The Effects of Bacterial Inoculants, Beet Pulp, and Propionic Acid on Ensiled Wet Brewers Grains

The Effects of Bacterial Inoculants, Beet Pulp, and Propionic Acid on Ensiled Wet Brewers Grains SCHNEIDER, J. J. H. HARRISON, HARRISON, and and K. A. A. LONEY R. M. SCHNEIDER, Department of of Animal Animal Sciences Dairy Forage Facility Washington State University Puyallup 98371 98371

ABSTRACT

The objective of of this research was to determine whether microbial inoculants, propionic acid, or beet pulp would enfermentation of of wet brewers hance the fermentation grains under short-term or long-term treatments (constorage. In trial 1, three treatments of lactic acid bactrol and two amounts of teria) were applied to wet brewers grains ensiled for 1, 1, 2, 3, 28, and 57 d. Each added amount of lactic acid bacteria faster, increased increased initial decreased pH faster, concentration of lactate, and reduced initial concentrations concentrations of acetate and butyrate compared with the control. control. In trial 2, four treatments treatments (control, (control, lactic acid bacteria, bacteria, lactic acid bacteria plus enzymes, propionic acid) enzymes, or propionic acid) were applied to wet brewers grains ensiled for 1, 1, 2, 4, 6, 6, 8, 8, and 90 d. d. Benefits Benefits of the treatments treatments were limited. same limited. Trial 3 utilized the same treatments treatments as as trial 2, and wet brewers brewers grains 40,55, 55, grains were were ensiled for for 10, 10, 25, 40, and 75 d. d. All treatments treatments increased increased production production of lactate, lactate, enhanced enhanced the the rate of pH decline, decline, and limited limited production production of butyrate. butyrate. In trial trial 4, wet brewers brewers grains grains were were ensiled ensiled for for 66 and 90 90 d as as four four treatments: 1) control, control, 2) 2) added added beet pulp treatments: 1) (15% 3) high moisture moisture (15% of wet weight), weight), 3) grain grain inoculant, inoculant, and and 4) 4) a combination combination of treatments treatments 2 and and 3. 3. Treatment Treatnient 4 resulted resulted in in the the lowest lowest concentration concentration of acetate, acetate, NH3 N H 3 N, N, and and pH and and the the greatest greatest concenconcentration tration of lactate lactate after after 90 90 d of storage. storage. Addition Addition of microbial microbial inoculants, inoculants, acid, acid, or beet beet pulp pulp to to wet wet brewers brewers grains grains was was beneficial beneficial in in promoting promoting aa more more efficient efficient fermentation fermentation during during long-term long-term storage; storage;

Received Received January January 6,6, 1994. 1994. Accepted Accepted December December 6, 6, 1994. 1994. 1995 1995 JJ Dairy Dairy Sci Sci 78:1096-1105 78:1096-1105

however, results for short-term storage were limited. (Key words: inoculants, propionic acid,wet brewers grains, fermentation) fermentation) key: BP = = beet pulp, ENZ == Abbreviation key: = cellulase and hemicellulase enzymes, HMI = high moisture grain inoculant, LAB = = lactic = wet brewers grains. acid bacteria, WBG = INTRODUCTION INTRODUCTION

patterns at breweries result in Production patterns (WBG) during May, excess wet brewers grains (WBG) shortage of WBG during June, and July and a shortage winter months. months. The periodic periodic (every (every 7 to 10 10 d) delivery of WBG is common, common, making anaeroanaerobic storage storage difficult and often resulting in mold development on the stored stored WBG. WBG.To provide a development continuous continuous supply of WBG to the dairies, dairies, excess cess WBG are often often ensiled in bunker silos silos for for use during during winter. winter. The typical typical management management practice practice for for storage storage of WBG in Washington Washington consists consists of applying applying rock salt to the surface surface of uncompacted uncompacted WBG and covering covering the silo with black plastic plastic secured secured with a minimal minimal number of tires. tires. Management Management methods methods to reduce reduce the the spoilspoilage age of WBG during during short-term short-term storage storage were were studied studied by Dixon and Combellas Combellas (6), (6), who who found found that neither NaCl NaCl (either (either mixed into into or sprinkled sprinkled on on the the surface surface of uncovered uncovered piles piles of WBG) WBG)nor nor NaOH NaOH addition addition was was able able to to preserve preserve WBG satisfactorily satisfactorily under under aerobic aerobic conditions. conditions. al. (2) reported reported a reduction reduction in in subsursubsurAllen et et aI. Allen face face deterioration deterioration of WBG stored stored uncovered uncovered for for 14 14 d with with addition addition of .40% .40% propionic propionic acid. acid. As with with other other ensiled ensiled feedstuffs, feedstuffs, itit is is critical critical As that that the the infiltration infiltration of of oxygen oxygen into into stored stored WBG be minimized. minimized. Failure Failure to to do do so so results results in in exexbe cessive cessive loss loss of of DM DM (I, (1, 2). 2). No No reports reports have have been been published published on on the the potential benefits benefits of of bacterial bacterial inoculation inoculation of of potential WBG. WBG. Research Research conducted conducted to to determine determine the the

