluted with a discontinuous phosphate buffer (. . . . ) in comparison to the elution pattern for soybean meal treated with 50% (vol/vol) ethanol ( --). Journal of Dairy Science Vol. 71, No. 9, 1988
PROTEOLYSIS OF A L C O H O L - T R E A T E D SOYBEAN MEAL PROTEINS
2421
TABLE 2. A m i n o acid composition o f protein fractions isolated from u n t r e a t e d soybean meal ( n m o l / m g total protein). Amino acid
Crude fraction ]
1
2
Fraction 3
4
Aspartic acid Threonine Serine Glutamic acid Proline Glycine AI anine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine Histidine Lysine Arginine
1526 422 729 2743 695 823 644 296 83 269 748 246 391 161 567 597
1763 910 1073 1486 538 2175 1480 325 ND 2 247 242 288 256 193 296 433
1178 247 314 2457 707 608 302 123 ND 112 154 37 94 183 509 491
1334 389 675 204.5
1463 284 820 8683
678
131
811 566 344 46 238 692 205 343 148 408 531
814 334 301 ND 300 816 203 497 119 538 600
I Water-soluble proteins extracted from soybean meal with water at 40°C. 2 ND = Not detectable.
ii 1
2
| 3
Figure 4. The SDS-PAGE patterns of the various separated protein fractions: 1) Fraction 1, 2) Fraction 2, 3) Fraction 3, and 4) Fraction 4.
0
4
8
12
24
Figure 5. The SDS-PAGE patterns of soybean meal protein after 0, 4, 8, 12, and 24 h digestion with Bacteroides amylopbilus H18 extract at 25°C. Journal o f Dairy Science Vol. 71, No. 9, 1988
2422
LYNCH ET AL.
A
ill
I
i
II
I
._ . _ , ,
t
0
I
4
8
12
24
Figure 6. The SDS-PAGE patterns of soybean meal protein after 0, 4, 8, 12, and 24 h digestion with
Bacteroides rurninicota B, 4 extract at 25°C.
surable amounts o f methionine were detected. Fraction 4 was extremely rich in glutamic acid but low in proline. Band patterns, as determined b y SDS-PAGE, of the pooled treatments are shown in Figure 4. Fraction 1 did not contain determinable proteins. Electrophoresis of fraction 2 resulted in a wide, vague band with no distinguishable protein bands. This was probably caused b y the presence of one or more glycoproteins. The SDS-PAGE pattern of fraction 3 showed several sharp bands. These bands all had molecular weights less than 35,000. Fraction 4 contained four major protein subunits and various minor ones with molecular weights ranging from 35,000 to 75,000 daltons.
0
I
2
4
10
30
60
Figure 7. The SDS-PAGE patterns of soybean meal protein after 0, 1, 2, 4, 10, 30, and 60 min trypsin digestion at 25°C. Journal of Dairy Science Vol. 71, No. 9, 1988
Exposure of water-extractable SBM proteins to B. amytopbilus H18 t resulted in a gradual degradation of protein fraction subunits represented in bands A, B and C after 24 h digestion (Figure 5). A slight accumulation of lower molecular weight proteins could also be noted (band E). Digestion of water-extractable proteins by B. ruminicola B14 resulted in SDS-PAGE patterns shown in Figure 6. Disappearance of bands was slower than noted for B. amylopbilus. None o f the bands had completely disappeared after 24 h of digestion. However, a reduction of bands representing the highest molecular weight subunits (band A) was again noted. Hydrolysis with trypsin resulted in rapid digestion of the higher molecular weight protein subunits (Figure 7). Intensity of bands A and B, representing proteins with molecular weights of 78,000 and 72,000 daltons, respectively, began to diminish 1 min after adding the trypsin. After 10 min, band C began to lose intensity. None of the other protein bands appeared to be affected by this enzyme during these short incubation times. The SDS-PAGE patterns during pepsin digestion are shown in Figure 8. In this assay, none of the bands completely disappeared. Bands representing the two highest molecular weight subunits (bands A and B) had lost intensity after 60 min of pepsin digestion. No significant interactions between protein fraction rate of hydrolysis and alcohol treatment were detected. Changes in affinities of
0
I
2
4
10
30
60
Figure 8. The SDS-PAGE patterns of soybean meal protein after 0, 1, 2, 4, 10, 30, and 60 min pepsin digestion at 25°C.
PROTEOLYSIS OF ALCOHOL-TREATED SOYBEAN MEAL PROTEINS
2423
TABLE 3. Effects of various alcohol treatments on rate of hydrolysis of soybean meal protein by various proteases. Alcohol treatment Protease
Untreated
30% (vol/vol) isopropanol
40% (vol/vol) propanol
50% (vol/vol) ethanol
B. ruminicola Bt 4
.0081 (.0018) 2
.0065 (.0018)
.0063 (.0023)
.0058 (.0018)
B, amylopbitus H 18
.0182 (.0016)
.0213 (.0020)
.0245 (.0020)
.0225 (.0020)
Pepsin a
1.98 (.39)
2.30 (.48)
2.31 (.62)
2.58 (.57)
Trypsin 3
9.76 (3.10)
16.49 (2.92)
11.86 (4.82)
15.13 (4.25)
1 Data are expressed as grams glycine equivalents released --i .milligrams enzyme protein -1 .minute-1. 2 Standard error of the mean values. aObtained from Sigma Co., St. Louis, MO.
p r o t e a s e s f o r specific p r o t e i n f r a c t i o n s were similar a m o n g t r e a t m e n t s . T a b l e 3 p r e s e n t s results o f various a l c o h o l t r e a t m e n t s o n rate o f h y d r o l y s i s of SBM p r o t e i n b y proteases. A l c o h o l t r e a t m e n t s did n o t a f f e c t SBM p r o t e i n availability as m e a s u r e d b y s u s c e p t i b i l i t y o f SBM p r o t e i n to d e g r a d a t i o n b y proteases. H o w e v e r , p r o t e o l y s i s b y B. amylophitus e x t r a c t was faster t h a n t h a t b y B. ruminicola e x t r a c t . R a t e s o f h y d r o l y s i s b y t h e various e n z y m e s
were n o t statistically c o m p a r a b l e because t h e y were d e t e r m i n e d u n d e r d i f f e r e n t c o n d i t i o n s . In T a b l e 4, rates of p r o t e o l y s i s of various p r o t e i n f r a c t i o n s are p r e s e n t e d . F r a c t i o n s 2 a n d 3 h a d m o r e ( P < . 0 5 ) s - a m i n o g r o u p s released per u n i t o f t i m e b y B. ruminicoIa t h a n did t h e c r u d e fraction. F r a c t i o n 3 was h y d r o l y z e d f a s t e r ( P < . 0 5 ) b y B. amytopbilus t h a n e i t h e r f r a c t i o n 1 or t h e c r u d e f r a c t i o n . F r a c t i o n s 3 a n d 4 were h y d r o l y z e d faster ( P < . 0 1 ) b y p e p s i n
TABLE 4. Rate of hydrolysis of soybean meal protein fractions by various proteases)
Protease
Crude fraction
1
Fraction 3
2
4
B. ruminicola B~ 4
.0016 a (.0020) 3
ND 2
.0134 b (.0020)
.0159 b (.002)
.0033 ab (.0020)
B. amylopbilus H 18 Pepsin
.0184 ab (.0020)
.0147 a (.0022)
.0231 bc (.0020)
.0278 c (.0020)
.0241 bc (.0020)
1.46 a (.57)
ND 2
1.40 a (.44)
2.90 b (.53)
4.443b (.47)
4.91 a (4,05)
ND a
21.72 b (4.50)
8.51 a (3.37)
18.10 b (2.95)
Trypsin
a'b'CValues in the same row with different superscripts differ (P<.05). 1Data are expressed as grams glycine equivalents released -1 ,milligrams enzyme protein -1 .minute -1 . 2 Variation in assay results made values obtained unreliable and were therefore considered as nondeterminable (ND). Standard error of the means. Journal of Dairy Science Vol. 71, No. 9, 1988
2424
LYNCH ET AL.
