STORAGE TRIALS WITH WET BREWERS’ GRAINS Thesis Ior Hm Degree oI M. S. MICHIGAN STATE UNIVERSITY TeImo B. OIeas I977 ._ IIII'? :I‘Qt' 1- F . '\ \N 6/0203] ABSTRACT STORAGE TRIALS WITH WET BREWERS' GRAINS By Telmo B. Oleas Studies were conducted during the summer to determine how best to store and preserve wet brewers' grains. Several methods of preservation were studied. Thirty pounds of wet brewers' grains were stored for 32 and 76 days in plastic buckets. Complete preservation was achieved by sealing with plastic foam. Mixing the grain with yeast (10%) decreased spoilage. There was formation of acetic, propionic and butyric acid and changes in the protein fraction. In a second experi- ment, 300 pounds of wet brewers' grains were stored for 32 and 60 days in steel barrels. Covering the grains with a plastic bag filled with water or adding 2% propionic acid or 1.4% I formic acid plus 0.1% paraformaldehyde resulted in complete preservation. Ethanol and lactic acid were the main fermen— tation products. Wet brewers' grains can be stored success- fully under anaerobic conditions, by adding pr0pionic acid or by adding formic acid and paraformaldehyde. STORAGE TRIALS WITH WET BREWERS' GRAINS By Telmo B. Oleas A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy Science 1977 ACKN OWLEDG MEN TS The author wishes to express his most sincere gratitude to his major professor, Dr. Robert M. Cook, whose continuous academic guidance and personal support made the present work possible. He also wishes to thank Dr. J. William Thomas for his assistance, helpful ideas and valuable criticism. Appreciation is extended to Mr. Nirmal K. Sinha from the Department of Food Science, Michigan State University, for the microbiological work and to Ms. Laurie J. Allison, Ms. Mary T. Araiza, Ms. Sherry L. Sholts and Mr. Edgar O. Bautista for their skillful technical assistance. The author's graduate studies at Michigan State University were financed by the Swiss Technical Mission in Ecuador and by the "Instituto Nacional de Investigaciones Agropecuarias" (INIAP). The interest and encouragement on the author's work and academic advance expressed by Dr. Toni Rihs and Mr. Jean Th. Spiro of the Swiss Mission and by Dr. Enrique Ampuero P., General Director of INIAP, are very much appreciated. The author wishes finally to give thanks to Dr. Kim A. Wilson from the Dairy Science Department of Michigan State University for personal support and friendship during his stay in East Lansing. ii TABLE OF CONTENTS Page LIST OF TABLES . ......................................... v LIST OF FIGURES .............................. I I I I I I I I I I I ix INTRODUCTION ......................................... ... 1 LITERATURE REVIEW . ..................................... . 3 Brewers' grains used as animal feed .. .............. .. Poultry . .......... . ............................... 6 Swine . . .......................................... . 7 Beef and dairy ........... ..................... .... 8 Preservation of brewers' grains . ..................... 10 MATERIALS AND METHODS . ........................... . ..... . 15 Trial 1 I 000000000000000000 I 00000000000000 I ..... I I I I I I 15 Trial 2.. ............................................ 18 Analytical methods ............................. ..... 19 RESULTS AND DISCUSSION .............................. .... 23 Trial 1 I I ooooooooooooooooooooooooooooooooo I I I I I I I I I I 23 Spoilage . ...................................... . . 23 pH I oooooooo I I I I I I I I I oooooooooooooooooooooooooooo I 26 Temperature I 000000000000 I I I I I' I I I I IIIIIIII I I I I I 32 Dry matter and protein . ................... . 39 Acid detergent fiber and acid de tergent insoluble nitrogen ... .......... . ........... ... 39 EthanOl I I I I I ooooooo I ...... I I I I I I I I IIIIIIIIIIIII I I “'2 Acetic acid . ..... . ............................. . . 48 Propionic acid ..... . .................. ........... 57 Butyric acid ...... ...... ............ ........ ..... 63 Lactic acid ............................... ..... .. 68 Ammonia ................................ ..... ..... 74 Correlations among measured constituents ......... 78 TrialZIIooIIIIIoIIII IIIIIIIIIIII I IIIIIIIIIIII IIIIII 90 Recovery ................. ..................... ... 90 pH .................... ...... ....... ...... ........ 95 Temperature ...... ..... ........................... 97 Dry matter and protein ..... ....... ........... 102 Acid detergent fiber and acid detergent insoluble nitrogen ............................ 105 Ethanol .... ...... . ........................... .... 108 Acetic acid ..... ....... . .......... ............... 110 Propionic acid .. ....... . ..... ..... ............. .. 110 Butyric acid ..... ........ . .................... ... 112 Lactic acid .................. ......... . ....... ... 112 Ammonia nitrogen ......................... ..... ... 116 Microbiological examination ...................... 118 Correlations among measured constituents in Trial 2 ......................... ........ ... 122 Summary of Trial 2 ......... ...... . ..... .......... 128 Comparison of Trial 1 with Trial 2 . ............ ..... 130 Recovery .......... ............................ ... 130 Fermentation pattern ...... ................. ...... 131 Type of additive ..... .......... .... ......... ..... 132 Type of silo .................. ............... .... 134 Practical considerations ................... ..... .... 136 CONCLUSIONS ..................... ............. . ....... .. 138 LITERATURE CITED ................... ...... ... ..... ...... 139 iv Table 10 11 12 LIST OF TABLES Nutrient composition of brewers' grains, corn silage, corn and soybean meal (% DM) List of additives or method used to ensile wet brewers' grains. Trial 1 ....... List of treatments used to ensile wet brewers' grains. Trial 2 .................. Percentage of spoiled material after ensiling wet brewers' grains. Trial 1 ... Changes in pH with time in wet brewers' grains ensiled with different additives. Trial]-.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Analysis of variance of the pH of ensiled brewers' grains. Trial 1 ................... Changes in temperature (F) with time in wet brewers' grains ensiled with different additives. Trial 1 ......................... Analysis of variance of the temperature of ensiled brewers' grains. Trial 1 .......... Dry matter and protein content of ensiled wet brewers' grains. Trial 1 .............. Acid Detergent Fiber (ADF) and Acid Detergent insoluble Nitrogen (ADN) content of ensiled wet brewers' grains. Trial 1 ............ Changes in ethanol concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial]-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Analysis of variance of ethanol content of ensiled wet brewers' grains. Trial 1 .... Page 5 17 20 24 27 29 33 36 40 43 45 47 Table 13 14 15 16 17 18 19 20 21 22 23 24 Changes in acetic acid concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial 1 Analysis of variance of acetic acid content of wet brewers' grains. Changes in propionic acid concentration (percent on a wet basis) with time in wet Trial 1 brewers' grains ensiled with different additives. TriallIIIIIIIIIIIII Analysis of variance of propionic acid content of wet brewers' grains. Changes in butyric acid concentration (percent in a wet basis) with time in 'wet brewers' grains ensiled with different additives. Analysis of variance of butyric acid Trial 1 content of wet brewers' grains. Changes in lactic acid concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Analysis of variance of lactic acid Trial 1 Trial Trial content of ensiled wet brewers' grains. Trial 1 .................. Change in ammonia concentration (mg nitrogen/100 g wet grains) with time in wet brewers' grains ensiled with different additives. Trial 1 Analysis of variance of ammoniacal nitrogen content of ensiled brewers' grains. Average spoilage, pH, temperature and TriallIIIIIIII composition of wet brewers' grains ensiled for 32 and 76 days ...... ..... ... Correlation coefficients among measured constituents of ensiled wet brewers' grains. vi Trial 1 Page 52 54 6O 62 64 69 72 75 76 81 83 85 Table 25 26 27 28 29 3O 31 32 33 34 35 36 Recovery of ensiled wet brewers' grains. Trialz.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII...‘ Changes in pH with time in wet brewers' grains ensiled with different additives. Trialz.