1096 1096

FERMENTATION BREWERS GRAINS FERMENTATION OF ENSll..ED ENSILED WET BREWERS

effects effects of microbial microbial inoculants inoculants on the fermenfermentation tation characteristics characteristics of high moisture moisture corn has been inconclusive. inconclusive. Birkelo Birkelo and Sorenson Sorenson (5) (5) found found that addition addition of Lactobacillus plus Streptococcus species species (l (1 x lOS I@ cfulg c N g of wet com) corn) to to coarsely coarsely cracked corn ensiled in plastic tic silage silage bags was was effective effective in preserving preserving the high moisture corn at 27.2% moisture, moisture, but not at al. (12) at 22.4% 22.4% moisture. moisture. Wardynski Wardynski et al. treated (24% moisture), moisture), treated high moisture moisture corn (24% ground ground through through a 1.6-mrn 1.6-nun screen, screen, with a liquid 6 cfulg inoculant inoculant (2 (2 xx 10 106 c N g of wet com) corn) containcontaining Lactobacillus, Streptococcus, Streptococcus, and Pediing Lactobacillus, ococcus species. species. The corn was ensiled ensiled in an upright upright concrete concrete silo. silo. The results indicated indicated that inoculation inoculation lowered lowered pH and the concentrations concentrations of acetate acetate and and ethanol. ethanol. The The practice practice of ensiling ensiling WBG with beet pulp (BP)isis an an alternative alternative storage storage method pulp (BP) adopted adopted to to minimize minimize effluent production. production. Results Results (11) (11) indicated indicated that only the incorporaincorporation of straw straw results results in less effluent production production than shreds per than 150 150 kg kg molassed molassed sugar beet shreds tonne tonne of WBG. WBG. However, However, the molassed molassed sugar % higher organic beet feed feed resulted resulted in aa 31 31% organic digestibility than treatment treatment with WBG matter digestibility plus plus straw, straw, as as measured measured in vitro vitro (11). (11). Few studies studies have have been conducted conducted to deterdetermine the effects effects of additives additives on the preservapreservamine tion of WBG when stored stored under anaerobic anaerobic conditions. This study study was conducted conducted to deterdeterconditions. mine whether whether microbial microbial inoculants, inoculants, propionic mine acid, or BP would would enhance enhance the fermentation fermentation of acid, WBG under short-term short-term (3 (3 to 10 10 d) d) or long-term long-term WBG (60 to to 90 90 d) d) storage. storage. (60 MATERIALS AND METHODS MATERIALS Trial 1 Trial

samples of WBG were delivered Unstored samples directly from from the brewery and were randomly randomly directly assigned in a 3 x 5 factorial factorial design design to three three assigned additive treatments treatments and five five ensiling times. additive kg) of treated WBG were ensiled Quantities (l (1 kg) Quantities in glass glass jars with a screw-cap screw-cap lid and mainin tained at 20·C. 20'C. Duplicate Duplicate silos silos per additive treatment were were opened after 1, 1 , 2, 2 , 33,, 228, 8 , and 57 treatment ensilage. The additive additive treatments treatments were 1) 1) dd of ensilage. control, receiving receiving 25 25 ml of waterlkg waterkg of WBG, WBG, control, 2) lactic lactic acid bacteria (LAB) (LAB) at 1I x lOS I d cfulg cWg 2) 6 cfulg WBG, or 3) 3) LAB LAB at 11 xx 10 lo6 cWg WBG. The WBG,

1097

LAB were Lactobacillus plantarum (provided Burns, Inc., Inc.. Tacoma, WA). The by Curtice Bums, LAB LAB were applied as a liquid solution at 25 mlIkg of WBG. The microbial characterization characterization mVkg of 50-g porconducted by aseptic transfer of was conducted tions of WBG to stomacher bags, diluted in 450 ml of saline, and agitated for 3 min in a laboratory 3500; laboratory blender (Stomacher Model 3500; Tekmar Co., Cincinnati, Cincinnati, OH). Serial dilutions were prepared for pour plating. The LAB were enumerated enumerated using Rogosa agar (Difco Labora30·C withtories, Detroit, Detroit, MI) and incubated aat t 30'C of 3 out modified atmosphere atmosphere for a minimum of d. Yeasts were enumerated using potatodextrose agar (Difco Laboratories). Laboratories). IdentificaIdentification of LAB was performed on samples obtained throughout the trial using the APT rapid CHL kit (Analytab NY). (Analytab Products, Plainview, NY). Trial 2

Samples of unstored WBG were delivered directly directly from the brewery and were randomly assigned in a 4 x 6 factorial design with four of ensiladditive additive treatments and six durations of ing. The additive treatments ing. treatments were 1) control, 2) LAB (1174 LAB (1 174 silage inoculant; Pioneer Hi-Bred 1.1 x lo6 106 International, IA) at 1.1 International, Inc., Johnston, LA) cfu/g of WBG, 3) LAB plus cellulase and cfulg hemicellulase + hemicellulase enzymes (ENZ; (ENZ; Grasszyme A + Farrnline International, International, Schaumburg, Schaumburg, IL) n.,) at B; Farmline 1.2 x ld N g of WBG, or 4) propionic acid lOS ccfulg 1.2 (Feed Curb Endox; Kemin Industries, (Feed Industries, Inc., Des Moines, IA) at .5% of wet weight. Pioneer Moines, 1174 silage inoculant contained L. L. plantarum 1174 and Streptococcusfaecium. Streptococcus faecium. Grasszyme A + B contained Lactobacillus and Pediococcus Pediococcus baccontained teria plus cellulase, cellulase, xylanase, cellobiase, and glucose oxidase. The enzymes were applied as a mixture mllkg of of WBG. The mixture at a rate of .275 mVkg enzyme activity was cellulase, cellulase, 23,300 hydroxyethyl cellulose units/ml; xylanase, 5800 IU/ lUI ml; rnI; cellobiase, cellobiase, 55 IU/ml; and glucose oxidase, 83 IUlml. IU/ml. Treatments 2 and 3 were applied as 83 of WBG. In a liquid solution at 10 mlkg mllkg of treatment 3, the solution of of 10 mVkg mllkg WBG included .275 ml of the enzyme mixture. Nine kilograms kilograms of WBG were ensiled in triplicate 1, 2, 4, 6, 8, and 90 d of of for opening opening after 1, ensilage. Silos were 19-L buckets that were ensilage. double lined with plastic bags and stored at 16 double to 20'C. 20·C. Joumal 5, 1995 Journal of Dairy Science Vol. 78, No. No.5.