= 1.00 ~O =r
8 E
i
c ¢o UJ
/i/I/i/
O m
/// .27
.03 | 100
300
500
Effluent volume (ml)
Figure 9. Absorbance (276 nm) pattern of untreated soybean meal soluble proteins ( - - ) and soluble protein from soybean meal treated with 70% ethanol at 80°C (. . . . . ) applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient (. . . . ).
than was fraction 2 or the crude fraction. The crude fraction applied to the hydroxylapatite column and fraction 3 were hydrolyzed more slowly (P<.05) by trypsin than were fractions 2 and 4. Elution pattern of soluble proteins from untreated SBM and SBM treated with 70% ethanol at 80°C was similar to that of the other SBMs (Figure 9). Elution patterns of soluble proteins collected at the omasum of steers fed untreated SBM, SBM treated with 70% ethanol at 80°C, or urea-casein were all similar (Figure 10). All three N supplements resulted in increases in peak 1 with no detectable traces of peaks 3 or 4. Separation of samples applied to the hydroxylapatite column by SDS-PAGE produced no determinable proteins (data not shown). DISCUSSl ON
Soluble proteins extracted from untreated SBM had an absorbance pattern at 276 nm similar to that reported previously (6, 28). The fraction eluting at .03 M phosphate (peak 1) is composed predominantly of trypsin inhibitors Journal of Dairy Science Vol. 71, No. 9, 1988
and metabolic proteins (18). Peak 2, eluting at .03 to .27 M phosphate, is possibly composed of a mixture of storage and metabolic proteins (18). The fraction eluting from hydroxylapatite at .27 M phosphate (peak 3) was identified as the major soybean storage protein, glycinin (6). Fisher et al. (6) identified the fraction eluting between .4 and .7 M phosphate (peak 4) as conglycinin but did not report a major peak eluting from the column when the phosphate buffer concentration was between .18 and .23 M as was observed in our work. The greatest denaturation of glycinin and conglycinin was observed for the 40% propanol treatment (Figure 2). Previous reports (7) showed that treatment with 40% propanol resulted in more protein denaturation than did treatment with 50% ethanol. The 40% propanol treatment was more effective in denaturing conglycinin than was propanol at any other concentration (26). Comparison of SDS-PAGE patterns of SBM proteins following exposure to bacterial protease showed that hydrolysis of protein subunits occurred faster when B. amylopbilus H18
PROTEOLYSIS OF ALCOHOL-TREATEDSOYBEAN MEAL PROTEINS
2425
"o ~r o "0 ~r ¢¢
E e4
i
LO0
UJ
i¢ o
i ffj
.27
~e
,03 100
300
500
Effluent volume (ml)
Figure 10. Absorbance (276 nm) pattern of omasal fluid from steers fed untreated soybean meal ( ), ), or urea-casein-corn ( ) applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient (. . . . ).
soybean meal treated with 70% ethanol at 80°C (. . . . .
extract was used than when B. ruminicola Bt 4 was used. Distinct disappearance of these proteins was noticeable after 4 h with B. arnylopbilus (Figure 5), whereas bands were still visible after 24 h when SBM protein was incubated with B. rurninicola extract. Enzymatic hydrolysis of SBM protein, as examined by SDS-PAGE, showed that the highest molecular weight subunits (71,000 and 78,000 dattons) were always the most rapidly digested. Comparison of pattern of pepsin and trypsin digestion over time (Figures 7 and 8) showed that proteolysis by trypsin was faster than that by pepsin and was probably due to the difference in specific activity of the enzymes used. At 25°C, glycinin was hydrolyzed more rapidly by pepsin than by trypsin (11). We were not able to confirm this finding. However, in our experiment, as well as in the previous work (11), protein subunits with
the highest molecular weights were the most rapidly digested. Rate of in vitro 0~-amino group release and the change in SDS-PAGE patterns show conformity. In both assays, the most rapid rate of digestion was found for trypsin. Comparison of the two bacterial proteases showed that B. amylopbilus extract had a greater specific activity than did B. ruminicola extract. Based on changes in SDS-PAGE patterns, we expected that fraction 4 (conglycinin) would be the most rapidly digested of all fractions tested. This deviation from expected results is possibly due to variability in the in vitro protease assays. Therefore, we think the results of the SDSPAGE assays are the most reliable of the two data sets. Rate of in vitro ammonia release in ruminal fluid has been directly related to the composition of SBM proteins (26). It has been Journal of Dairy Science Vol. 71, No. 9, 1988
2426
LYNCH ET AL.