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII... Changes in temperature (F) with time in wet brewers' grains stored with different additives or methods of ensiling. Trial 2 ..... Analysis of variance of temperatures of ensiled wet brewers' grains. Trial 2 .. ..... ... Dry matter and protein content of ensiled wet brewers' grains. Trial 2 .................. Acid Detergent Fiber (ADF) and Acid Detergent insoluble Nitrogen (ADN) content of ensiled wet brewers' grains. Trial 2 .................. Changes in ethanol concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial 2 ..... Changes in acetic acid concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial 2 ........................................ Changes in propionic acid concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial 2 ............................ Changes in butyric acid concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial 2 . ...................... ..... Changes in lactic acid concentration (percent on a wet basis) with time in wet brewers' grains ensiled with different additives. Trial 2 ... ....... ........ Changes in ammonia concentration (mg nitrogen/100 g wet grain) with time in wet brewers' grains ensiled with different additives. Trial 2 .. ....... .... vii Page 91 96 98 101 103 106 109 111 113 114 115 117 Table 37 38 39 Microbiological tests on wet brewers' grains ensiled during 60 days (micro- organisms per gram of ensiled wet brewers' grains). Trial 2 ........ Average recovery, pH, temperature and composition of wet brewers' grains ensiled for 32 and 60 days. Trial 2 .... Correlation coefficients among measured constituents of ensiled wet brewers' grains. Trial 2 ............. viii Page LIST OF FIGURES Figure Page 1 Change in pH of wet brewers' grains ensiled with or without yeast. Trial 1 ................. 31 2 Change in temperature of wet brewers' grains ensiled with or without yeast. Trial 1 .. ..... .. 38 3 Change in ethanol content of wet brewers' grains ensiled with or without yeast. Trial].IIIIIIIIIIIIIIIIIIIIICIIIIIIIIIII IIIII II. 50 4 Change in acetic acid content of wet brewers' grains ensiled with or without yeast. Trial 1 ................ ..... ....... .......... ... 56 5 Change in propionic acid content of wet brewers' grains ensiled with or without yeast. TriallIIIIIIIIIIIIIIIIIIII IIIIIIIIII III 59 6 Change in butyric acid content of wet brewers' grains ensiled with or without yeast. Trial 1 ........................ ..... .... 67 7 Change in lactic acid content of wet brewers' grains ensiled with or without yeast. Trial 1 ...................... ........ ... 71 8 Change in ammoniacal nitrogen content of wet brewers' grains ensiled with or without yeast. Trial 1 ......................... 80 ix INTRODUCTION By-products from the processing of various plant materials used in the manufacture of products for human consumption can be used for livestock feeds. In this practice these products are not wasted but rather transformed to a superior kind of food suitable for human consumption. Brewers' grains are a product of the beer industry that can be incorporated in sig— nificant pr0portions in the diets of animals, particularly ruminants. The production of this grain is greatest in spring and summer, when pastures are green, and least in fall and winter when demand for feed is great. Consequently, market- ing wet brewers' grains as livestock feed presents a problem, especially during summer. Wet brewers' grains can be dried to about 10% moisture which assures a stable, storable product. However, this drying process requires considerable energy because of the high water content. Also, there is a pollution problem caused by dust, odor, smoke, etc. On the other hand, the fresh product sold now by the breweries spoils in less than a week if not stored properly. Spoiled grains can cause serious health problems when fed to animals. A practical way to store wet brewers' grains on the farm needs to be found. 2 The objectives of this research were: (1) to study methods of storage of wet brewers' grains using small model silos, and (2) to describe the chemical and nutritional changes that occur during storage. LITERATURE REVIEW The National Academy of Sciences (1971) defined brewers' grains as the coarse, insoluble residue from brewed malt, and classified them as protein supplements. During 1973 there were 138,445,000 barrels of beer produced in the United States (World Beer Production: 1971-1974). Brewers dried grains production during the same year was 348,000 tons (U.S.D.A. Agricultural Statistics, 1975). This figure does not account for brewers' grains sold on a wet basis, estimated at 37% of the total production (Hunt, 1969). Therefore, the total pro- duction of brewers' grains on a dry basis was about 552,000 tons. Brewers' grains result from a process that involves solubilization and isolation of part of the starch from barley. Barley contains little or no amylase in the ripe seed. Thus, for making beer the cereal is allowed to germinate to synthe- size enzymes and then dried and stored until needed. Such germinated dried barley is known as malt. During germination the starch in the malt is only slightly hydrolysed since it is physically protected from amylase action by the cellular structure in the seed. Accordingly, the first step in brewing is the grinding of the malt and its suspension in water so as to permit hydrolysis of the starch. After saccharification 3 4 has reached the desired stage the mixture is boiled to stop further enzymatic action, then filtered. The solid wet filtrate is called brewers' grains (Stainer, 1970). HOpS are added to the malt liquor to give beer a bitter flavor and later filtered. The filtered hops are mixed with the brewers' grains in a proportion of about 3% following pressing the wet material to reduce moisture. The grains can be dried in a rotatory oven to about 90% dry matter. This product is stable. The composition of brewers' grains compared with corn silage, corn and soybean meal is listed in Table l. Brewers' grains are relatively high in protein, they have about three times as much digestible protein as corn, but a much lower energy content. Digestible and metabolizable energy values are comparable to those in corn silage. Potassium content is very low. This low potassium level results from the high solubility of potassium salts in the malt and they remain in the filtrate. Barley contains 0.52% potassium, wort sediment 0.90%, yeast 1.96% and beer 0.62%, on dry matter basis (Pomeranz and Dikeman, 1976). Nitrogen free extract in brewers' grains consists mainly of pentosans. They make up 25.2% of the dry matter since most of the starch from the barley grain has been hydrolyzed to glucose and removed in the brew liquid (National Academy of Sciences, 1971). 5 Table 1. Nutrient Composition of Brewers' Grains, Corn Silage, Corn and Soybean Meal (% DM).a 335:2. com 81:2?“ Ref. No. 5-02-141 3-02—822 4-02—879 5-04-600 Ash 4. 2 5 .7 1 .6 6 .7 Crude fiber 16.1 21.6 2.4 6.8 Ether extract 7'2 3'1 “'7 5'2 N_free extract 44.2 58.5 80.3 34.6 Protein 28.3 8.2 10.9 46.7 12351.22?“ 3'8 7'5 39” T D N (Cattle) 66.3 67.6 88.8 84.9 Calcium 0.30 0.50 0.05 0.31 Phosphorus 0.53 0.20 0.35 0.65 Sodium 0.28 -—-- 0.34 0.27 Potassium 0.10 0.88 0.80 1.93 figaiaggle 2.92 2.98 3.92 3.74 figaiafigle 2.40 2.44 3.21 3.07 a National Academy of Sciences, 1971 6 Brewers' grains used as animal feed. Brewers' grains are used as feed for several species of animals. They are a good source of protein. The relatively high crude fiber (16%) is characteristic of roughages. Poultry. Both dried and wet brewers' grains have been used successfully in poultry rations. Laying hens fed brewers' dried grains at levels of 5 and 10% of the diet did not have a significant difference in feed intake or in body weight gain or in the number or weight of eggs when compared to the hens consuming a commerical ration. The diets had 2500 to 2800 cal/g metabolizable energy and crude protein was 17 to 18% (Laurent and Vanssay, 1971). However, others have reported that addition of 10% brewers dried grains plus yeast to a corn-soybean meal diet resulted in an increase in egg weights and egg numbers: interior egg quality was also improved (Eldred gt a1., 1975). Levels of brewers' grains as high as 20% of the total ration have been suggested for laying hens (Couch, 1976). An experiment was conducted with starters (0 to 8 weeks) and growers (8 to 18 weeks) of a commercial egg producing strain of chickens. For Optimal performance the diet should not exceed 10% brewers dried grains for starters or 30% for growers. These experimental rations, with and without brewers' grains had about 22% protein and similar energy contents (Ademosun, 1973). For