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SCHNEIDER ET AL. AL. SCHNEIDER

Trial 3 Trial

Samples Samples of unstored WBG were delivered directly randomly directly from from the brewery and were randomly assigned assigned in a 4 x 5 factorial factorial design with four additive treatments and five durations durations of ensilensiling. Treatments Treatments were ensiled in triplicate triplicate using double lined with plas19-L buckets buckets that were double tic bags, stored at 20·C, 2 0 T , and opened on d 10, 25, 40, 40, 55, and 75 of ensilage. ensilage. The additive additive treatments treatments were I) 1) control, control, 2) LAB at l.l 1.1 x I()6 106 cfulg WBG, 3) LAB LAB + ENZ at 1.2 1.2 x lOS Id cfu/g cNg propionic acid at .5% WBG, or 4) propionic .5% of wet weight. weight. Treatments 1 to 3 were applied as a solution at 10 m1Ikg mVkg of WBG. liquid solution Trial 4 Trial

Samples Samples of unstored WBG were delivered delivered directly directly from from the brewery and were randomly assigned in a 4 x 2 factorial factorial design with four assigned additive additive treatments treatments and two durations durations of ensilensiling. Treatments Treatments were ensiled in triplicate triplicate using ing. 19-L 19-L buckets buckets that were double double lined with plastic bags, stored stored at 20·C, 20'C. and opened on d 6 and ensilage. The additive additive treatments treatments were I) 1) 90 of ensilage. control, 2) BP pellets added at 15% 15% of wet weight, 3) 3) high moisture grain inoculant inoculant (lIMI) (HMI) applied at a rate of 10 mlJkg mVkg of WBG (2.0 (2.0 x applied 104 10" cfu/g c N g of WBG) WBG)and mixed prior to ensiling ensiling (1189 (1189 high moisture corn inoculant; inoculant; Pioneer 4) 15% 15% BP plus Hi-Bred International, International, Inc.), Inc.), or 4) HMI (BP + + HMI). The HMI contained contained L. L. plantarum and S. S. faecium faecium bacteria. Chemical Analysis Chemical

Chemical Chemical analysis analysis included included DM in a 60·C 60'C forced-air oven, oven, pH with a Corning Coming 155 155 pH/ion pWion forced-air meter (Corning (Coming Science Science Products, Products, Medfield, Medfield, MA) using a 1: 1:lI (wtIvol) (wt/vol) ratio of WBG to water, water, and VFA and lactic lactic acid by gas-liquid gas-liquid chromatography chromatography (Bulletin (Bulletin 749C; Supelco, Supelco, Inc., Bellefonte, Bellefonte, PA). PA). In addition, addition, WBG were anaanawater-soluble carbohydrates carbohydrates (7), 0, NNH3 H3 lyzed for water-soluble N (3), (3), NDF, and ADF using the method for forage fiber analysis analysis with Na2S03, forage Na2S03, and of Goering Goering and Van Soest (8), (8), and total N by macro-Kjeldahl macro-Kjeldahl (4). (4). Statistical Analysis

The model used for analysis analysis of data in trials 1 to 4 was Journal of Dairy Dairy Science Science Vol. 78. 78, No. 5, 1995 Journal No.5.

Yij = p

+ Ti + Dj + T,f

x DAj

+ Eij

where

mean, mean, treatment (i = I1 ... . . . n), = 11 ... . . . n), day (j(i = interaction between treatment treatment interaction and day, day, and Eij = error (10). (10). Ejj

p = tJ. = Tj Ti = = = Dj = Dj (T x D)jj D>ij = = (T

Data were analyzed by SAS SAS (9), (9), and the least squares squares means procedure was used to separate separate differences differences among among treatment means. means. Significance nificance was declared declared at P < .05 .05 unless otherotherwise noted. noted. RESULTS AND DISCUSSION DISCUSSION RESULTS

Trial 1 Trial

Yeasts and LAB LAB were measured during the first first 3 d of the experiment experiment to monitor fermenfermentation. The number of LAB in WBG on d 0 was <103
1099

ENSll..ED WET BREWERS GRAINS FERMENTATION OF ENSILED TABLE 1. The pH of of wet brewers grains (WBG) (WBO) ensiled (LAB) (trial with or without addition of lactic acid bacteria (LAEI) 1). 1).

Treatment' Treatment 1 Control

10' 1@ LAB

l()6 1Oa LAB

6.38 6.09· 6.091 5.58· 5.588 4.46" 4.46" 44.00c .w

pH PH 6.31 5.68b 5.68b 4.76b 4.76b 4.02b 4.02b 4.l8·b 4.181b

6.27 5.lOC 5.1OC 44.460 .w 33.82 . ac ~ 4.08bc 4.08"

(d) (6)

11 2 3 28 57

&.b.cMeans within a row with unlike superscripts superscripts differ 8~b.CMeans (P < .05). .05). (P l()6 cfdg cfulg of WBG; WBG; SEM = !LAB 1LAB applied at 10' 105 and 106 .0204. .0204.