observed that fractions relatively rich in conglycinin and acidic subunits of glycinin released the greatest amounts of ammonia, van der Aar et al. (26) hypothesized that conglycinin was more susceptible to degradation by ruminal bacterial proteases than the other proteins. The preferential hydrolysis of conglycinin by B. amylopbilus (Figure 5) and to a lesser extent by B. rurninicola (Figure 6) support this hypothesis. Lack of differences in hydroxylapatite elution patterns of omasal soluble proteins from steers fed untreated SBM, SBM treated with 70% ethanol at 80°C, and urea-casein-corn indicate that the protein material is of predominantly bacterial origin. It was hypothesized that some of the soluble glycinin and conglycinin, once denatured by alcohol treatment, would escape ruminal degradation and flow out of the rumen. Because only 5 to 10% of protein from alcohol-treated SBM was soluble (13, 27), diets were formulated to allow N supplements to provide 70% of dietary CP. We thought this would provide measurable soluble proteins if alcohol treatment resulted in soluble proteins resistant to ruminal degradation. Because over 80% of SBM N is waterextractable at pH 6.5 (28), the potential exists for significant amounts of soybean proteins to be solubilized in the rumen. Mahaevan et al. (14) reported that in vitro degradation rates of soluble and insoluble SBM proteins were identical. Because previous work showed increased ruminal outflow of SBM N when SBM was treated with ethanol and heat (13, 27), it was hypothesized that soluble proteins from SBM treated with 70% ethanol at 80°C may also escape the rumen. If the proteins escaping the rumen could be captured and identified, this information could be useful in determining what qualities would be advantageous for the ruminal escape of proteins. The soluble proteins from steers fed urea-casein-corn are thought to be of bacterial origin, since ruminal half-life of casein is less than 22 min (15) and very small amounts of protein from the corn would be soluble. Conglycinin is both rapidly hydrolyzed by proteases and easily denatured by aqueous alcohol solutions (16). The molecular threedimensional structure is responsible for these properties. Conglycinin is glycosylated (2) and thus has hydrophilic characteristics. These Journal of Dairy Science Vol. 71, No. 9, 1988
hydrophilic properties cause the molecule to be less compact when solubilized in water (7). This loose structure promotes denaturation by aqueous alcohol solutions and enzymatic hydrolysis. Although changes due to alcohol treatment reduce degradation of insoluble proteins, they do not seem to provide significant structural changes to prevent degradation of the soluble fraction in vivo. It appears that increases in ruminal escape of alcoholtreated SBM may be due to the proteins reduced solubility, since proteins that are rapidly hydrolyzed by ruminal proteases are also most susceptible to alcohol dentaturation. ACKNOWLEDGMENTS
Appreciation is expressed to M. P. Bryant, Department of Animal Sciences, University of Illinois, for supplying the bacterial cultures, and to D. R. Nelson, Department of Veterinary Clinical Medicine, University of Illinois, for cannulating the steers. REFERENCES
1 Baker, E. C., and D. A. Sullivan. 1983. Development of a pilot-plant process for the extraction of soy flakes with aqueous isopropyl alcohol. J. Am. Oil Chem. 60:1261. 2 Beachy, R. N. 1982. Molecular aspects of legume seeds storage protein synthesis. Page 87 in CRC Crit. Rev. Food. Sci. Nutr. CRC Press, Boca Raton, FL. 3 Carmer, S. G., and M. R. Swanson. 1973. An evaluation of ten pair-wise multiple comparison procedures by Monte Carlo methods. J. Am. Stat. Assoc. 68:66. 4 Cuthbertson, D. P., and M. I. Chambers. 1950. utilization of a casein supplement administered to ewes by ruminal and duodenal fistulae. Biochem. J. 46:17. 5 Eldridge, A. C., K. Warner, and W. J. Wolf. 1977. Alcohol treatment of soybeans and soybean products. Cereal Chem. 54:1229. 6 Fisher, C. E., 1. B. Leach, and P. Wilding. 1976. Improved separation of the major water-soluble proteins of soya meal by a single step chromatographic procedure. J. Sci. Food Agric. 77:1039. 7 Fukushima, D. 1969. Denaturation of soybean proteins by organic solvents. Cereal Chem. 46:156. 8 Fukushima, D. 1969. Enzymatic hydroysis of alcohol-denatured soybean proteins. Cereal Chem. 46:405. 9 Glimp, H. A., M. R. Karr, Co. O. Little, P. G. Woolfolk, G. E. Mitchell, Jr., and L. W. Hudson. 1967. Effect of reducing soybean solubility by dry heat on protein utilization of young lambs. J. Anita. Sci. 26:858. 10 Lowry, O. H., N. J. Rosebrough, A. H. Farr, and R.
PROTEOLYSIS OF ALCOHOL-TREATED SOYBEAN MEAL PROTEINS
11
12
13
14
15
16
17
18 19
J. Randall. 1951. Protein measurement with the Folin reagent. J. Biol. Chem. 193:265. Lynch, C. J., C. K. Rha, and N. Catsimpoolas. 1977. Note on the rapid proteolysis of glycinin by pepsin and trypsin. Cereal Chem. 54:1282. Lynch, G. L., L. L. Berger, and G. C. Fahey, Jr. 1987. Effects of ethanol, heat and(or) lipid treatment of soybean meal on nitrogen utilization by ruminants. J. Dairy Sci. 70:91. Lynch, G. L., L. L. Berger, N. R. Merchen, G. C. Fahey, Jr., and E. C. Baker. Effects of ethanol and heat treatment of soybean meal and ruminal salt infusion on ruminal escape of soybean meal protein in steers. J. Anim. Sci. 65:1617. Mahadevan, S., J. D. Erfle, and F. D. Sauer. 1980. Degradation of soluble and insoluble proteins by Bacteroides amylopbilus protease and by tureen microorganisms. J. Anim. Sci. 50:723. Mangan, J. L. 1972. Quantitative studies on nitrogen metabolism in the bovine rumen. Br. J. Nutr. 27:261. Mann, R. L , and D. R. Briggs. 1950. Effect of solvent and heat treatments on soybean proteins as evidenced by electrophoretic analysis. Cereal Chem. 27:258. Mickelson, R., N. L. Fish, and T. J. Claydon. 1967. Some chemical and flavor characteristics of a milk proteolysate of Pseudomonas fluorescens. J. Dairy Sci. 50:172. Nielsen, N. C. 1985. Structure of soybean proteins. New Protein Foods 5:27. Peter, A. P., E. E. Hatfield, F. N. Owens, and U. S. Garrigus. 1971. Effects of aldehyde treatments of soybean meal on in vitro ammonia release, solubility and lamb performance. J. Nutr. 101:65.
2427
20 Plegge, S. D., L. L. Berger, and G. C. Fahey, Jr. 1982. Effect of roasting on utilization of soybean meal by ruminants. J. Anita. Sci. 55:395. 21 Schaefer, D. M., C. L. Davis, and M. P. Bryant. 1980. Ammonia saturation constants for predominant species of rumen bacteria. J. Dairy Sci. 63:1248. 22 Spears, J. W., E. E. Hatfield, and J. H. Clark. 1980. Influence of formaldehyde treaunent of soybean meal on performance of growing steers and protein availability in the chick. J. Anim. Sci. 50:750. 23 Uden, P., P. E. Colucci, and P. J. Van Soest. 1980. Investigation of chromium, cerium and cobalt as markers in digesta rate of passage studies. J. Sci. Food Agric. 31:625. 24 van der Aar, P. J., L. L. Berger, and G. C. Fahey, Jr. 1982. The effect of alcohol treatments on solubility and in vitro and in situ digestibilities of soybean meal protein. J. Anita. Sci. 55:1179. 25 van der Aar, P. J., L. L. Berger, G. C. Fahey, Jr., and S. C. Loerch. 1983. Effects of alcohol treatments on utilization of soybean meal by lambs and chicks. J. Anita. Sci. 57:511. 26 van der Aar, P. J., L. L. Berger, K. M. Wujek, I. Mastenbroek, and G. C. Fahey, Jr. 1983. Relationship between electrophoretic band patterns and in vitro ammonia release of soluble soybean meal protein. J. Dairy Sci. 66:1272. 27 van der Aar, P. J., L. L. Berger, G. C. Fahey, Jr., and N. R. Merchen. 1984. Effect of alcohol treatments of soybean meal on ruminal escape of soybean meal protein. J. Anim. Sci. 59:483. 28 Wolf, W. J., and D. A. Sly. 1965. Chromatography of soybean proteins on hydroxylapatite. Arch. Biochem. Biophys. 110:47.