broilers a ration that was 32% wet Brewers' grains silage plus 10% molasses or 42% wet brewers' grains silage was tested. Weight at the end of 7 seven weeks was the same in the control and in the test groups (1600 g). The silage was preserved with propionic acid at the level of 2% (Wegner, 1973). ‘Swing. In contrast to poultry, no studies using wet brewers' grains in swine feed were found. When 15% brewers dried grains was included in the pre- starting diet for pigs until they reached 15 kg, and then 20% until their weight was 95 kg, satisfactory results in weight gain and carcass quality were obtained (Branckaert and Vallerant, 1972). Young and Ingram (1968) conducted an experi— ment in which brewers dried grains furnished 0, 25, 50, 75 or 100% of the supplemental protein in a corn-soybean meal diet for growing-fattening pigs. They found no difference in growth rate or carcass quality up to 50% of the supplemental protein. The digestible energy was 52.3% for brewers dried grains alone and 55.8% for brewers dried grains plus 5% yeast. The estimated metabolizable energy was 2.38 and 2.50 kcal/g, respectively (Kornegay, 1973). For comparison digestible energy in corn for swine is 3.44 kcal/g and metabolizable energy 3.22 kcal/g (National Academy of Sciences, 1971). Reproductive performance of sows was very acceptable when either 20 or 40% of the diet was derived from brewers dried grains. The diets were readily consumed and palatability was not a problem. The rations with and without brewers' grains were formulated to have 15% protein and equal levels of lysine and metabolizable energy (Wahlstrom and Libal, 1976). 8 Beef and dairy. The major proportion of the brewers' grains produced is fed to beef and dairy cattle. Some farmers feed brewers' grains on a regular basis. The level may be as much as 20% of the diet (Bullock, 1974; Stephens, 1976). Increased nitrogen retention was reported when brewers dried grains plus 5% brewers dried yeast were added to a high urea semipurified diet for fattening steers (Hatch 23 31., 1972) . The net energy value of brewers dried grains for beef cattle maintenance was determined to be 2.3 kcal/g and was 1.4 kcal/g for gain (Preston gt a1., 1973). The incidence of rumen parakeratosis and abcessed livers was low for beef cattle fed brewers dried grains when compared with other low roughage rations (Johnson, 1973; Preston, 1973). In other experiments dairy cow rations low in protein were supplemented with distillers dried grains, brewers dried grains or urea. Dis- tillers dried grains and brewers dried grains gave similar effects on milk yield, milk fat, weight gain and feed intake. These rations were superior to the ratios that had urea or low protein (Loosli and Warner, 1968). Griffiths (1971) found that for cows in mid lactation milk production and composition were the same when a 18.5% crude protein concen- trate was diluted 2:1 with brewers dried grains. Wet brewers' grains silage has been fed successfully to cattle. In one trial with dairy cows 15 kg lucerne silage were replaced by 11.5 kg wet brewers' grains silage. Milk yield was not affected but the fat content was reduced and the iodine number of the fat increased. Average production 9 of the cows fed brewers' grains was 16.6 kg of 4% milk per cow daily (Axelsson and Hellberg, 1941). Studies have been conducted to compare milk production when brewers' grains silage or silage made from sugar beet tops was fed to cows. Milk production was the same, but the milk contained less dry matter and fat when brewers' grains silage was fed. However, no adverse effects were observed when this milk was fed to infants (Mollenbach and Larsen, 1953). Orth and Kordts (1965) conducted a study to determine the effect of feeding 10 kg of wet brewers' grains silage on milk quality. Taste, smell or bacterial counts of milk were not affected. However, butter was softer and the iodine number higher. There was no decrease in milk yield, milk fat or milk protein. Also, the cows were in good health. The nutritive value of brewers' grains silage was reported by Hashimoto gt a1. (1971). Digestibility coefficients for dairy cows were 73% for crude protein, 28% for crude fiber and 67% for nitrogen free extract. Porter and Conrad (1971) compared the nutritive value of wet brewers' grains, brewers dried grains, distillers dried grains and solubles and a com- bination of wheat bran and soybean oil meal for milk production. These grains made up 20% of the concentrate mixture on a dry matter basis. Milk yields were the same for all concentrates. Cows ate less dry matter when wet brewers' grains were fed, but the digestibility was higher. A product called "Maltlage" that is marketed consists of 65% wet brewers' grains, 32.75% corn and 2.25% of a vitamin-mineral supplement. Rakes and 10 Davenport (1975) fed this product to lactating dairy cows at the level of 0, 40 or 50% of the total ration. The control diet was 71.6% corn silage, 6.5% soybean meal, 21% corn and 0.9% vitamin-mineral supplement. There were no differences in milk production. There has been a considerable number of studies that demonstrate the utility of brewers dried grains in livestock feeding. Although less effort has been devoted to similar studies with wet brewers' grains silage, they are apparently equally useful. Preservation of brewers' grains Wet brewers' grains produced by the breweries have about 80% moisture. They can be dried to about 10% moisture. This product is stable, but the process of drying implies high energy cost, and current restrictions on atmospheric con— taminants (dust, odor, smoke, etc.) result in large costs for capital, operation and maintenance. Equipment malfunction is an additional problem (Linton, 1973). To reduce the cost of drying, the grains are pressed to reduce their moisture con- tent. The resulting press water or effluent containing both suspended and soluble solids, can present a serious disposal problem (Finley gt a1., 1976). Brewers' grains liquor have a biological oxygen demand (B.O.D.) of 22,500 milligrams per liter. This liquor may account for 30 to 60% of the B.O.D. and suspended solids generated by a brewery (Hang gt al., 1975) - 11 Fresh brewers' grains are a highly perishable product due to their high moisture content, nutrient composition and the microbial contamination to which they are exposed. When the grains cannot be fed to the animals within a period of a few days, they need to be stored. Bad storage conditions result in losses due to spoilage. Fritzch and Abadjieff (1967) attributed several cases of illness in cattle to moldy brewers' grains silage. Several additives have been used to assure good preser- vation of wet forages stored in silos (Watson and Nash, 1960). In the case of brewers' grains several methods of storage and the use of different additives have been tested. Wet brewers' grains ensiled with no additive had a dry matter loss of 17.5%. With three liters of a 2N AIV solution per 100 kg the dry matter loss was 11.6% and with five liters 6.4% at pH 2.3. The quality of the silage was similar in all cases but the loss of dry matter was reduced by rapid acidi- fication (Krinstad and Ulvesli, 1951). Wet brewers' grains stored in a water proof concrete silo has 12.2% loss of organic matter. In an earth pit the losses were higher. The silo silage was of better quality than the silage from the earth pit and had less butyric acid content (Dijstra, 1955). A positive correlation was established between dry matter loss and butyric acid concentration in wet brewers' grains ensiled in round concrete silos and kept from four to eight months (Schoch, 1956). Silage from brewers' grains, as evaluated by butyric acid content, was unsatisfactory without 12 additive or with 0.20 to 0.27% sodium chloride, 4.2 to 8.4% dried beet slices or 4 to 10% dried pear residue. Good results were obtained when 10 to 15% dried apple residue, 5 liters 1.1N formic acid or 5 liters of 2N AIV solution per 100 kg of grain were added. Draining off the juice from the start of the ensiling period reduced losses of dry matter. The composition of fresh brewers' grains and of the silages with or without additives was similar (Schoch, 1957). Three and one half tons of wet brewers' grains were stored under anaerobic conditions in a wooden silo lined with polyethylene. Eleven pounds of sodium chloride were added per ton of grain. After three weeks 23 gallons of seepage had been gathered. Dry matter losses were 10.9% after 20 weeks. During storage the pH fell from 4.7 to 3.9, but in the spoiled material pH increased to 