was also significant significant for concentrations concentrations of laclactate, tate, acetate, acetate, and butyrate butyrate (Table 2). 2). The concentration centration of lactate increased increased from from d 2 to 28 for all treatments treatments but was considerably considerably lower by d 57 of fermentation. fermentation. The concentration concentration of lactate lactate significantly significantly increased increased with each each supsupplemental plemental amount amount of added LAB LAB for d 3 and 28 of fermentation. fermentation. The concentration concentration of aceacetate was negligible 3, after after which all negligible until d 3, treatments 57, the treatments increased. increased. By d 28 and 57, control control WBG had 80 to 100% 100% and 295% 295% more more acetate ()6 acetate than did the the 1 x 105 lo5 and I1 x I106 treatments, treatments, respectively. respectively. Butyrate concentraconcentration was minimal 28, when the control control minimal until d 28, had 1.32% 1.32% of DM, DM, and both LAB LAB treatments treatments contained contained ~.23%. <.23%.All treatments treatments had increased increased

of butyrate by d 57 of of fermentation; fermentation; content of 106 treatment contained the however, the 1 x lo6 least, 1 .23 versus 1.90 and 2.48% 1.23 2.48% of DM. The concentration concentration of of soluble carbohydrates carbohydrates (milligrams per gram of of DM) from d decreased (milligrams treatments: 61 to 10 for 3 to d 56 for all treatments: 18 for 1 x lo5 105 treatment, and 36 control, 35 to 18 to 19 19 for 1 1 x 106 106 treatment. The low concentrations concentrations of of acetate and butyrate for all treatments prior to d 28 indiof an undesirable fermentafermentacate the absence of satisfaction. Although all treatments were satisfactorily preserved through d 28, additional LAB fermentation than promoted a more efficient fermentation did the control, as indicated by a faster pH decline, higher concentration concentration of lactate, and decline, concentrations of acetate and butyrate. lower concentrations butyrate. Allen and Stevenson Stevenson (1) stated that the high temperature temperature during the brewing process process would not eliminate eliminate spore-forming spore-forming clostridia. clostridia. FurtherFurthermore, more, data of Allen and Stevenson Stevenson (1) showed showed that clostridia clostridia remained remained active active in WBG at a pH as low as as 3.9. The fermentation fermentation profile of as indicates longer periods periods indicates that WBG stored for longer (57 d) d) may undergo undergo an undesirable undesirable fermentafermentation, resulting resulting in large amounts amounts of butyric acid, acid, lactate, and increased increased pH. reduced lactate, Trial Trial 2

Trial Trial 2 was designed designed to look at fermentation fermentation changes changes of WBG under short-term short-term (up (up to 8 d) d) or long-term (90d) d) storage. storage. These two storage storage long-term (90 periods periods are are the most common common in Washington. Washington. The 30% DM The mean composition composition of WBG was 30%

TABLE TABLE 2. 2. Concentration Concentration of of lactic lactic acid acid and and VFA VFA in in wet wet brewers brewers grains grains (WBG) (WBG)ensiled ensiled with with or or without without added added lactic lactic acid acid bacteria bacteria (LAB) (LAB) (trial (trial 1). 1). Treatment! Treatment1

105 LAB LAB 10'

Control Control Lactate Lactate

Acetate Acetate

Butyrate Butyrate

Lactate Lactate

Butyrate

Lactate

Acetate Acetate

Butyrate

.06 .06 .02 .02 .oa .08 b .l2 .12b 1.90. 1.900 .062 .062

.3a .38 .78' .78· b 1.95b 1.95 b 2.37b 2.37 .3aa .38· .061 .061

.03 .03 .OS .08 .22 .22 b .57b .57 b .7Ob .70 .140 .140

.05 .05 .04 .04 .I4 .14 b .23b .23 b 1.23b 1.23 .062 .062

(% (% of of OM) DM)

(d) (6)

11 22 33 28 2a 57 57 SEM SEM

Acetate Acetate

l()6 106 LAB LAB

.45 .45 b .26 .26b .300 .30. .85· .8S .300 .30. .MI .061

.03 .03 .03 .03 .07 .07 I.14· 1.141 2.07& 2.078 .140 .140

.06 .06 .04 .04 .06 .06 1.32· 1.321 2.48· 2.488 .062 .062

.45 .45 b .35 .35b c 1.43 1.43C C 1.47 1.47= b .lob .10 ,061 .061

.03 .03 .03 .03 .26 .26 b .63 .63b b .70 .70b .140 ,140

•.b.cMeans 1.b.CMeans within within aa row. row, for for each each VFA VFA component component between between treatment, treatment, with with unlike unlike superscripts superscripts differ differ (f' (P << .05). .05). 10' and 1()6 cfulg of WBO. lLAB applied at 'LAB applied at 105 and I06 cfdg of WBG. Journal Journal of of Dairy Dairy Science Science Vol. Vol. 78, 78, No.5, No. 5, 1995 1995

1100 1100

SCHNEIDER SCHNEIDER ET ET AL, AL.