Journal of Dairy Science Vol. 71, No. 9, 1988
Bacteroides amylophilus,
Bacteroides ruminicola,
Pepsin, T r y p s i n , and in t h e R u m e n of Steers G. L. LYNCH, P. J. van der AAR, 1 L. L. BERGER, G. C. FAHEY, JR., and N. R. MERCHEN Department of Animal Sciences
University of Illinois Urbana 61801 ABSTRACT
untreated soybean meal or soybean meal treated with 70% ethanol at 80°C indicated that no detectable soluble glycinin or conglycinin escaped ruminal degradation.
Sodium dodecyl sulfate-gel electrophoresis and cation exchange chromatography were used to examine degradation of treated and untreated soybean meal protein fractions by Bacteroides amylopbilus H181, Bacteroides ruminicola Bx4, pepsin, trypsin, and intraruminally. Soybean meal treatments consisted of 30% vol/vol isopropanol, 40% propanol, or 50% ethanol at 22°C or 70% ethanol at 80°C. Water-soluble protein fractions were applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient of .03 to .27 and then .27 to 1.0 M. The four protein fractions with the highest absorbance at 276 nm were dialyzed against distilled water prior to being subjected to enzymatic hydrolysis. Soybean meal treated with 40% propanol had the greatest reduction in absorbance of all effluents at 275 nm, followed by soybean meal treated with 50% ethanol or 30% isopropanol. Comparison of electrophoretie patterns over time showed that B. amylopbilus H181, degraded protein subunits more rapidly than B. ruminicola B14. Protein subunits with the highest molecular weights were the most rapidly degraded by B. arnylopbilus H181, B. ruminicola B14, pepsin, and trypsin. Hydroxylapatite chromatography of omasal fluid from steers supplemented with
INTRODUCTION
Soybean meal (SBM) is a common protein source in ruminant diets and is rapidly degraded by ruminal microorganisms. Ruminal digestion of protein is less efficient than postruminal digestion (4) because some of the degraded protein N is lost from the rumen as ammonia. Ammonia is converted into urea in the liver and much of it is subsequently excreted in the urine. Various treatments have been examined for their ability to improve resistance of SBM protein to ruminal microbial proteases. Formaldehyde (19) and heat (9) treatments reduced degradation of SBM protein by ruminal microbes. However, excessive treatment resulted in depressed postruminal availability as measured by chick growth bioassay (20, 22). Soybean meal treatments involving alcohols, with and without addition of heat, depressed ruminal protein degradation (13, 24) but did not affect the nutritive value for chicks (12, 25). Development of a commercial process to extract soy flakes with aqueous isopropanol (1) generated interest in the effect of these solutions on susceptibility of soy protein to enzymatic degradation. In vitro fermentation of different feed proteins with Bacteroides amylopbilus indicated wide variation in the degradation rates of different soluble proteins, and the relationship between degradation rate of soluble and insoluble proteins is inconsistent (14). Reduced N disappearance in situ was observed Received July 1, 1987. when SBM was treated with aqueous alcohols Accepted March 14, 1988. (5, 12, 24); however, hydrolysis of alcohol1CLO -- Instituut Voor de Veevoeding "De Scho- treated SBM protein by Aspergillis sojae octhorst", Meerkoeten Weg 26, 8218 NA Lelystad, curred faster than that of untreated SBM (8). The Netherlands. 1988 J Dairy Sci 71:2416-2427
2416
PROTEOLYSIS OF ALCOHOL-TREATED SOYBEAN MEAL PROTEINS Effect of alcohol treatments on proteolysis of SBM proteins by ruminal microbial and mammalian proteases and on in vivo degradation of specific SBM proteins has not been reported before. The purpose of this study was to determine effects o f various alcohol treatments on different SBM protein fractions and to measure proteolysis of these fractions by microbial proteases, pepsin, trypsin, and within the rumen. MATERIALS AND METHODS Soybean Meal Treatment
Hexane-extracted, dehulled SBM was treated with various aqueous alcohol solutions at two temperatures. Treatments with 30% vol/vol isopropanol, 40% vol/vol propanol, or 50% vol/vol ethanol were conducted at 22°C. These concentrations were chosen because earlier work (24) indicated that they resulted in the greatest depression in ruminal degradation. One hundred gram SBM were soaked in 250 ml of alcohol solution for 30 min. After soaking, SBM was filtered through Whatman No. 41 filter paper and air-dried. The heat processed treatment involved adding 22.7 kg SBM to 76.8 L of 70% vol/vol ethanol heated to 80°C. The mixture was stirred for 30 min, after which the liquid was drained and the SBM dried at 77°C for 12 h. Soluble proteins were isolated from untreated SBM treated at 22°C with 30% isopropanol, 40% propanol, or 50% ethanol from SBM treated with 70% ethanol at 80°C, or from omasal fluid from steers fed untreated SBM, SBM treated with 70% ethanol at 80°C, or ureacasein. Forty-gram SBM samples were suspended in 200 ml distilled water and agitated for 1 h in a 40°C water bath prior to centrifugation for 20 min. The supernatant was exhaustively dialyzed against .03 M potassium phosphate buffer containing .1% 0~-mercaptoethanol, followed by gel filtration over a Sephadex G-25 column (Pharmacia Fine Chemicals, Inc. Milwaukee, WI). An amount of extract containing 200 mg protein (10) was applied to a hydroxylapatite column (2 × 30 cm). The column was eluted with a discontinuous phosphate gradient of .03 to .27 M then .27 to 1.0 M (6) using an Ultragrad (LKB-Produkter AB, S-16125 Bromma, Swed.) gradient mixer. Proteins eluting from the column were detected
2417
by monitoring column effluent at 276 nm with a Unicord S monitor (LKB-Produkter AB). The four largest fractions from untreated SBM and SBM treated with 30% isopropanol, 40% propanol, and 50% ethanol at 22°C were extensively dialyzed against distilled water, frozen, and stored. Soluble proteins in omasal fluid from steers fed untreated SBM, 70% ethanol-treated SBM at 80°C, or urea-casein were prepared by compositing samples b y treatment, followed by centrifugation at 35,000 × g, with the supernatant dialyzed against .03 M phosphate buffer containing .1% 0~-mercaptoethanol. Proteins were separated as above by applying 24 mg protein to the hydroxylapatite column. Omasal fluid was collected as described previously (13). Amino Acid Analysis
Fractions isolated from untreated SBM were hydrolyzed with 6N He1 for 22 h at 110°C prior to analysis for amino acid composition with a Beckman 119CL amino acid analyzer (Beckman Instruments, Inc., Palo Alto, CA). Release of ~-amino groups was determined as described by (17). Digestion rates of various protein fractions were expressed as micrograms glycine equivalents released per milligram enzyme protein - 1 • minute - 1 • In Vitro Bacterial Digestion
The bacteria, Bacteroides amylophilus strain H181 and Bacteroides ruminocola strain B14, were grown and maintained on media described by Schaefer et al. (21). Eighteen hours after transfer to fresh media, bacteria were centrifuged at 8000 × g. The pellet containing the bacteria was suspended in 1 ml .05 M phosphate buffer (pH 7.6). Bacterial cells were disrupted using a French press cell (20,000 psi) and suspended in 25 ml .05 M phosphate buffer (pH 7.6). Three milliliters of water-soluble protein extract were incubated with 1 ml bacterial suspension. After 1, 3.5, and 6 h of digestion, 1-ml samples were immediately analyzed for free (~-amino groups. Rates of protein hydrolysis for the protein fractions were determined b y linear regression. In Vitro Trypsin and Pepsin Digestion
Three milliliters of water-soluble protein extract from untreated SBM and 30% isoJournal of Dairy Science Vol. 71, No. 9, 1988
2418
LYNCH ET AL.