8.4. In order to reduce the losses still further, higher levels of salt and better sealing were recommended (Myers and Ollier, 1962). The use of airtight silos has given successful results for the storage of wet brewers' grains mixed with supplemental feeds. In this case wet brewers' grains were pressed to reduce their moisture content to a 68—72 percent range. Addi- tion of dry grain and a mineral-vitamin mixture reduced the moisture content further to 49-54% (Anonymus, 1969; Anonymus, 1976). Using 200 ml test tubes as model silos, Allen and Stevenson (1975) showed that addition of 0.50 and 0.75% formic acid, and 0.75% of a formic-propionic acid mixture resulted in good quality silage. l3 Formic acid or propionic acid at 0.40% or a mixture of formic and propionic acid at 40% reduced spoilage in uncovered piles of wet brewers' grains. Depth of discoloration and spoilage after 14 days of storage was from 5 to 7.5 cm in the grain treated with the formic-propionic acid mixture and 23.5 cm in the untreated grain. Addition of 2% molasses did not have beneficial effects on the conservation of the grains (Allen gt 31., 1975). In conclusion, storage and conservation of brewers' grains depends primarily on the characteristics of the silo where the grains are to be kept. Good results are to be expected under strict anaerobic conditions. In this way the growth of lactic acid forming bacteria is assured. The acid- ity produced by these bacteria will inhibit the growth of putrefactive molds. If the grains are not stored in airtight silos, the addition of preservatives must be considered. To enhance an active lactic acid fermentation that will inhibit other microbial growth, initial acidification of the mass and the addition of readily available carbohydrates for the lactic acid bacteria can be tested. It is possible that an increase of the lactic acid bacteria population by means of an inoculation could inhibit or stop the growth of other unde— sirable microorganisms. The silage obtained must not have lost the nutritional characteristics of fresh feed. During the ensiling process toxic substances must not be formed. Additives used must be innocuous when fed to the animal. The final product must be palatable and acceptable for the animal. 14 Additives must be easy to handle and harmless for the persons using them and for the storage structures. Furthermore, they must be economical. MATERIALS AND METHODS Two storage trials were conducted with wet brewers' grains and brewers' yeast obtained by truck from the Strohs Brewery in Detroit. Both trials were conducted during hot weather. Trial 1 Fresh brewers' grains alone and grains mixed with yeast (10%) were placed in five gallon plastic buckets. Dimensions of the buckets were: bottom diameter 10 in., top diameter 13 in. and height 14 in. A thermocouple was placed in the center of the mass to monitor temperature changes during the ensiling period. Samples of grain were taken on days 4, 8, 15 and 22 for pH, ethanol, volatile fatty acids (acetic, propionic and buytric), lactic acid and ammonia. These samples were taken from the unspoiled part of the grain. Samples of the original material and of the material ensiled for 32 and 76 days were taken for analysis of dry matter, protein, ammonia, acid detergent fiber, acid detergent insoluble nitrogen, ethanol, volatile fatty acids and lactic acid. These samples were taken after emptying the barrels from the part of the material that was not spoiled. Spoilage was separated by hand and its weight determined. 15 16 Four buckets were used for each treatment, two with wet brewers' grains alone and the other two with wet brewers' grains plus 10% autolized wet brewers' yeast. One of the buckets to which yeast was added and one of the buckets with- out yeast were removed and emptied to determine the amount of spoiled material after 32 days and the other two after 76 days. The various chemicals were mixed with 60 1b of brewers' grains in a horizontal mixer and then 30 1b divided into each bucket. The exact weight of the content of each bucket was recorded. Lactobacillus cultures were grown in sterilized milk autoclaved at 130°C for 30 minutes. Two hundred ml of this culture were mixed with 60 lb of grain. The buckets were placed in a heated room, around 76°F, to simulate hot weather conditions. Table 2 gives a description of the type of ensiling method and material used. A split-plot design with repeated measurement was used to analyse the results of this experiment. Seventeen different additives or ensiling methods and the presence or absence of yeast formed 34 treatment combinations. The periods were the days after the initiation of the experi- ment in which the data were collected. Each treatment combination had two repetitions. Bonferroni's "t" test was used to evaluate differences of group treatment means. Correlations were determined between the averages of the measurements or composition of 32 and 76 days silage. 17 Table 2. List of additives or method used to ensile wet brewers' grains. Trial la Treatments # buckets 1 None 4 2 Propionic acid (0.5%) " 3 Propionic acid (1.0%) " 4 Ammonium propionate (0.5%) " 5 Ammonium isobutyrate (0.5%) " 6 Ammonia (0.3% nitrogen) " 7 Paraformaldehyde (0.1%) " 8 Formic acid to pH 3.2 plus paraformaldehyde (0.1%) " 9 Potassium carbonate (1.5%) " 10 Foam sealants " 11 Potassium carbonate (1.5%) plus propionic acid (0.5%) " 12 Sulfuric acid to pH 3.6 " l3 Formic acid to pH 3.6 " 14 Dried molasses (3%) n 15 Sucrose-starch mix (1:1) (3%) " 16 Sodium benzoate (0.1%) " l7 Lactobacillus casei plus L; bulgaricus culture " 18 Lactobacillus casei culture in grain without yeast 2 a The numbers in parenthesis are the concentrations of the additive mixed with the wet grains, except where indicated. l8 Trial 2 Seventeen treatments with three replications each were used. The experimental units were 55 gallon steel barrels containing 300 1b of wet brewers' grains. The grain was mixed with the additives in a mixer, 300 lb at the time, and placed immediately in the barrels lined with plastic bags (38 x 65 in., .004 in. gauge). Two barrels of the three repe- titions were emptied at the end of 32 days and samples collected. These samples, as well as the original fresh material were analysed for dry matter, total nitrogen, ammonia, acid deter- gent fiber, acid detergent insoluble nitrogen, ethanol, volatile fatty acids and lactic acid. Thermocouples were placed in the center of the mass of the barrel to monitor temperatures. Samples of treatments 1, 2, 3, 4, 5, 6, 11, 13, 14, 15, 16 and 17 were placed in 100 ml test tubes at room temperature and frozen on days 5, 7, 10 and 13 after the initiation of the experiment. Analysis for volatile fatty acids, lactic acid, ethanol and ammonia were made on these samples. Spoiled material was separated by hand and samples from the unspoiled material were taken from different sections at the silo. Calcium sulphate was added as a slurry to cover the grain. About 10 1b of limestone, dried molasses, liquid molasses or ground corn were used for treatments 7, 9, 10 and 12, respectively to form a seal about 1 cm thick on tOp of 19 the grain. A commercial product called "Super Silo-Zime"* was the lactic acid bacterial culture used, and 340 g were mixed with 300 1b of grain. This is the equivalent to the 5 lb per ton recommended by the manufacturer. The barrels were in a large building where air circulated freely. A split-plot design with repeated measurement was used for statistical analysis of the results of this experiment. The number of treatments was 17 and the periods were the days after the initiation of the experiment in which the data were collected. Each treatment had three replications. Bonferroni's "t" test was used to evaluate differences of treatment means. Correlations were determined between averages of measurements or composition of 32 and 60 days silage. (Table 3) Analytical methods Dry matter was determined by drying samples in an oven at 10590 overnight. Total nitrogen was determined using the Kjeldahl method (AOAC, 1965). Copper sulphate was used as the catalyst. Acid detergent fiber and acid detergent insoluble nitrogen were determined using the Van Soest method (Goering and Van Soest, 1970). Samples for these analysis were dried for 48 hr at 4580 in an air forced oven. Samples of the grain were diluted 1 in 10 with water, homogenized and filtered through four layers of cheesecloth. pH values were taken from this filtered homogenate. After centrifugation at 27,000 g * Biochemical Corporation of America, Salem, VA 24153 