and 30% 30% CPo CP.Analysis Analysis of of the the propionic propionic acid acid and treatment at at dd 00 showed showed .88% .88% of of DM DM as as proprotreatment pionate, which which is is slightly slightly greater greater than than our our pionate, desired rate rate of of addition addition of of .5%. .5%. The The results results of of desired the pH pH and and VFA VFA analyses analyses are are presented presented in in the Table 3. 3. The The interaction interaction of of additive additive with with time time Table was significant significant for for pH, pH, lactate, lactate, and and acetate. acetate. was The concentration concentration of of butyrate butyrate (not (not presented) presented) The was negligible negligible throughout throughout the the 90-d 90-d study study for for was all treatments, treatments, indicating indicating that all all treatments treatments all were well well preserved. preserved. The The control control treatments treatments were the lowest lowest pH at at all all time time points points ~ Id 6; 6; had the propionic acid acid was was intermediate intermediate on d 11 and and 2. 2. propionic The treatments treatments with LAB LAB and and propionic propionic acid The the highest highest pH on d 4 and and 6. 6. After 90 90 d of had the fermentation, the LAB LAB + + ENZ treatment treatment had fermentation, the lowest lowest pH, pH, the LAB LAB treatment treatment and control the and the propionic propionic acid acid treathad the highest, and intermediate and not different from from ment was intermediate concentration of lactate was the control. The concentration control treatment treatment on d 1, 1, as as greater for the control expected from the lower pH. The LAB + + ENZ expected treatment had a significantly greater concentraconcentratreatment lactate than did the other treatments treatments on tion of lactate d 6 of fermentation. fermentation. In contrast, on d 8, 8, the treatment had the greatest propionic acid treatment amount of of lactate. Between d 8 and 90, the concentration concentration of lactate lactate decreased for all treatments by 40 to 83%. 83%. Acetate concentrations concentrations remained <1.2% of DM OM for all treatments <1.2% of through d 8. Results at 90 d indicated that the treated WBG contained significantly higher concentrations acetate than did the control, control, concentrations of acetate averaging averaging 68% higher. Except for the LAB + + ENZ treatment on d 90, the pH, lactate, and acetate data suggest that, in this study, use of of additives had no beneficial effects over those of of the control. control. This treatment had a significantly lower pH on d 90 than did the control and an equal concentration concentration of of lactate. Allen and Stevenson (1) (I) packed WBG into 160-ml test tube silos and stored them for 90 d. Their 90-d control treatment had a higher pH and butyrate butyrate content than our control and a lower concentration of of lactate. The amount of of acetate at 90 d for control treatments in our study was simiIar that of of Allen and Stevenson (1). No No similar to that explanation is evident for the response response of of the control treatment treatment in our study; however, however, Allen Allen and and Stevenson Stevenson (1) (1) contended contended that that sufficient sufficient LAB LAB are are inherently inherently present present to to initiate initiate the the priprimary mary fermentation. fermentation. The of length length of of storage storage on on NDF, NDF, The effect effect of ADF, ADF, soluble soluble carbohydrates, carbohydrates, and and NH3 NH3 N N conconJournal airy Science 5, 1995 Journal of of D Dairy Science Vol. Vol. 78, 78, No. No,S, 1995

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1101 1101

FERMENTATION FERMENTATION OF OF ENSILED ENSILED WET WET BREWERS BREWERS GRAINS GRAINS

tent of WBG is is presented presented in in Table Table 4. 4. The The tent concentration of NDF decreased decreased from from d 8 to concentration 90 of fermentation fermentation in all all treatments treatments except except 90 propionic acid; acid; the LAB LAB + + ENZ treatment treatment was was propionic significantly lower lower in NDF than than control and significantly propionic treatments treatments on d 90. 90. Length Length of storstorpropionic age had a minimal minimal effect effect on concentrations concentrations of age ADF; the propionic propionic acid treatment treatment contained contained ADF; significantly more more ADF on d 90 90 than than did the significantly control. An interaction interaction of additive additive and time time control. was observed observed for for soluble soluble carbohydrates carbohydrates and was NH3 N. The The amount amount of soluble soluble carbohydrates carbohydrates NH3 decreased substantially substantially for for all treatments treatments durdurdecreased fermentation period, and the LAB LAB ing the 90-d fermentation + ENZ treatment treatment contained contained significantly significantly more + soluble carbohydrates carbohydrates on d 90 90 than did the soluble decrease in NDF and an treatments. A decrease other treatments. increase in soluble soluble carbohydrates carbohydrates would be increase expected because because the enzymes enzymes used in Trial Trial 2 expected specific for structural structural carbohydrates. carbohydrates. The were specific concentration of NH3 N H 3 N increased increased in all all treatconcentration ments from from d 8 to 90, 90,and the LAB LAB treatment treatment ments N H 3 N on d 90. 90. The contained the most NH3 contained H 3 N present at d 90 was small small NH3 amount of N significance. and probably of little biological significance. fermentation data of trial 2 indicate that a The fermentation fermentation occurred occurred between 8 and shift in fermentation storage. Evidence Evidence of this was the 90 d of storage. reduction in increase in pH and acetate and the reduction lactate. lactate. Stevenson (1) suggested suggested that a Allen and Stevenson decrease in number of LAB and production production of decrease lactic acid after several several days of fermentation fermentation of lactic deficiency of carbohycarbohyWBG may be due to a deficiency drate or to low pH. In our study, study, the LAB + + ENZ treatment resulted in the lowest pH and

the greatest greatest content content of residual residual soluble soluble carbocarbothe hydrates at d 90. 90. This This result supports supports the the thethehydrates proposed by Allen and Stevenson Stevenson (1) (1) that ory proposed carbohydrate may limit the the extent of fermentafermentacarbohydrate during long-term long-term storage storage of WBG. WBG. tion during Trial 3 Trial