propanot, 40% propanol, and 50% ethanoltreated SBM were incubated with .10 mg trypsin in 1 ml .05 M phosphate buffer (pH 7.6) at 25°C. After 1, 4, and 7 min, 1-ml samples were taken and digestion was stopped by adding .1 ml of 1 N HC1. Prior to analysis for free a-amino groups, .1 ml 1 N NaOH was added to the tubes. Rates o f protein hydrolysis were determined by linear regression. To evaluate susceptibility to pepsin, 3 ml of water-soluble protein extract fractions were incubated with .4 mg pepsin in 1 m l . 1 N HC1 at 30°C. After 1, 9, and 17 min, 1-ml samples were taken, neutralized with .2 N NaC1 and analyzed for free a-amino groups. Rates of protein hydrolysis were determined by linear regression. Electrophoresis
Composition of protein fractions isolated from untreated SBM extract was examined using SDS P A G E (26). Also, effects of hydrolysis by various proteases on electrophoretic patterns of SBM protein over time were studied. Hydrolysis conditions were similar to those described previously. The molecular weight of subunits was determined with a standard protein solution consisting of lysozyme (Mr 14,300), trypsinogen (M r 24,000), ovalbumin (M r 45,000), bovine plasma albumin (M r 66,000), and phosphorylase a (M r 92,500). Pepsin and trypsin digestions were stopped after 0, 1, 2, 4, 10, 30, and 60 min. Bacterial digestions were stopped after 0, 4, 8, 12, and 24 h of digestion. Ruminal Protein Degradation
Three mature Hereford steers (average 508 kg) and three mature Simmental steers (average 594 kg) fitted with ruminal cannulae were used to measure soluble SBM protein outflow from the rumen. Treatments consisted of three N supplements: untreated SBM, SBM treated with 70% ethanol at 80°C, and a urea-caseincorn mix. Nitrogen supplements were added to a basal diet (Table 1) to achieve a 14.4% CP final diet, with approximately 70% of the dietary CP provided by N supplements. Steers were fed daily at 0800 and 2000 h and were tethered in solid floor stalls in a temperaturecontrolled (18.3°C) room under continuous lighting. Journal of Dairy Science Vol. 71, No. 9, 1988
Each experimental period was 14 d in length. Days 1 through 10 were for diet adaptation with d 11 through 14 used for collection of omasal fluid samples. Cobalt EDTA (23) was continuously infused into the rumen to serve as omasal fluid flow marker. A p p r o x i m a t e l y 75 ml of omasal fluid were collected five times daily; collection times were randomized so that omasal samples were collected every 70 min of a 24-h period. Omasal samples were collected by inserting a polyethylene tube (.95 cm i. d., 1.59 cm o. d.) into the omasum through the reticulo-omasal orifice. A plastic strainer was attached to the omasal end of the tube and held approximately 12 cm into the omasum by 2 x 10 cm rubber flanges positioned on each side of the reticulo-omasal orifice. Fluid samples were collected by attaching the opposite end of the tube to a filter flask and applying negative pressure with a vacuum pump. Omasal samples were composited, centrifuged at 14,000 x g, and frozen at - 2 0 ° C prior to protein isolation. Statistical Analysis
Data on rate of digestion were analyzed b y analysis of variance. When significant differences were observed, treatment means were compared by the F test-protected, least significant difference method (3).
RESULTS
Elution patterns resulting from separation of water-extractable proteins on the hydroxylapatite column from untreated SBM or from SBM treated with 30% isopropanol, 40% propanol, and 50% ethanol are presented in Figures 1, 2, and 3. Four major and two minor peaks were observed. The first fraction eluted with .03 M phosphate buffer, the second between .18 and .23 M phosphate, the third with .27 M phosphate, and the fourth between .4 and .7 M phosphate. Two minor peaks were observed between .08 and .12 M and .23 and .25 M phosphate. The relative absorbance at 276 nm of soluble protein fractions from SBM treated with 30% isopropanol is superimposed on the pattern found for untreated SBM in Figure 1. The isopropanol-treated sample contained more proteins that eluted at .03 M and between .18 and .23 M phosphate and less of the proteins
PROTEOLYSIS OF ALCOHOL-TREATEDSOYBEAN MEAL PROTEINS TABLE 1. Ingredient and chemical composition of basal diet (dry basis). Item
(%)
Ingredient composition Corncobs, ground Cornstarch grits Prairie hay, ground Dextrose Casein Urea Dicalcium phosphate Limestone Vitamin mix ~
45.0 30.14 12.5 11.0 .5 .4 .2 .16 .1
Chemical composition Dry matter Crude protein Acid detergent fiber
79.7
4.31 26.4
Contains 3,300,000 IU vitamin A, 330,000 IU vitamin D, and 22,000 IU vitamin E/kg.
1
2
2419
that eluted in fractions 3 and 4. The same observations were made for the samples from SBM treated with 40% propanol (Figure 2) and 50% ethanol (Figure 3). Reduction in peak 3 and 4 proteins was largest for the 40% propanol treatment. Fifty percent ethanol treatment resulted in a reduction in size of these peaks intermediate between isopropanol and propanol. Four fractions of the water-extractable proteins from untreated SBM were obtained as shown in Figure 1. The amino acid composition of these fractions is presented in Table 2. Fraction 1 contained high concentrations of threonine, alanine, and glycine and low concentrations of leucine relative to the crude SBM protein fraction applied to the column. Fraction 2 contained relatively lower concentrations of serine, leucine, tyrosine, and phenylalanine. Fraction 3 was the only one in which mea-
3
4
qD z
:E z
0 "o -I-
(D ',N
.,4
m
U.I
0
r-
..... i 100
300
EFFLUENT VOLUME
500
127o3
-I -¢
700
(ML)
Figure 1. Absorbance (276 nm) pattern of soybean meal proteins ( ) applied to an hydroxylapatite column and eluted with a discontinuous phosphate buffer (. . . . ) in comparison to the elution pattern for soybean meal treated with 30% isopropanol ( ). Journal of Dairy Science Vol. 71, No. 9, 1988
2420
LYNCH ET AL. 1
2
3
4
Z O
:E Z
i/:,,)l/},J / ,,,,'://
gO UJ
j
m O r-
-4 .<
jj~ jl ....
~'
.03
100
300
5~
EFFLUENT
VOLUME
700
(ML)
Figure 2. Absorbance (276 nm) pattern of soybean meal proteins ( - - ) applied to an hydroxylapatite column and eluted with a discontinuous phosphate buffer (. . . . ) in comparison to the elution pattern for soybean meal treated with 40% (vol/vol) propanol ( --).