20 Table 3. List of treatments used to ensile wet brewers' grains. Trial #2a 1 Control, no additive 2 Propionic acid (1%) 3 Propionic acid (2%) 4 Formic acid (1.4%)b plus paraformaldehyde (0.1%) 5 Sulfuric acid (0.3%)C 6 Butylated hydroxyanisole (B H A) (200 ppm) 7 Ground limestone on top of the grain 8 Calcium sulphate on top of the grain 9 Dried molasses on t0p of the grain 10 Liquid molasses on tOp of the grain 11 Liquid molasses (7%) 12 Ground corn on tOp of the grain 13 Ground corn (10%) 14 Lactic acid culture, 340 g/300 1b grain 15 Lactic acid culture, 340 g/300 1b grain plus liquid molasses (7%) 16 Lactic acid culture, 340 g/300 1b grain plus ground corn (10% 17 Sealed with a plastic bag full of water on top a The numbers in parenthesis represent the concentration of the additive mixed with the wet grains, except where indicated. b 2200 ml 85% formic acid/300 1b grain c 1000 ml 40% sulfuric acid/300 1b grain 21 for 15 min to precipitate the proteins. The supernatant was used for volatile fatty acids, ethanol and lactic acid determination. Ammonia was determined by a colorimetric method used for blood ammonia (Okuda gt a1., 1965) and modified by Kulasek (1976). The method of Barker and Summerson (1941) was used for lactic acid analysis. Volatile fatty acids (acetic, pr0pionic and butyric) and ethanol were measured using a Hewlett-Packard gas liquid chromatograph model 5730A with flame ionization detector. A glass column (6 ft x 2 mm ID) was packed with 3% Carbowax 20 M, 0.5% H3PO4 on 60/80 Carbopack B (Supelco, Inc. 1-1825). Nitrogen was the carrier gas at a flow rate of 60 ml/min. The temperature program used was two minutes beginning at 140qC with a temperature increase of 4qC/min for ten minutes, and finally eight minutes at 180qC. Prior to the injection the samples were acidified with a drOp of 9N H2804. Injection volume was 3 microliters. Concentrations were calculated relating the areas under the peaks for the standards with the areas under the peaks of samples. Total microbial count on the grain was made using agar plates. The dilutions considered for plating were 10-3, 10’“, 10-5 and 10-6. Plates were incubated at 320 for 48 hours. For coliform bacteria the medium used was violet red bile 2, 10'3 and 10’“, incubated agar with plate dilutions of 10- at 37qC for 24 hours. Yeast and mold determinations were made using acidified potato dextrose agar, pH 3.5. The 22 plates were incubated at room temperature for five days for the dilutions of 10-2, 10"3 and 10-4. A lactobacillus broth (pH 5.4) was used for lactobacillus estimation with dilutions of 10-2, 10'3 and 10'” incubated at room temperature for three days. RESULTS AND DISCUSSION Trial 1 Four days after the beginning of the experiment most of the buckets had developed colonies of mold on the surface. After six days, flies started to grow beneath the surface of the grain of treatment no. 9 (potassium carbonate). Two days later grain in buckets treated with propionic acid (0.5%), ammonium isobutyrate and sodium benzoate started to spoil. After 32 and 76 days when the buckets were emptied, all the treatments but the one sealed with foam had the surface layer of the grain decomposed. Flies were observed growing in the spoiled part of the grain. Digging into the mass to take sam- ples from the unspoiled part of the grain hastened spoilage. Spoilage. All buckets except those sealed had consid- erable spoilage (Table 4). Eighteen percent of the grain was spoiled in one bucket covered with foam that had a leak between the foam and the edge of the bucket. However, the grain in the other three sealed buckets had no spoilage. This demonstrates that when anaerobic conditions are main- tained spoilage can be prevented. Other treatments that reduced spoilage were propionic acid (0.5%). formic acid plus paraformaldehyde, sucrose-starch mix and bacterial culture. These averaged 24.3% spoilage compared to 30.5% for 23 24 m.:m o.m: o.mm ARMV mommmHoE Uofimm m.mm m.H: o.mm o.m mg on need oafihom 0.00 0.50 0.Hm 0.0 mm 0p sommm 0.00 0.00 0.00 Aem.0v 0fiom oflcoflmopm + Aem.av m000M m.d o.m o.oo mpsmammm swom 0.00 0.00 0.00 0%.: 000001 m.dm m.am 0.0H ARH.OV muzsmUHmEMOMmmmm + m.m mm op 000m caenom m.mm o.mm n.5N A&H.ov mahgocHMEMOanmm 0.00 0.0: 0.00 A2 em.0v mmz m.mm m.:m o.mm ARm.ov mpmnhpsnoma adenoas< m.mm m.m: o.mm ARm.ov mpmcowmonm Esflsoee< 0.0m o.m: m.om ARO.HV uflom cacowmoum o.wm o.mm 0.3m A&m.ov Baum oMGOHmoum m.om o.mm o.mm achpcoo M made on mzmu mm memSpmmme nopmm umafiomm R nopmm Umaflomm R .mcflmhw .mnmzmnp Pm; mCHHflmcm Hmpmm Hmflhmpme wmaflomm mo mwMPcmohom .: wands mosam> mm Mo owmnm>m o ozam> 620 n mosam> 039 %o mMMho>m m :.om H.mm w.mm mmmnm>¢ M0 m.mm m.mm m.mm opmmmh &0H + mcflmum .mnmzonp Pm; o.mm 3.0m 0.0m opmmmh Psonpwz mcwmnm .mthmhp Pm; wqmm o.mn o.mm nammmo m:aawodpopomg 0.00 0.00 0.0a msoflnmmasp 4m + H0000 msHHflomnopomq 0.Hm 0.00 0.0m ARH.0V memoNcmp esfleom m.mm 0.0m m.~m Aemv sohmpmummoeosm many on mzmu mm M hoped cmafiomm & mopwm umawomm & mPQmSPmons A.U.PCO0V a mdnme 26 the control. Addition of yeast reduced spoilage from 32.0 to 28.9%. The unspoiled grain treated with propionic acid (1%) and sulfuric acid was darker than the unspoiled grain from all the other treatments. The odor of the unspoiled grain was objectionable in all, except the sealed buckets, due in part to the contact with the spoiled material. pH. Fresh brewers' grain had a pH of 5.3. After the second day of ensiling pH had decreased to values close to 4.0 in all the treatments except in those containing potassium carbonate. Addition of ammonia did not prevent the usual decrease in pH (Table 5). Analysis of variance for pH on days 2, 8, l5 and 22 indicated that effects of additives and days were highly significant. The effect of presence or absence of yeast was not significant. The interactions of additives, yeast and days, were significant (Table 6). Figure 1 shows that the pH increased in day 15 and decreased after day 22 due to the addition of yeast. Comparison of treatments means from day 2 to 22 indicated the following: The pH of the acid treatments propionic (0.5 and 1.0%), formic acid, formic acid plus paraformaldehyde and sulfuric acid was not significantly different from the pH values of the control. Addition of potassium carbonate increased average pH from a control value of 4.03 to 5.06 (P<.01). The pH of the grain treated with propionic acid alone was lower than the pH of the grain treated with ammonium propionate or potassium carbonate plus prOpionic acid, but 27 00.: 0:.: 00.0 00.0 00.: 00.0 00.: 00.0 0000000E 00000 00.0 0:.: 00.0 00.0 00.: 00.0 00.0 0.0 00 00 0000 000000 00.0 00.: 00.0 00.0 00.: 00.0 00.0 0.0 00 00 :0000 00.: 00.: 00.: 00.: 0:.0 0:.: 00.: 00.0 0000 00000000000 + 00.0 00000 m0.m 00.: ~0.m mpcwammw 800m 00.0 00.0 00.: 0:.0 :0.0 00.0 0:.0 00.0 00000 mm.m mm.m m0.m mm.m 0m.m mm.m mm.m 00.0 0003000050000009 + 0.m mm op 0000 00800m 00.: 00.: 00.0 00.0 00.: :0.0 00.: 00.0 0000000000000000 00.0 0:.: 00.0 00.0 :0.: 00.0 00.0 00.0 002 00.: mm.: mm.m 00.: 00.0 mm.m 00.0 0m.o 00000959000 SS0£088¢ 00.: 0:.: 00.: mm.: mm.: mm.: 00.: $n.o opmco0monm 850coas< 00.: 00.0 00.0 00.: 00.: 00.: 00.: 00.0 0000 000000000 00.0 0:.: 00.0 00.0 00.0 :0.0 00.: 00.0 0000 000000000 00.: 00.: 00.0 00.0 00.0 00.0 00.: 0000000 W mm. mm Mama m0 m N 0.088.0on9 .mm>000000 pCmHmMMHU £003 umHHQO mfl000m .0003009 903 C0 0800 £003 mm Gflmwowmwmw .m 00909 28 00500> on 00 00w000>0 o 00500> 030 00 m0w000>0 p 00500> 05cm Ho 00w0n0>0 0 00.: 0:.: 00.0 :0.: 00.: 00.: :0.: 00000>¢ 00.: 00.: 00.0 00.: 00.: 00.: 00.: 000000 000 + 000000 .0002000 003 00.: mm.: 00.: mm.: 00.: 00.: 00.: 000000 0500003 000000 .0003000 003 00.: 00.: 00.: 0:.: 00.0 00.: 00.0 000000 0000000000000 00.: 00.: 00.: :0.: 00.: :0.0 00.0 00000000sp_4m + 00000 0000000000000 :0.: 00.: 00.0 00.: 00.: 00.0 :0.: 00.0 00000000 0000000 om.m 00.: mm.m mm.m :m.m mm.m 00.m Ro.m N08 c0000m|0woposm 0.0. 