Trial Trial 33 was was designed designed to to determine determine at what 90 d of fermentation fermentation point between 8 and 90 (short-term (short-term and long-term long-term storage) storage) the the shift shift in fermentation that was was observed observed in trial trial 2 would fermentation occur. The mean composition composition of WBG was occur. 18.5% DM and 34% CPo CP. The data of pH, 18.5% lactate, acetate, acetate, NH3 N H 3 N, and soluble soluble carbohycarbohylactate, 5 . Interaction Interaction of drate are presented in Table 5. drate additive time was was significant significant for for pH, lactate, lactate, additive x time acetate, NH3 N H 3 N, and soluble soluble carbohydrates. carbohydrates. acetate, declined through through d 55 for for all treattreatThe pH declined ments ments and increased increased slightly slightly by d 75. 75. All treatments resulted resulted in a lower pH at each time treatments control, except for for the propoint than did the control, treatment on d 10. 10. The concentraconcentrapionic acid treatment lactate was greater greater in treated WBG than tion of lactate the control control on d 25, 40, and 55 of fermentafermentation. propionic acid 75 d of storage, storage, the propionic tion. After 75 treatment had the greatest content of lactate. lactate. treatment No differences differences occurred in the concentration concentration of acetate on d 10 10 and 25; however, acetate however, during the remainder of the study, the LAB + + ENZ treattreatment had the highest acetate. acetate. Butyrate was was negligible (not presented) treatments. negligible presented) for all treatments. concentration of NH3 N H 3 N increased increased from from an The concentration average of .02 .02 to .04% of DM from from d 10 10 to 75; 75; average + ENZ treatment contained the most the LAB + by d 75. The treatments treatments with LAB + + ENZ and propionic acid had significantly more soluble

TABLE 4. Chemical composition of wet brewers grains (WBG) ensiled during short-term and long-term long-term storage (trial 2). NDF Treatment 1 Treatment1

d 8

d 90 d90

ADF

dd90 90

d 8

d 8

d 9900

SEM

63.44 65.17 61.43 63.32

62.09be 62.09b 59.97 cd 59.91Cd 55.89d 55.89d 65.431b 65.43ab 1.602 1.602

26.3 26.3 26.9 26.9 27.1

19.9 19.9" 23.2k 23.2be 20.8be 20.8b 25.6.b 25.61b 1.80 1.80

d 8

d 90

- (mg/g (mglg of Dh4) DM) -

(% of DM)

Control LAB LAB + ENZ Propionic acid

SC2 sc2

N H3 N NH3

.07cd .07Cd ,090 .091 .08be .08k .06d

.03 .03 .02 .02 .03 .003

7.1 b 7.1b .6 b .6b 19.41 19.4' 7.0b 7.0b

25.10 25.1 1 7.8b 7.8 b 226.oa 60 27.21 27.2a 2.40

a.b.C.dMeans l.b.c.dMeans within a column with unlie unlike superscripts differ (P (J' < .05). 'LAB hemicelluJase enzymes. enzymes. lLAB = Lactic acid bacteria; ENZ = cellulase and hemicellulase 2Soluble 2Soluble carbohydrates. carbohydrates. Journal of airy Science Vol. 78, No. 5, 1995 of D Dairy No.5,

1102

o

N

TABLE 5. Chemical composition of wet brewers grains (WBG) ensiled with or without addition of lactic acid bacteria (LAB), LAB plus enzymes (ENZ),I or propionic acid (trial 3). Treatment

pH

Lactate2 Acetate

Control LAB LAB + ENZ Propionic acid SEM

3.62" 3.45b 3.39b 3.58" .017

1.{)6l 1.13"

Control LAB3 LAB + ENZ Propionic acid SEM

3.22" 3.11 b 3.03° 3.05< .02

2.21 b 3.00a 3.25" 3.16" .12

NH3 N

SC3

pH

Lactate

.024" .02Ob .015° .025" .0013

56.02° 59.61 c 78.14b 100.84" 3.104

3.42" 3.32b 3.16d 3.23c .017

l.44d

.026b .025b .036" .033.0013

14.71 c 23.85b 38.49& 39.09& 3.10

3.33" 3.2Ob 3.23b 3.Q9C .02

d 10

.99" .63b .120

.19 .15 .28 .33 .252

.21 b 1.92"

.94b .25

NH3 N

SC

pH

Lactate

.033b .034b .041" .038" .0013

28.9Qd 42.17° 55.99b 58.91" 3.104

3.36" 3.26b 3.17° 3.22b .02

2.64b 3.18" 2.95" 3.25" .12

1.97° 2.22bc 2.31 ab .120

.11 .24 .57 .60 .252

.033b .036b

20.75b 15.97b 38.40& 33.73" 3.10

d 75

l,b,c,dMeans within a column with unlike superscripts differ

cr < .05).

leeUulase and hemicellulase enzymes. 2Lactate, acetate, and NH 3 N are expressed as percentages of DM. 3SC = Soluble carl>ohydrates, milligrams per gram of DM.

l.72b 1.9Qb 1.78b 2.84" .12

1.55b 2.03b 3.47"

Acetate

NH3 N

SC

.028° .0290 .031 bc .034ab .0013

13.75b 21.38b

d40

d 25

d 55

.44b

Acetate

l.48b

.048" .033b

.25

.0013

.I9b .4Qb

1.70& .68b .25

48.72" 40.38" 3.10

SCHNEIDER ET AL.