1
2
3
4
l\
\ :E Z
III I
\
I
I
I
I
1.00
"u
O
A
gO N
I
I
/
UJ
/
/
"u -I-
/
-4 m O
/ ._/ 11
.27
Iij 100
-4
J
.03
300 EFFLUENT
500 VOLUME
700
(ML)
Figure 3. Absorbance (276 nm) pattern of soybean meal proteins ( ) applied to an hydroxylapatite column and eluted with a discontinuous phosphate buffer (. . . . ) in comparison to the elution pattern for soybean meal treated with 50% (vol/vol) ethanol ( --). Journal of Dairy Science Vol. 71, No. 9, 1988
PROTEOLYSIS OF A L C O H O L - T R E A T E D SOYBEAN MEAL PROTEINS
2421
TABLE 2. A m i n o acid composition o f protein fractions isolated from u n t r e a t e d soybean meal ( n m o l / m g total protein). Amino acid
Crude fraction ]
1
2
Fraction 3
4
Aspartic acid Threonine Serine Glutamic acid Proline Glycine AI anine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine Histidine Lysine Arginine
1526 422 729 2743 695 823 644 296 83 269 748 246 391 161 567 597
1763 910 1073 1486 538 2175 1480 325 ND 2 247 242 288 256 193 296 433
1178 247 314 2457 707 608 302 123 ND 112 154 37 94 183 509 491
1334 389 675 204.5
1463 284 820 8683
678
131
811 566 344 46 238 692 205 343 148 408 531
814 334 301 ND 300 816 203 497 119 538 600
I Water-soluble proteins extracted from soybean meal with water at 40°C. 2 ND = Not detectable.
ii 1
2
| 3
Figure 4. The SDS-PAGE patterns of the various separated protein fractions: 1) Fraction 1, 2) Fraction 2, 3) Fraction 3, and 4) Fraction 4.
0
4
8
12
24
Figure 5. The SDS-PAGE patterns of soybean meal protein after 0, 4, 8, 12, and 24 h digestion with Bacteroides amylopbilus H18 extract at 25°C. Journal o f Dairy Science Vol. 71, No. 9, 1988
2422
LYNCH ET AL.
A
ill
I
i
II
I
._ . _ , ,
t
0
I
4
8
12
24
Figure 6. The SDS-PAGE patterns of soybean meal protein after 0, 4, 8, 12, and 24 h digestion with
Bacteroides rurninicota B, 4 extract at 25°C.
surable amounts o f methionine were detected. Fraction 4 was extremely rich in glutamic acid but low in proline. Band patterns, as determined b y SDS-PAGE, of the pooled treatments are shown in Figure 4. Fraction 1 did not contain determinable proteins. Electrophoresis of fraction 2 resulted in a wide, vague band with no distinguishable protein bands. This was probably caused b y the presence of one or more glycoproteins. The SDS-PAGE pattern of fraction 3 showed several sharp bands. These bands all had molecular weights less than 35,000. Fraction 4 contained four major protein subunits and various minor ones with molecular weights ranging from 35,000 to 75,000 daltons.
0
I
2
4
10
30
60
Figure 7. The SDS-PAGE patterns of soybean meal protein after 0, 1, 2, 4, 10, 30, and 60 min trypsin digestion at 25°C. Journal of Dairy Science Vol. 71, No. 9, 1988
Exposure of water-extractable SBM proteins to B. amytopbilus H18 t resulted in a gradual degradation of protein fraction subunits represented in bands A, B and C after 24 h digestion (Figure 5). A slight accumulation of lower molecular weight proteins could also be noted (band E). Digestion of water-extractable proteins by B. ruminicola B14 resulted in SDS-PAGE patterns shown in Figure 6. Disappearance of bands was slower than noted for B. amylopbilus. None o f the bands had completely disappeared after 24 h of digestion. However, a reduction of bands representing the highest molecular weight subunits (band A) was again noted. Hydrolysis with trypsin resulted in rapid digestion of the higher molecular weight protein subunits (Figure 7). Intensity of bands A and B, representing proteins with molecular weights of 78,000 and 72,000 daltons, respectively, began to diminish 1 min after adding the trypsin. After 10 min, band C began to lose intensity. None of the other protein bands appeared to be affected by this enzyme during these short incubation times. The SDS-PAGE patterns during pepsin digestion are shown in Figure 8. In this assay, none of the bands completely disappeared. Bands representing the two highest molecular weight subunits (bands A and B) had lost intensity after 60 min of pepsin digestion. No significant interactions between protein fraction rate of hydrolysis and alcohol treatment were detected. Changes in affinities of
0
I
2
4
10
30
60
Figure 8. The SDS-PAGE patterns of soybean meal protein after 0, 1, 2, 4, 10, 30, and 60 min pepsin digestion at 25°C.
PROTEOLYSIS OF ALCOHOL-TREATED SOYBEAN MEAL PROTEINS
2423
TABLE 3. Effects of various alcohol treatments on rate of hydrolysis of soybean meal protein by various proteases. Alcohol treatment Protease
Untreated
30% (vol/vol) isopropanol
40% (vol/vol) propanol
50% (vol/vol) ethanol
B. ruminicola Bt 4
.0081 (.0018) 2
.0065 (.0018)
.0063 (.0023)
.0058 (.0018)
B, amylopbitus H 18
.0182 (.0016)
.0213 (.0020)
.0245 (.0020)
.0225 (.0020)
Pepsin a
1.98 (.39)
2.30 (.48)
2.31 (.62)
2.58 (.57)
Trypsin 3
9.76 (3.10)
16.49 (2.92)
11.86 (4.82)
15.13 (4.25)
1 Data are expressed as grams glycine equivalents released --i .milligrams enzyme protein -1 .minute-1. 2 Standard error of the mean values. aObtained from Sigma Co., St. Louis, MO.
p r o t e a s e s f o r specific p r o t e i n f r a c t i o n s were similar a m o n g t r e a t m e n t s . T a b l e 3 p r e s e n t s results o f various a l c o h o l t r e a t m e n t s o n rate o f h y d r o l y s i s of SBM p r o t e i n b y proteases. A l c o h o l t r e a t m e n t s did n o t a f f e c t SBM p r o t e i n availability as m e a s u r e d b y s u s c e p t i b i l i t y o f SBM p r o t e i n to d e g r a d a t i o n b y proteases. H o w e v e r , p r o t e o l y s i s b y B. amylophitus e x t r a c t was faster t h a n t h a t b y B. ruminicola e x t r a c t . R a t e s o f h y d r o l y s i s b y t h e various e n z y m e s
were n o t statistically c o m p a r a b l e because t h e y were d e t e r m i n e d u n d e r d i f f e r e n t c o n d i t i o n s . In T a b l e 4, rates of p r o t e o l y s i s of various p r o t e i n f r a c t i o n s are p r e s e n t e d . F r a c t i o n s 2 a n d 3 h a d m o r e ( P < . 0 5 ) s - a m i n o g r o u p s released per u n i t o f t i m e b y B. ruminicoIa t h a n did t h e c r u d e fraction. F r a c t i o n 3 was h y d r o l y z e d f a s t e r ( P < . 0 5 ) b y B. amytopbilus t h a n e i t h e r f r a c t i o n 1 or t h e c r u d e f r a c t i o n . F r a c t i o n s 3 a n d 4 were h y d r o l y z e d faster ( P < . 0 1 ) b y p e p s i n
TABLE 4. Rate of hydrolysis of soybean meal protein fractions by various proteases)
Protease
Crude fraction
1
Fraction 3
2
4
B. ruminicola B~ 4
.0016 a (.0020) 3
ND 2
.0134 b (.0020)
.0159 b (.002)
.0033 ab (.0020)
B. amylopbilus H 18 Pepsin
.0184 ab (.0020)
.0147 a (.0022)
.0231 bc (.0020)
.0278 c (.0020)
.0241 bc (.0020)
1.46 a (.57)
ND 2
1.40 a (.44)
2.90 b (.53)
4.443b (.47)
4.91 a (4,05)
ND a
21.72 b (4.50)
8.51 a (3.37)
18.10 b (2.95)
Trypsin
a'b'CValues in the same row with different superscripts differ (P<.05). 1Data are expressed as grams glycine equivalents released -1 ,milligrams enzyme protein -1 .minute -1 . 2 Variation in assay results made values obtained unreliable and were therefore considered as nondeterminable (ND). Standard error of the means. Journal of Dairy Science Vol. 71, No. 9, 1988
2424
LYNCH ET AL.