00 00 0.0000 00 0 0 00000.00 0 a A.U.Pcoov m 0HQ0B 29 Table 6. Analysis of variance of the pH of ensiled brewers' grains. Trial 1 Degrees Mean F Source Of Variance of Freedom Square Statistic Additives 15 0.2881 45.52** Yeast 1 0.1526 1.62 (Additives) (Yeast) 15 0.5435 5.77** Duplicates/trt. combination 32 0.09#2 Days 3 0.9768 11 .63M (Additives) (Days) 45 0.1356 1.61* (Yeast) (Days) 3 0.8149 9.70** (Additives) (Yeast) (Days) 45 0.1706 2.03** Residual error 96 0.08h0 * Significant P<.05 ** Significant P<.Ol 30 pmmoh Rea msam msflmnw .mpmzmsp pm; Aolllllov pmdmz PSOQPHS mswmpm .msozmnn v63 Aolllllov .H aways .Pmmmz psonpwz so spa; anflmCo msflmpm .mumzonn Pm; mo mm CH mwcmzo .H mpswflm 31 02:57; am..L( m><0 ...: a fin «N n. o N u n w W .. o.n In 0“ 1 MT! o.V .. o.n 32 higher than the pH of the grain treated with formic acid (P<.01). Formic acid plus paraformaldehyde lowered the pH more than formic acid alone (P<.01). Molasses and the sucrose- starch mix did not affect the pH when compared to the control and did not differ among themselves either. The pH of sodium benzoate and Lactobacillus culture treated grain was 4.04 and 4.11, respectively, which was not different from that of the control (4.03). In all treatments, except the potassium carbonate treat- ment, pH was low enough to maintain silage in good condition provided anaerobic conditions were maintained. Temperature. Temperatures of brewers' grains at different intervals during ensiling process are presented in Table 7. Buckets containing brewers' grains were stored at 700 to 84°F. Temperature of the grain inside buckets was above room temperature until day 8, and usually near room temperature thereafter. Analysis of variance for temperature (Table 8) indicated that the effects of additives, yeast and days, and their interactions were highly significant. Temperatures of the grains with yeast added (76.60F) were lower than temperatures of the grains without yeast (79.70F). Treatment with yeast decreased temperature after day 8 (Figure 2). Temperatures in buckets sealed with foam were signifi- cantly lower than in control (76.2 vs 79.80F) (P<.05). Temperatures of the grains with basic treatments did not differ significantly from the control or from the acid 33 0.05 0.00 0.05 0.05 0.00 0.00 0.H0 0.H0 0.55 0.05 0.05 0.05 0.05 0.55 A50.Hv mpmsopnwo sswmmmpom 0.05 0.00 0.05 0.55 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.00 0.05 0.05 A5a.0v 000000 IHMEhomwme + N.m mm op 0000 cashew 0.05 0.50 0.55 0.05 0.00 0.H0 0.05 0.05 0.05 0.05 0.05 0.05 0.55 0.05 50H.00 owhnouamsuommumm 0.05 0.00 0.05 0.H0 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Anemonpflc 00.00 wagoss¢ :.05 0.00 0.05 m.om 0.30 m.Hw 0.05 0.05 m.m5 0.05 0.m5 o.H5 0.55 0.35 500.0v opmnhpsnomw eswcoss¢ H.00 0.0m N.55 N.5m 0.30 0.00 m.mm 0.05 m.m5 N.m5 0.55 0.05 0.55 0.05 550.0v opmsowmong sswcoss< 0.05 0.00 0.05 0.00 N.H0 0.00 0.00 N.H0 0.05 0.05 0.05 0.05 0.05 0.50 550.Hv 0000 cascamonm 5.05 0.50 0.55 0.05 0.00 0.H0 0.00 0.05 0.05 0.00 0.05 0.05 0.05 0.05 A00.0V 0000 owcowmonm 0.05 0.00 0.05 0.00 0.00 0.00 0.00 0.H0 0.00 0.05 0.05 0.05 0.05 0.05 Honpcoo m 0.05 00 00 0H 0H 0H wmw 0 0 is 0 m a 020200009 0H H0099 .mo>090000 pawn00000 £903 coawmso mcflwmm .0903099 903 CH mEHP spas Amy oHSPMQoQSop CH mmmcmso .5 magma 34 0.05 0.00 0.05 0.05 0.00 0.05 0.55 5.05 0.05 0.05 0.05 0.05 0.05 0.05 000000 000 + 00000 0.05 0.50 0.05 0.00 0.00 0.00 0.00 0.00 0.05 0.05 0.05 5.05 0.55 0.05 000000 0000000 00000 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.00 0.05 0.00 0.05 0.05 0.05 0.05 000000 0500000no0000 0.55 0.00 0.05 0.05 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0000000000_1m .+00000 0500000po0000 0.05 0.00 0.05 0.00 0.00 0.00 0.05 0.05 0.55 0.05 0.05 0.05 0.05 0.05 000.00 00000C0p 8:000m 0.55 0.00 0.55 0.05 0.00 0.05 0.55 0.05 0.05 0.05 0.05 0.05 0.05 0.55 A000 000000-0000000 0.55 0.00 0.05 0.05 0.00 0.00 0.05 0.55 0.05 0.05 0.05 0.05 0.05 0.05 00000000 00000 0.55 0.50 0.05 0.05 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.0 00 00 0000 00Ehom 5.55 0.00 0.05 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.0 00 00 0000 00000000 0.05 0.00 0.55 0.05 0.00 0.00 0.05 0.05 0.55 0.00 0.05 0.05 0.05 0.05 000.00 0000 00Qo0mo0m + moomM 0.05 0.00 0.05 0.55 0.00 0.05 0.05 0.55 0.05 0.55 0.00 0.05 0.05 0.05 00000000 0000 m 0.05 00 00 00 00 00 0mm 0 0 l0 0 0 0 000000000 0.0.00000 5 00000 35 00500> 00 Mo mmm000>0 0 00500> 030 mo mmw000>0 n 00500> msow Mo 00w000>0 0 N.©5 0.55 0.:5 0.35 0.0m 0.:w o.Nm 0.0m 0.05 o.m5 0.35 o.m5 0.05 o.N5 0050000m800 Soom 0.05 0.00 0.55 0.00 5.00 0.00 0.05 0.05 0.05 0.55 0.05 0.05 0.05 0.05 0000000 0 0.05 00 mm 00 00 00 wmm 0 0 00 0 m 0 000000000 0.0.00000 5 00000 36 Table 8. Analysis of variance of the temperatures of ensiled brewers' grains. Trial 1 Degrees Mean F Source Of Variance of Freedom Square Statistic Additives 16 83.7108 7.14** Yeast l 2055.1777 l75.29** (Additives) (Yeast) 16 47.5162 4.05** Duplicates/trt. combination 34 ll.72#3 Days 11 892.0u31 311.54** (Additives) (Day) 176 9.4740 3.31** (Yeast) (Day) 11 27.5814 9.63** (Additives) (Yeast) (Day) 176 5.877# 2.05** Residual error 37h 2.8633 ** Significant P<.Ol 37 pmmmz Rea msam mflwdpm .mhmzohn Po; Aolllllov Pmmmz Facspflz msflmnm .mhoSoHQ Pm; Acillllov .H Adana .Pmmoz pzonpflz no QPHB umafimco mcflmmm .mhmzmhn #03 mo whopwnmmSoP CH owcwnv .N muswflm 38 02....me «31.3. m>4a ..— d -'V ~00 "N l l .mk ,vk 0k 93 on Y- ”a QUOLVHBJWQL (:l) 39 treatments. Acid treated grains had higher average tempera- tures than those sealed with foam (78.10 vs 76.20F) (P<.lO). Temperatures of the grains treated with propionic acid, ammonium propionate or potassium carbonate plus propionic acid did not differ. Addition of formic acid lowered tempera- ture more than did propionic acid (76.90 vs 79.30F) (P<.05). Temperature of the grains treated with formic acid or with formic acid plus paraformaldehyde were similar, 77.50 and 76.3OF. Temperatures of grains treated with molasses (77.10F) or sucrose-starch mix (77.u°F) did not differ significantly from those sealed with foam (76.20F), nor among themselves. Grains treated with sodium benzoate had lower temperatures than did the control (78.10 vs 79.70F) (P<.lO). There was no significant difference in average temperature between grains sealed with foam and those treated with Lactobacillus culture (76.2° vs 77.3°F). Dry matter and protein. The average dry matter content on day 32 was 22.#% and decreased to 20.5% after 76 days (Table 9). Only sodium benzoate treated grains did not have this decrease. During this interval the protein content of the dry matter increased in every treatment except in the one with formic acid plus paraformaldehyde. The average was 3#.3% on day 32 and 40.9% on day 76. The changes in dry matter and protein were similar for the grains alone or grains plus yeast. Acid detergent fiber and acid detergent insoluble nitrogen. Average acid detergent fiber increased from 22.8% 40 0.00 0.00 0.00 0.00 0.00 5.00 .0m.o. 0000 0000m00m0 + Asm.av 00 a 0.50 0.00 5.0m 5.00 0.00 0.00 00200000 0000 0.00 5.00 0.00 0.00 0.00 0.00 000.00 00000 m.mm m.mm 0.mm 0.m0 0.00 5.00 000.0. 00000000500000n0 + 0.0 mg 00 0000 000000 0.00 5.00 0.50 m.m0 0.00 0.00 .00.ov mvhnmoamsnommnmm 0.00 0.00 0.00 0.00 0.00 5.00 .0000000: 00.0. MHCoEE< 0.00 0.00 0.00 0.00 0.00 5.00 .0m.0. mpmnhpsnomH 850coss< 0.00 0.00 0.00 0.00 0.00 0.00 .00.00 mpmco0mopn ss0coss< 0.00 0.00 m.mm 0.00 0.00 0.m0 000.0. 0000 00c00mopm o.mm m.mm m.mm 0.00 0.00 5.00 Afim.o. 00cm 00:00Qoum 0.00 5.00 0.00 5.00 5.00 0.00 0000000 M 900095 000.00 90.00 mm 000.00 m. 20.00 mK. 0000.00 90.00 mm 03.00 0000005000.... ~w0mmp 000008 000. c0mpopm\& 009908 000 R H HGHHB .mC000w .mpmzmhn no; UmHMmCm mo PCmPCOO Camposm 0cm poppms 500 .m manna 41 mmsaw> mm 00 mw000>0 o 0500> 080 mmsam> QB» mo mm080>0 M 0.50 0.00 0.00 0.00 m.o0 0.00 0000000 0.50 0.00 0.00 5.00 0.00 0.00 000000 000 + m8000m .mnozmup 003 m.5m m.0: 0.2m 0.00 m.om m.00 000000 050:003 m8000w .mumsmpn 003 0.0: N.m: m.mm N.om N.mH H.0N nwmmmo msaafiomnopomq 0.0: m.H: n.5m o.mH m.wa 3.0N EdowmwwHSQ 4% + 00000 0800000900000 n.5m m.mm n.0m N.NN :.NN m.HN 0&H.ov 00000809 8:000m 0.5m 0.0: o.mm 0.00 n.00 0.00 ..Ro.m. £0000mu000005m o.mm o.mm 0.0m N.mm o.mm m.:m Afio.mv mmmmmao8 U¢00Q m.mm 0.mm 5.mm m.mm 3.00 :.mm w.m mm on 0000 008pom 0.00 0.0: 5.00 0.00 0.00 0.00 0.0 mm 00 00000 .x 0000 05 00000 0000 mm 00000 00 0800 m5 00000 mhwu mm 80000 “00009 000008 and. 8000008.