Journal of Dairy Science Vol. 78, No. 5, 1995

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1103 1103

FERMENTATION OF OF ENSn..ED ENSILED WET WET BREWERS BREWERS GRAINS GRAINS FERMENTATION

carbohydrates on on dd 10 10 than than did did the the other other treattreatcarbohydrates ments, and and this this trend remained remained throughout throughout the the ments, study. The The concentration concentration of soluble soluble carbohycarbohystudy. drates tended tended to to decrease decrease through through dd 40 40 and and drates ENZ decreased through through dd 55 55 with the the LAB LAB ++ ENZ decreased treatment. Because Because the the treated treated WBG had aa treatment. lower pH and and higher higher lactate lactate concentration concentration lower throughout the the experiment, experiment, results results indicate indicate a throughout preserved product product than than the the control. control. better preserved The results results of trial 3 would indicate indicate that the the The shift in fermentation fermentation of stored stored WBG occurred occurred shift somewhere after d 55. 55. pH and and acetate acetate insomewhere creased, and lactate lactate decreased, decreased, between between d 55 creased, 75 of storage. storage. Although Although no microbial microbial anaanaand 75 lyses were conducted. conducted, the fermentation fermentation profile profile would suggest suggest that, that, after after the the initial initial fermentafermentawould tion, the the microbial microbial population population shifted shifted away away tion, from that of a homofermentative homofermentative LAB. LAB. These These from data would suggest suggest that storage storage :S;2 52 mo would data desirable. be desirable. Trial 4 Trial

pellets were used in trial 4 bebeBeet pulp pellets cause shredded shredded BP is not commonly available cause has in Washington, Washington, and previous previous research research (11) has conducted with shredded shredded BP. The conconbeen conducted preensiled centration of NDF measured in preensiled samples averaged averaged 66% of OM DM for inoculated inoculated samples treatments and 56% 56% of OM DM for treatments treatments contreatments DM content content (Table 6) was taining BP. The OM significantly higher at all times for the BP treatments than for control control or HMI treatments. treatments The concentration concentration of OM DM decreased decreased an average 7% for control and HMI and 4.6% for the of 7% BP treatments of fermentation. fermentation. The treatments by d 90 of level of of of CP (Table 6) decreased an average of 24% with the addition addition of BP. The lower con24%

centrations NDF and and CP CP and and greater greater OM DM in in centrations of of NDF the treatments treatments containing containing BP BP are are due due to to the the the lower NOF NDF and and CP CP and and greater greater OM DM content content of of lower the the BP. BP. The The interaction interaction of of additive additive and and time time was was significant significant for for OM DM and and CPo CP. By dd 90, 90, the the concentration concentration of of CP CP had increased increased slightly slightly for for the the control control and and HMI HMI treatments treatments but remained remained the same same for for the the BP treatments, treatments, resulting in an an the average of 31 31% less CP CP in the the BP treatments treatments average % less control or HMI HMI treatments treatments on on d 90. 90. than in control The The pH, pH, lactate, lactate, NH3 N H 3 N, and and soluble soluble carbocarbohydrate hydrate data data are are presented presented in Table Table 7. 7. The The significant between additive additive interaction was significant for pH. pH, acetate, acetate, NH3 N H 3 N, and soluble soluble and time for carbohydrates. All treatments treatments underwent underwent extenextencarbohydrates. sive fermentation fermentation by d 6 of storage, storage, resulting resulting sive low pH; pH; however, however, the the HMI HMI treatment treatment in low resulted in the the lowest lowest pH, pH, 3.49. 3.49. The The treatments treatments resulted containing containing BP had the lowest lowest pH on d 90; 90; BP + HMI had the lowest, lowest, pH 3.62, and HMI had the highest, pH 4.22. The control control and BP + + treatments had the greatest concentration concentration HMI treatments lactate on d 6, 6, and, and, after after 90 90 d of storage. storage, the of lactate + HMI treatment treatment still still had the greatest greatest BP + concentration concentration of lactate lactate (1.51% (1.51% of OM). DM). The concentration concentration of acetate was low for all treattreatments ments on d 6. 6, but, by d 90, 90, the acetate acetate had 5-, 12-, 12-, and 2-fold for the increased by 6-, 6-, 5-, increased control, BP, and BP + + HMI treatments, HMI, control, treatments containing containing BP had respectively. The treatments concentrations of N H3 N NH3 significantly lower concentrations than did the control at d 6 and 90; 90; BP + + HMI had the least on d 90. 90. The treatments treatments containcontainsoluble carbohycarbohying BP had significantly more soluble drates on d 6 and 90 than did the control control and of HMI treatments. The greater amount of carbohydrates was related to residual soluble carbohydrates the lower pH of of these treatments treatments and likely provided additional sugar for the LAB.

TABLE 6. Change in concentration of DM n wet brewers OM and CP i in brewers grains grains (WBG), (WBG), ensiled beet pulp (SP), (BP), high moisture inoculant (HMI), (HMI), or BP + + HMI (trial 4). 4).

DM OM Treatment

d 6

Control BP HMI BP + + HMI SEM

29.5OC 40.281 40.28" 29.03C 29.03c 38.12b 38.12b

CP d90 d 90

d 6

27.42b 27.42b 37.21. 37.21" 26.97b 26.97b 37.631 37.63"

29.081 29.08" 21.63b 21.63b 29.151 29.15" 22.45b 22.45b

(% of of DM) OM) (96

(%)

.192

d 90 31.87a 31.87" 22.86b 22.86b 32.361 32.36" 22.44b 22.44b .344

cb.cMeans P < .06 for DM; a.b.CMeans within a column with unlike superscripts superscripts differ (if' OM; P < .05 for CP). loumal of of Dairy Science Vol. 78, No. No.5, Journal 5, 1995

SCHNEIDER SCHNEIDER ET ET AL. AL.