= 1.00 ~O =r
8 E
i
c ¢o UJ
/i/I/i/
O m
/// .27
.03 | 100
300
500
Effluent volume (ml)
Figure 9. Absorbance (276 nm) pattern of untreated soybean meal soluble proteins ( - - ) and soluble protein from soybean meal treated with 70% ethanol at 80°C (. . . . . ) applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient (. . . . ).
than was fraction 2 or the crude fraction. The crude fraction applied to the hydroxylapatite column and fraction 3 were hydrolyzed more slowly (P<.05) by trypsin than were fractions 2 and 4. Elution pattern of soluble proteins from untreated SBM and SBM treated with 70% ethanol at 80°C was similar to that of the other SBMs (Figure 9). Elution patterns of soluble proteins collected at the omasum of steers fed untreated SBM, SBM treated with 70% ethanol at 80°C, or urea-casein were all similar (Figure 10). All three N supplements resulted in increases in peak 1 with no detectable traces of peaks 3 or 4. Separation of samples applied to the hydroxylapatite column by SDS-PAGE produced no determinable proteins (data not shown). DISCUSSl ON
Soluble proteins extracted from untreated SBM had an absorbance pattern at 276 nm similar to that reported previously (6, 28). The fraction eluting at .03 M phosphate (peak 1) is composed predominantly of trypsin inhibitors Journal of Dairy Science Vol. 71, No. 9, 1988
and metabolic proteins (18). Peak 2, eluting at .03 to .27 M phosphate, is possibly composed of a mixture of storage and metabolic proteins (18). The fraction eluting from hydroxylapatite at .27 M phosphate (peak 3) was identified as the major soybean storage protein, glycinin (6). Fisher et al. (6) identified the fraction eluting between .4 and .7 M phosphate (peak 4) as conglycinin but did not report a major peak eluting from the column when the phosphate buffer concentration was between .18 and .23 M as was observed in our work. The greatest denaturation of glycinin and conglycinin was observed for the 40% propanol treatment (Figure 2). Previous reports (7) showed that treatment with 40% propanol resulted in more protein denaturation than did treatment with 50% ethanol. The 40% propanol treatment was more effective in denaturing conglycinin than was propanol at any other concentration (26). Comparison of SDS-PAGE patterns of SBM proteins following exposure to bacterial protease showed that hydrolysis of protein subunits occurred faster when B. amylopbilus H18
PROTEOLYSIS OF ALCOHOL-TREATEDSOYBEAN MEAL PROTEINS
2425
"o ~r o "0 ~r ¢¢
E e4
i
LO0
UJ
i¢ o
i ffj
.27
~e
,03 100
300
500
Effluent volume (ml)
Figure 10. Absorbance (276 nm) pattern of omasal fluid from steers fed untreated soybean meal ( ), ), or urea-casein-corn ( ) applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient (. . . . ).
soybean meal treated with 70% ethanol at 80°C (. . . . .
extract was used than when B. ruminicola Bt 4 was used. Distinct disappearance of these proteins was noticeable after 4 h with B. arnylopbilus (Figure 5), whereas bands were still visible after 24 h when SBM protein was incubated with B. rurninicola extract. Enzymatic hydrolysis of SBM protein, as examined by SDS-PAGE, showed that the highest molecular weight subunits (71,000 and 78,000 dattons) were always the most rapidly digested. Comparison of pattern of pepsin and trypsin digestion over time (Figures 7 and 8) showed that proteolysis by trypsin was faster than that by pepsin and was probably due to the difference in specific activity of the enzymes used. At 25°C, glycinin was hydrolyzed more rapidly by pepsin than by trypsin (11). We were not able to confirm this finding. However, in our experiment, as well as in the previous work (11), protein subunits with
the highest molecular weights were the most rapidly digested. Rate of in vitro 0~-amino group release and the change in SDS-PAGE patterns show conformity. In both assays, the most rapid rate of digestion was found for trypsin. Comparison of the two bacterial proteases showed that B. amylopbilus extract had a greater specific activity than did B. ruminicola extract. Based on changes in SDS-PAGE patterns, we expected that fraction 4 (conglycinin) would be the most rapidly digested of all fractions tested. This deviation from expected results is possibly due to variability in the in vitro protease assays. Therefore, we think the results of the SDSPAGE assays are the most reliable of the two data sets. Rate of in vitro ammonia release in ruminal fluid has been directly related to the composition of SBM proteins (26). It has been Journal of Dairy Science Vol. 71, No. 9, 1988
2426
LYNCH ET AL.