& 000008 and & 080800009 A.U.PCOOV m manme 42 on day 32 to 25.3% on day 76. The highest value was for the treatment with dried molasses (Table 10). Addition of yeast decreased the acid detergent fiber content in the grains. Acid detergent insoluble nitrogen increased from 0.92% on day 32 to 0.97% on day 76 after ensiling. Addition of yeast decreased the acid detergent insoluble nitrogen of the grains by an average of 9%. The highest values were for the treatments that had paraformaldehyde. These treatments had from 17 to 30% higher acid detergent insoluble nitrogen con- tent than controls. This is due to the fact that formaldehyde binds proteins making them insoluble. In the case of ruminants this complex is not digested in the rumen but may be digested in the abomassum. Ethanol. Ethanol content of the silage attained maximum concentration after 15 days of ensiling. From day 15 to day 32 ethanol concentration decreased, but increased on day 76 to a level of 48% of that on day 15. Ethanol content of the silage with yeast (0.62%) was greater than without yeast (0.48%) (P<.05) (Table 11). The interaction of additives with days and of days with yeast was significant (Table 12). The control did not differ from acid or basic treatments in ethanol content. The ethanol content of silages treated with acids or bases was similar. Treatments with propionic acid or ammonium propionate had an average ethanol concen- tration not different from the control (0.29% vs 0.34%). Formic acid alone was similar to formic acid plus para- formaldehyde in its effect on ethanol formation When compared 43 mm.o mm.o om.o m.mm 5.:N m.om_ A&m.ov uwom cacoanonm + ARm.HV moomm mm.o mo.H mm.o m.:m m.mm m.mm mvcmamwm swam oo.H oo.H oo.H m.mm 0.5m H.mm Aam.av moomx 0N.H :m.a ma.a o.mm m.:m :.NN ARH.OV mahzwcawshommnmm . + N.m mm op vwom owahom MH.H mm.a :o.a m.mm o.mm m.mm A&H.ov ovznwuamaHOanmm Hm.o :0.0 mm.o m.mm N.mm m.mm. AGmonPHC Rm.ov manoea¢ om.o no.0 mm.o m.:m 0.0N m.HN Agm.ov mpdhhpsnomw aswcoss¢ mm.o mm.o om.o H.mm u.mm 3.3m A&m.ov mpmcowmona azacoee< :m.o No.0 mm.o m.mm d.:m N.mm A&o.dv Uwom owcowgonm :m.o mo.H om.o :.mm m.mm N.Hm Agm.ov ufiom aflcowmoum Hm.o mm.o 3m.o m.mm 5.:N m.HN Honpcoo _m mzmcymm umpmm mzmu mm pmpmm .un mzmw on hwpmw wand wm nwpmm Asa RV zm< R “Em &v mm< &\ pCmSmeua ma Hawks .mnwmpw..mnm3mhp pm; cmHHmCm ho pampcoo‘fizm mm mo mwmnm>m o msam> mac n mwsaw> 03p mo mwmnm>m m :m.o no.0 No.0 _H.:m m.mm. m.mm mmmnm>¢ mm.o oo.H dm.o m.dm m.mm m.nm pmwmh pzonpws mcwmuw .mpmsmpp p03 mm.o mm.o om.o m.mm m.:m m.mm opmmmm §oa + mcwmnw .mumsmhn Pm; Ho.a mm.o mo.a o.mm :.om n.3N nmmmmo msaaflownopomq mm.o um.o Hm.o n.nm 0.3m m.mm msoflummasp 4m + ammmo msaawownopomg om.o om.o no.0 3.3m N.mm w.mm ARH.oV mpmoucmn azwuom mm.o om.o mm.o m.mm w.:m m.mm A&o.mv soumpmnmmouosm :m.o um.o No.H H.5N m.mm m.mm A&0.mv mmmwmaos umfiha om.o om.o mm.o o.NN m.mm m.HN w.m mm op dwom owahom mm.o mo.a Ha.o m.mm H.:m m.am o.m mg op :ommm .x mhmv wm hwpmm mhmu mm umpmm an mzmv on nmpmm mhmc mm umpmm Asa §mxza< R Pamsvwmua Cam “3 he» mm A.U.Paoov 0H manna 45 00.0 m0.0 50.0 mm.0 N0.H mm.0 dm.a A&0.mv mommmaoa oopum 0m.0 0m.0 00.0 00.0 0m.H BH.0 00.0 0.m mm op opow upshom 00.0 0m.0 00.0 00.0 00.H no.0 00.H 0.0 mm 0p 00mmm 0:.0 3m.0 :0.0 0N.0 mm.0 0m.0 50.0 ARm.0v Upow opcopaonm + A&m.av moowm m0.0 0H.H 0N.0 mpcwamom Soon 00.0 00.0 0H.0 :0.0 N0.0 00.0 00.0 Aam.av 00000 nm.0 mm.0 HH.0 mm.0 00.0 mm.0 NH.0 ARH.0V ouhnoUHMSMommnmm + 0.m mm op 0How opepom 0:.0 mH.0 0H.0 00.0 0:.0 mm.0 mm.0 ARH.0V oohnooamsnommnmm 0:.0 mm.0 0H.0 mm.0 mm.0 0:.0 :0.0 Acowohppc Rm.0v manoss¢ mm.0 0m.0 0H.0 00.0 00.0 00.0 m0.0 “Rm.0v mpmnmpspomfl ssflcoae< ma.0 m:.0 :H.0 mm.0 :0.0 0H.0 :0.0 ARm.0v opmcopgong eupqoss< mH.0 0:.0 HH.0 Hm.0 no.0 00.0 00.0 Ago.av 0000 cacopmoum :m.0 dm.0 mm.0 mo.a mm.0 m0.H mH.0 A&m.0v opom opcowmonm :0.0 00.0 00.0 0m.0 mm.0 00.0 00.0 Hoppaoo m 900 9mm mm ma 0 m pomapmmpe ham ma Hawks .mo>pppoom pconopmpo app; coawmco mcwmnw .mnozmup pm; mp ospp app; Ampmwp pm; N no pcoogomv noppmppcmozoo Hogwapo up mowcwso .HH oanma 46 mosaw> 00 mo mowwgo>m o mosam> 03p Mo mommno>w Q mosam> poop po mommno>w m mm.0 m:.0 HH.0 mm.0 00.0 H0.0 00.0 mwmpm>< No.0 Hm.o 00.0 mm.o 00.0 :0.0 00.0 opmmoz Roa + mCHmnm .mpozonn pm; 0:.0 mm.0 mH.o 0:.0 00.0 0:.0 wm.o opmMoh poonppz mcpmnw .mnoSoHQ pm: mm.0 0:.0 0m.o 0m.0 :0.0 50.0 3H.0 nwommo msaapownopoma mm.o 00.0 no.0 mm.o 00.N mm.0 HN.H wSOHhmmHSQ_4m + Momma mSHHMOMDOpomQ 00.0 0:.0 mo.o 0:.0 mm.H 00.0 00.0 A&H.ov opdounop sopoom 0N.H :0.0 no.0 :0.0 om.m mm.H NH.N A&o.mv Nae nouwpmnomohosm m 900 pmm mm ma 0 m £05089 and A.U.p¢oov HH wands 47 Table 12. Analysis of variance of ethanol content of ensiled wet brewers' grains. Trial 1 Degrees Mean F Source Of Variance of Freedom Square Statistic Additives 15 2.6554 7.05** Yeast 1 1.8735 4.98* (Additives) (Yeast) 15 0.1851 0.49 Duplicates/trt. combination 32 0.3765 Days 3 1.6652 13.28** (Additives) (Days) 45 0.8335 6.65** (Yeast) (Days) 3 0.5013 4.00* (Additives) (Yeast) (Days) 45 0.1547 1.23 Residual error 96 0.1254 * Significant P<.05 ** Significant P<.01 48 until day 22 of ensiling. During the same period of time grains treated with sulfuric acid had a higher ethanol content than did control (P<.05). Addition of molasses or sucrose- starch mix increased the ethanol content over the control (P<.01). The treatment with the sucrose-starch mix had a higher ethanol content than the treatment with molasses (P<.05). The addition of sodium benzoate did not increase the ethanol concentration when compared with the control, but lactic acid culture inoculation resulted in an increased ethanol formation (P<.05). After 32 days of ensiling ethanol content of the grains had decreased to levels close to 0.10% for all treatments, except 0.5% propionic acid and Lactobacillus casei culture which had concentrations of 0.28 and 0.36%, respectively. At the end of 76 days the grains that had most ethanol were those sealed with foam (1.10%). Grains to which molasses, a sucrose—starch mix or a Lactobacillus casei plus L; bulgaricus culture was added averaged more ethanol than did control (0.63% vs 0.49%). Low levels were observed at this time in the grains treated with paraformaldehyde (0.17%), formic acid plus paraformaldehyde (0.23%) or sulfuric acid (0.28%) when compared to the average of all treatments (0.42%). Grains with yeast had a higher ethanol concentration than grains without yeast (0.51% vs 0.33%) (Figure 3). Acetic acid. Analysis of variance for acetic acid values on days 2, 8, l5 and 22 indicated that the effects of addi— tives, yeast and days, and their interactions were significant 49 pmmom &0H moan mapwnw .mpoaohp p03 Aolllllov pmwoz psosppz mzpmnw .mnoSonn pm; Aolllllov .H aways .pmwom poosppz no app; ooapmco mcpmpw .mpoSonp pm; po pcopcoo Hozmnpo cw mmcmno .l . .m opswpm 50 0k 02 3.0.20 3:? 0:0 «0 \L./ «N d n— o.— lHOIEM 13M :IO 1N3383d 51 (Table 14). Comparisons of the treatment means on these days indicated the following: Grains treated with acids (propionic, formic and sulfuric) had lower acetic acid content when com— pared to the control (P<.01). Basic treatments (ammonia and potassium carbonate) resulted in lower concentrations than the control (P<.01), but higher than acid treatments (P<.05). Grains treated with propionic acid alone did not differ from those treated with ammonium propionate, potassium carbonate plus propionic acid or formic acid. Formic acid alone had a similar effect than formic acid plus paraformaldehyde. However, the grains treated with formic acid were lower in acetic acid content than the control (P<.01). Addition of molasses, a sucrose—starch mixture or sodium benzoate did not change the acetic acid content of the silage when compared to the control. The grains to which a Lactobacillus casei plus L; bulgaricus culture was added had a lower concentration than the control (P<.05). During the first 15 days acetic acid concentrations were about the same for grains with and without yeast, but from day 22 the grains with yeast had higher concentrations (Figure 4). On day 32 grains without yeast had 0.52% acetic acid and grains with yeast 0.30%. The concentration increased after 76 days of ensiling, but again grain without yeast had more acetic acid than grain with yeast (0.74% vs 0.41%). After 32 days of ensiling grains (Table 13) treated with ammonium propionate, potassium carbonate, dried molasses and a sucrose-starch mix had higher acetic acid concentrations 52 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000.00 00000000 00000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0.0 00 00 0000 000000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0.0 00 00 00000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000.00 0000 000000000 + 000.00 00000 00.0 om.o NN.0 mpcmawmm 8000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000.00 00000 00.0 0m.0 00.0 00.0 00.0 00.0 00.0 000.00 0000000000000000 + 0.0 00 00 0000 000000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000.00 0000000000000000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000000000 00.00 0000000 0N.o om.0 00.0 0N.0 0m.0 00.0 00.0 00m.0v mpwhhpsnom0 8:0:oae< 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000.00 0000000000 00000000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000.00 0000 000000000 00.0 «0.0 00.0 00.0 00.0 00.0 00.0 000.00 0000 000000000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0000000 m 000 000 mm 00 0 0 000000000 000 00 00009 .mm>0p0000 p00000000 0p03 omHHmCm 0:000m .mhmzmpp p03 00 080p np03 Am0mmn pm; 0 co pamo0mmv COHpMMpCmocoo 0000 00pwom :0 mmmcmno .ma wands 53 wmzam> 00 Mo mmm00w>0 o mmsam> 03p mo mwm00m>m Q mmsam> 0300 Ho wmm00m>0 0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0000000 00.0 00.0 00.0 mm.o 0m.0 00.0 mm.0 000000 000 + 00000m 0003009 p03 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000000 0000000 000000 .0000000 000 0m.0 00.0 00.0 00.0 m0.o :m.0 00.0 QHmmMO mzaa0omnopomq om.0 mm.o 00.0 0N.0 00.0 0N.o 00.0 wso000m059 4m + mmwwo mSHHHomnopomq 0m.o 00.0 0m.0 mm.o N0.0 mm.0 0m.o 000.00 mpwoucmp 8500om mm.o 00.0 00.0 mm.o 00.0 0m.0 00.0 0&0.m0 000 no0mpmummo0osm m 000 nm0 mm 00 0 m pzmapmwua ham A.