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Journal airy Science Journal of of D Dairy Science Vol. Vol. 78, 78, No. No.5, 1995

Results Results from from trial trial I1 clearly clearly show show the the benefibeneficial cial effects effects of LAB LAB inoculants inoculants on on fennentation fermentation characteristics characteristics of WBG, as as indicated indicated by a faster faster pH decline, decline, increased increased concentration concentration of lactate, lactate, and reduced reduced concentrations concentrations of acetate acetate and butyrate butyrate than the control. control. The data also also show show that use of more LAB, LAB, I1 x 1()6 106 versus versus 1 x lOS, 1 6 , further further improves improves the fennentation fermentation of ensiled WBG,as as evidenced evidenced by a lower pH and ensiled WBG, a higher concentration concentration of lactate. lactate. Results Results from from trial 3 showed showed that the use of LAB, LAB, LAB LAB + ENZ, propionic acid significantly ENZ, or propionic significantly increased increased the initial production production of lactate and enhanced enhanced the rate of pH decline, resulting in a higher decline, resulting concentration quality preserved product. The concentration acetate, although not different from that for of acetate, control, was significantly significantly decreased by treatments with LAB and propionic acid compared treatment, with effects of the LAB + ENZ treatment, suggesting a more efficient fermentation. fennentation. The suggesting of LAB limited the production of of butyrate use of of fermentation. fennentation. The in trial 1I through 28 d of of inoculants are inconclusive for trial benefits of of the exceptionally exceptionally well-preserved well-preserved 2 because of treatment. Results from trial 4 indicate control treatment. short-tenn no major benefit from HMI for short-term of WBG. However, the use of of HMI + storage of BP pellets resulted in a lower pH, acetate, and of lactic acid NH3 N and increased content of long-tenn storage. under long-term of WBG is a viable option for Ensilage of dairy producers who want to store WBG for dairy of propionic acid, extended periods. The use of inoculants, or BP in conjunction with proper of the silo is beneficial in promoting sealing of more efficient fermentation fennentation during long-term long-tenn storage. REFERENCES REFERENCES

Allen, W. W. R., R., and and K. K. R. R. Stevenson. Stevenson. 1975. 1975. Influence Influence of of 1I Allen, on thc the ensiling ensiling process process of of wet wet brewers’ brewers' additives on additives Can. J. 1. Anim. Anim. Sci. Sci. 55:391. 55:391. grains. Can. grains. 2 Allen, W. W. R., R., K. K. R. R. Stevenson, Stevenson, and and J.1. G. G. BuchananBuchanan2Allen. Smith. 1975. 1975. Influence Influence of of additives additives on on short-term short-tenn Smith. uncopreservation of of wet wet brewers brewers grains grains stored stored iin n uncopreservation Anim. Sci. Sci. 555:609. vered piles. piles. Can. Can. J.J. Anim. vered 5609.

FERMENTATION OF OF ENSILED ENSILED WET WET BREWERS BREWERS GRAINS GRAINS FERMENTATION Official Analytical Analytical Chemists. Chemists. 1975. 1975. 33Association Association of Official Official Method Method of Analysis. Analysis. 12th 12th ed. ed. AOAC, AOAC. Official DC. Washington, DC. Washington, official Analytical Analytical Chemists. Chemists. 1980. 1980. 44Association Association of Official Official Methods Methods of Analysis. Analysis. 13th 13th ed. ed. AOAC, AOAC. Official Washington, DC. DC. Washington, 5 Birkelo, Birkelo, C. C. P., and D. D. Sorenson. Sofenson. 1991. 1991. Effect Effect of inoculants on high moisture com corn fennentation fermentation characcharacinoculants performance. Page 35 35 in in South South teristics and cattle cattle performance. teristics BmkDakota Beef Rep. Rep. South South Dakota Dakota State Univ., BrookDakota ings. ings. R., and J.J. Combellas. Combellas. 1983. 1983. A A note note on preserpreser66 Dixon, R.• grains. Trop. Trop. Anim. Prod. Prod. 8: 8: brewers grains. vation of wet brewers 151. 151. M.. K. K.A. A. Gilles, Gilles, J. Hamilton. Hamilton, P. P. Rebers, Rebers, and and 77 Dubois, M., F. Smith. Smith. 1965. 1%5. Colorimetric Colorimetric method for for determinadeterminaF. sugars and and related related substances. substances. Anal. Anal. Chern. Chem. 28: 28: tion of sugars 350. 350.

1105 1105

88GoeMg. Goering, H. K.. and and P. P. J.J. Van Van Soest. Soest. 1970. 1970. Forage Forage H. K., Fiber Fiber Analyses Analyses (Apparatus, (Apparatus. Reagents, Reagents, Procedures, Procedures, and and Some Some Applications). Applications). USDA USDA Agric. Agric. Handbook Handbook 379. 379. ARS-USDA, DC. ARS-USDA. Washington, Washington, DC. 99SAS/STAT@ SAS/STAT~ Users Users Guide: Guide: Statistics Statistics (Release (Release 6.04). 6.04). 1987. 1987. SAS SAS lnst., Inst., Inc., Cary, Cary. NC. NC. 10 Steel, R.G.D., and J. losteel, J. H. Torrie. Tome. 1980. 1980. Principles Principles and Procedures proctdurcs of Statistics: Statistics: A A Biometrical Biometrical Approach. Approach. 2nd ed. ed. McGraw-Hill McGraw-Hill Book Co., Co., New York, NY. NY. U 11 Trident Trident Feeds. Feeds. 1990. 1990. Molassed Molassed sugar beet beet feed feed ensiled ensiled with draff -- the the ideal ideal combination combination for for improved improved performance, performance, Tech. Tech. update. Trident Feeds. Feeds, PeterPeterborough, borough, England. England. 12 Wardynski, F., F.. S. S. Rust, and M. M. Yokoyama. Yokoyama. 1993. 1993. 12Wardynski. Effect of microbial microbial inoculation inoculation of high moisture moisture com corn on fennentation fermentation characteristics, characteristics, aerobic aerobic stability, stability, and cattle performance.J. Anim. Anim. Sci. Sci. 71:2246. 71:2246. cattle performance.

of Dairy Science Vol. 78, No. No.5, Journal of 5. 1995