observed that fractions relatively rich in conglycinin and acidic subunits of glycinin released the greatest amounts of ammonia, van der Aar et al. (26) hypothesized that conglycinin was more susceptible to degradation by ruminal bacterial proteases than the other proteins. The preferential hydrolysis of conglycinin by B. amylopbilus (Figure 5) and to a lesser extent by B. rurninicola (Figure 6) support this hypothesis. Lack of differences in hydroxylapatite elution patterns of omasal soluble proteins from steers fed untreated SBM, SBM treated with 70% ethanol at 80°C, and urea-casein-corn indicate that the protein material is of predominantly bacterial origin. It was hypothesized that some of the soluble glycinin and conglycinin, once denatured by alcohol treatment, would escape ruminal degradation and flow out of the rumen. Because only 5 to 10% of protein from alcohol-treated SBM was soluble (13, 27), diets were formulated to allow N supplements to provide 70% of dietary CP. We thought this would provide measurable soluble proteins if alcohol treatment resulted in soluble proteins resistant to ruminal degradation. Because over 80% of SBM N is waterextractable at pH 6.5 (28), the potential exists for significant amounts of soybean proteins to be solubilized in the rumen. Mahaevan et al. (14) reported that in vitro degradation rates of soluble and insoluble SBM proteins were identical. Because previous work showed increased ruminal outflow of SBM N when SBM was treated with ethanol and heat (13, 27), it was hypothesized that soluble proteins from SBM treated with 70% ethanol at 80°C may also escape the rumen. If the proteins escaping the rumen could be captured and identified, this information could be useful in determining what qualities would be advantageous for the ruminal escape of proteins. The soluble proteins from steers fed urea-casein-corn are thought to be of bacterial origin, since ruminal half-life of casein is less than 22 min (15) and very small amounts of protein from the corn would be soluble. Conglycinin is both rapidly hydrolyzed by proteases and easily denatured by aqueous alcohol solutions (16). The molecular threedimensional structure is responsible for these properties. Conglycinin is glycosylated (2) and thus has hydrophilic characteristics. These Journal of Dairy Science Vol. 71, No. 9, 1988
hydrophilic properties cause the molecule to be less compact when solubilized in water (7). This loose structure promotes denaturation by aqueous alcohol solutions and enzymatic hydrolysis. Although changes due to alcohol treatment reduce degradation of insoluble proteins, they do not seem to provide significant structural changes to prevent degradation of the soluble fraction in vivo. It appears that increases in ruminal escape of alcoholtreated SBM may be due to the proteins reduced solubility, since proteins that are rapidly hydrolyzed by ruminal proteases are also most susceptible to alcohol dentaturation. ACKNOWLEDGMENTS
Appreciation is expressed to M. P. Bryant, Department of Animal Sciences, University of Illinois, for supplying the bacterial cultures, and to D. R. Nelson, Department of Veterinary Clinical Medicine, University of Illinois, for cannulating the steers. REFERENCES
1 Baker, E. C., and D. A. Sullivan. 1983. Development of a pilot-plant process for the extraction of soy flakes with aqueous isopropyl alcohol. J. Am. Oil Chem. 60:1261. 2 Beachy, R. N. 1982. Molecular aspects of legume seeds storage protein synthesis. Page 87 in CRC Crit. Rev. Food. Sci. Nutr. CRC Press, Boca Raton, FL. 3 Carmer, S. G., and M. R. Swanson. 1973. An evaluation of ten pair-wise multiple comparison procedures by Monte Carlo methods. J. Am. Stat. Assoc. 68:66. 4 Cuthbertson, D. P., and M. I. Chambers. 1950. utilization of a casein supplement administered to ewes by ruminal and duodenal fistulae. Biochem. J. 46:17. 5 Eldridge, A. C., K. Warner, and W. J. Wolf. 1977. Alcohol treatment of soybeans and soybean products. Cereal Chem. 54:1229. 6 Fisher, C. E., 1. B. Leach, and P. Wilding. 1976. Improved separation of the major water-soluble proteins of soya meal by a single step chromatographic procedure. J. Sci. Food Agric. 77:1039. 7 Fukushima, D. 1969. Denaturation of soybean proteins by organic solvents. Cereal Chem. 46:156. 8 Fukushima, D. 1969. Enzymatic hydroysis of alcohol-denatured soybean proteins. Cereal Chem. 46:405. 9 Glimp, H. A., M. R. Karr, Co. O. Little, P. G. Woolfolk, G. E. Mitchell, Jr., and L. W. Hudson. 1967. Effect of reducing soybean solubility by dry heat on protein utilization of young lambs. J. Anita. Sci. 26:858. 10 Lowry, O. H., N. J. Rosebrough, A. H. Farr, and R.
PROTEOLYSIS OF ALCOHOL-TREATED SOYBEAN MEAL PROTEINS
11
12
13
14
15
16
17
18 19
J. Randall. 1951. Protein measurement with the Folin reagent. J. Biol. Chem. 193:265. Lynch, C. J., C. K. Rha, and N. Catsimpoolas. 1977. Note on the rapid proteolysis of glycinin by pepsin and trypsin. Cereal Chem. 54:1282. Lynch, G. L., L. L. Berger, and G. C. Fahey, Jr. 1987. Effects of ethanol, heat and(or) lipid treatment of soybean meal on nitrogen utilization by ruminants. J. Dairy Sci. 70:91. Lynch, G. L., L. L. Berger, N. R. Merchen, G. C. Fahey, Jr., and E. C. Baker. Effects of ethanol and heat treatment of soybean meal and ruminal salt infusion on ruminal escape of soybean meal protein in steers. J. Anim. Sci. 65:1617. Mahadevan, S., J. D. Erfle, and F. D. Sauer. 1980. Degradation of soluble and insoluble proteins by Bacteroides amylopbilus protease and by tureen microorganisms. J. Anim. Sci. 50:723. Mangan, J. L. 1972. Quantitative studies on nitrogen metabolism in the bovine rumen. Br. J. Nutr. 27:261. Mann, R. L , and D. R. Briggs. 1950. Effect of solvent and heat treatments on soybean proteins as evidenced by electrophoretic analysis. Cereal Chem. 27:258. Mickelson, R., N. L. Fish, and T. J. Claydon. 1967. Some chemical and flavor characteristics of a milk proteolysate of Pseudomonas fluorescens. J. Dairy Sci. 50:172. Nielsen, N. C. 1985. Structure of soybean proteins. New Protein Foods 5:27. Peter, A. P., E. E. Hatfield, F. N. Owens, and U. S. Garrigus. 1971. Effects of aldehyde treatments of soybean meal on in vitro ammonia release, solubility and lamb performance. J. Nutr. 101:65.
2427
20 Plegge, S. D., L. L. Berger, and G. C. Fahey, Jr. 1982. Effect of roasting on utilization of soybean meal by ruminants. J. Anita. Sci. 55:395. 21 Schaefer, D. M., C. L. Davis, and M. P. Bryant. 1980. Ammonia saturation constants for predominant species of rumen bacteria. J. Dairy Sci. 63:1248. 22 Spears, J. W., E. E. Hatfield, and J. H. Clark. 1980. Influence of formaldehyde treaunent of soybean meal on performance of growing steers and protein availability in the chick. J. Anim. Sci. 50:750. 23 Uden, P., P. E. Colucci, and P. J. Van Soest. 1980. Investigation of chromium, cerium and cobalt as markers in digesta rate of passage studies. J. Sci. Food Agric. 31:625. 24 van der Aar, P. J., L. L. Berger, and G. C. Fahey, Jr. 1982. The effect of alcohol treatments on solubility and in vitro and in situ digestibilities of soybean meal protein. J. Anita. Sci. 55:1179. 25 van der Aar, P. J., L. L. Berger, G. C. Fahey, Jr., and S. C. Loerch. 1983. Effects of alcohol treatments on utilization of soybean meal by lambs and chicks. J. Anita. Sci. 57:511. 26 van der Aar, P. J., L. L. Berger, K. M. Wujek, I. Mastenbroek, and G. C. Fahey, Jr. 1983. Relationship between electrophoretic band patterns and in vitro ammonia release of soluble soybean meal protein. J. Dairy Sci. 66:1272. 27 van der Aar, P. J., L. L. Berger, G. C. Fahey, Jr., and N. R. Merchen. 1984. Effect of alcohol treatments of soybean meal on ruminal escape of soybean meal protein. J. Anim. Sci. 59:483. 28 Wolf, W. J., and D. A. Sly. 1965. Chromatography of soybean proteins on hydroxylapatite. Arch. Biochem. Biophys. 110:47.
Journal of Dairy Science Vol. 71, No. 9, 1988