©.pCoov ma mHDwB 54 Table 10. Analysis of variance of acetic acid content of wet brewers' grains. Trial 1 Degrees Mean F Source Of Variance of Freedom Square Statistic Additives 15 0.2616 5.hO** Yeast 1 0.1506 3.11* (Additives) (Yeast) 15 0.1072 2.21* Duplicates/trt. combination 32 0.0485 Days 3 0.3251 14.67** (Additives) (Days) 45 0.0770 3.h9** (Yeast) (Days) 3 0.3607 16.28** (Additives) (Yeast) (Days) 45 0.0571 2.57** Residual error 96 0.0222 * Significant P<.10 ** Significant P<.01 55 0000h Rea 05am 0cflmnw .0003000 003 Aolllllov 00000 03on003 0:0000 .0003000 003 Aolllllov .0 00000 .0000» 0000003 00 £003 00H00C0 0:0000 .0003009 003 no 0Q00coo 0000 000000 CH 0wC0no .: mhswfim 56 1H8l3M 13M :IO 1N3383d 76 32 22 15 DAYS AFTER ENSILING 57 than the control (0.82% vs 0.56%). Low levels (0.14 to 0.22%) were found in the grains treated with ammonium isobutyrate, paraformaldehyde, formic acid plus paraformaldehyde, potas- sium carbonate plus propionic acid, sulfuric acid, formic acid and in the grain sealed with foam. After 76 days only the grains treated with potassium carbonate and sodium benzoate had acetic acid concentrations higher than the control (1.06 and 0.88% vs 0.72%). Treatment with formic acid plus paraformaldehyde resulted in a low acetic acid concentration (0.25%) which compares with 0.72% for the control and 0.50% for the sealed grains. In general, addition of acid or basic chemical compounds resulted in a decrease in acetic acid content in the silage, but the addition of sugars did not have any effect after 32 days. However, on day 76 only formic acid plus paraformalde- hyde lowered acetic acid concentration of the grain (Table 13). Propionic acid. Propionic acid was low in all grains during the first 15 days of the ensiling process, but started to increase at day 22 until day 76 when the average was 0.40% (Figure 5). Ensiled wet brewers' grains without yeast had greater propionic acid content (P<.01) than did grains to which 10% yeast was added (Table 16). On day 32 silage from grain treated with ammonia, formic acid, paraformaldehyde, formic acid plus paraformaldehyde, sulfuric acid, dried molasses, sucrose—starch mix, sodium ben- zoate, lactic acid culture or sealed with foam, had low concen- trations of propionic acid (0.02 to 0.08%). Higher (Table 15) 58 00000 000 05am 000000 .0003000 003 Aolllllov 00000 0000003 000000 .0003000 003 fielllllfv . .0 00000 .00000 0000003 00 0003 0000000 000000 .0003000 003 00 0C00000 0000 00000900m 00 000000 .m 005000 59 °.°9'°..V_C~g “4913M 13M so manna 76 32 22 15 DAYS AFTER ENSILING 60 00.0 00.0 00.0 00.0 00.0 00.0 00.0 A&o.mv 00000000 00000 no.0 00.0 00.0 no.0 00.0 00.0 00.0 0.m 00 00 0000 008000 :0.0 00.0 00.0 00.0 00.0 00.0 00.0 0.0 00 op 00000 m:.o 0m.o 00.0 mm.o m0.o mm.o 0m.o 00m.ov 0000 000000000 + ARM.HV momma 00.0 0m.o :0.0 00000000 0000 om.o 00.0 0m.o 0:.0 00.0 00.0 00.0 000.00 moowm no.0 00.0 :0.0 00.0 00.0 00.0 00.0 000.00 0000000000000000 + 0.0 00 00 0000 000000 00.0 mm.o No.0 no.0 No.0 00.0 00.0 000.00 0000000000000000 00.0 0:.0 50.0 00.0 No.0 00.0 Ho.o Afimwohpwc Rm.ov 00G088< 0m.o 00.0 om.o 0m.o 00.0 00.0 N0.o 00m.ov 00000000000 00000000 0N.o 00.0 0m.o 00.0 00.0 00.0 00.0 00m.ov 0000000000 00000000 00.0 00.0 No.0 :0.0 00.0 00.0 00.0 000.00 0000 000000000 00.0 00.0 om.o 00.0 00.0 00.0 00.0 000.00 0000 000000000 :0.0 mm.o ©N.O 0N.o 00.0 00.0 00.0 HOHPQOU m 000 pmm 00 m0 0 0 000000000 000 00 00009 .00>000000 000000000 0003 0000000 000000 .0003000 003 00 0000 0003 000000 003 0 00 00000000 0000000000000 0000 000000000 00 0000000 .m0 00000 61 00000> we 00 00w000>0 0 00000> 030 00 000000>0 0 00000> 0000 00 00000000 0 00.0 00.0 NN.0 m0.0 NH.o 00.0 00.0 0w000>< 00.0 0N.o m0.o 00.0 mo.o :0.0 :0.0 000000 000 + 000000 .0003000 003 mm.o 0m.o 00.0 mm.o 00.0 00.0 :0.0 000000 0000003 000000 .0003000 003 00.0 00.0 0N.o No.0 00.0 :0.0 00.0 000000 0000000000000 00.0 m:.0 00.0 m0.o 00.0 00.0 00.0 0000000000 44 + A0000 0000000000000 00.0 NN.0 00.0 00.0 00.0 00.0 00.0 000.00 00000000 000000 00.0 om.o :0.0 00.0 00.0 00.0 00.0 0&0.mv N00 000000I000000m m 000 000 00 m0 0 0 000000000 000 0.0.00000 00 00000 Table 16. Analysis of variance of propionic acid content of wet brewers' grains. Trial 1 . Degrees Mean F Source Of Variance of Freedom Square Statistic Additives 15 0.5984 219.76** Yeast 1 1.1732 430.83** (Additives) (Yeast) 15 0.0605 169.11** Duplicates/trt. combination 32 0.0027 Days 3 0.1095 18 . 24H (Additives) (Days) 45 0.0170 2.8#** (Yeast) (Days) 3 0.0106 1.76 (Additives) (Yeast) (Days) #5 0.0276 4.59** Residual error 96 0.0060 ** Significant P<.01 63 levels were found in the grains treated with prOpionic acid and in the control that had 0.26%. On day 76 grain treated with formic acid plus paraformal— dehyde had the lowest propionic acid content (0.08%), while high values were determined on samples from grains treated with propionic acid. Grain to which ammonium isobutyrate, ammonia, potassium carbonate or a lactic acid culture (L4 page; plus L; bulgaricus) was added had propionic acid concentrations from 0.00 to 0.79% while the control had 0.33%. Butyric acid. During the first 15 days of the experiment butyric acid concentrations in the grains were low (0.11% at day 15), but then started to increase and reached a maximum on day 76 (0.98%) (Table 17). Grains with yeast had less butyric acid than grain without yeast until day 32, neverthe- less, concentrations at the end of the experiment were similar (Figure 6). Butyric acid was not detected in grains ensiled with 1% prOpionic acid until day 32. However, on day 76 this silage had a concentration of 0.02%. This value was the lowest for all treatments. Low values on day 32 were also observed in the grains treated with 0.5% propionic acid, paraformaldehyde, potassium carbonate plus propionic acid, formic acid, sulfuric acid, dried molasses and sucrose-starch mix. On day 76 only the grains treated with 1% propionic acid had butyric acid contents lower than control (0.#2 vs 0.66%). Grains ensiled by sealing with foam had 0.82% butyric acid after 76 days. Despite their butyric acid content, 64 mm.0 0H.H :H.o no.0 00.0 50.0 00.0 A&0.mv mommmaos coflho mm.0 :0.H 0N.0 mm.0 00.0 50.0 00.0 0.0 mm op aflom oflspom ma.o No.0 oa.o mm.o no.0 mo.o oo.o o.m mm op sommm 0:.0 om.H wfl.o ms.o sm.o 0:.0 00.0 Asm.ov snow oaaoaaopa + Asm.av moons 0:.0 mm.0 0H.0 mp:MHMom Emom Hm.o :©.H oo.H om.a 00.0 ma.o oo.o Asm.av moumx Hm.0 05.0 m:.0 00.0 00.0 mH.0 00.0 A&H.0V muznmvamsnoMmumm + 0.m mm op vflom owanom 0m.0 mm.a HH.0 00.0 NH.0 00.0 00.0 ARH.0V ocmsmuamahommnmm dN.o mo.H om.o dm.o 00.0 00.0 00.0 Afiwwohpflfi Rm.ov NHQOES< :N.0 mm.a 0:.0 dm.0 MH.0 No.0 00.0 A&m.0v op¢hhp§pomw Esfizoee< 0N.0 00.0 dm.0 0m.0 00.0 No.0 00.0 A&m.0v opmcowmoum 85wcoae< no.0 N:.o 00.0 00.0 00.0 No.0 00.0 ARO.HV cwom OHQOHQon 0H.o ms.o mH.o HH.o Ho.o 00.0 00.0 Asm.ov chow cascamoum HN.O 00.0 3N.o mN.o :0.0 no.0 00.0 HOHPCOU m 205 9mm mm ma 0 N psmspmmna ham ma dawns .mm>wpwwcm Pamnwmmfiu spa; cmawmso mcwmnw .mnmsmhp pm; 2H asap spas Amanda pm; m cw pamohomv cowpmspsoocOo cwom ownzpsn CH momsmno .ma magma 65 mosam> 00 mo mowmnwbm o mosam> 039 Mo mommgo>m Q mosam> 930% Mo mommno>m m Nm.o mm.o Hm.o 0:.0 HH.O 50.0 00.0 mWMHm>< mm.0 00.0 NN.0 mm.0 00.0 :0.0 00.0 opmmoh fioa + mcwmpm.myozonn Po; 0m.0 00.0 00.0 mm.0 NH.0 00.0 00.0 opmmmh Psonpws mcfimnw .mumzmnp Pm; ::.o mm.o 0H.H 0:.0 No.0 ma.o oo.o pwmmmo msaawmmpopomq 0:.0 N0.H mm.0 $5.0 00.0 00.0 00.0 mooflnmeSQ 4m + wommo mSHHHompopomH 0m.0 00.0 mm.0 No.0 00.0 :0.0 00.0 A&H.0v mpmoacmp Edwuom m:.0 HN.H HN.0 m0.H 00.0 :0.0 00.0 ARO.MV was sonmvmuomonozm w nos 9mm mm ma m m pawspmmna awn A.0.pcoov ma magma 66 pmmoh §0H moan mcflMpw .mpmzmpp Pm; Aolllllov pmwoh Psonpfla mcfimnm .mpozmnp pm; Aolllllov .H awake .memz PSOQPHS 90 Spa; umHHmcm mcflmmw .mmmamMQ #03 mo Pampcoo vflom ownhpsp Cw mwcmzo .0 opsmwm 67 won» 13M :IO lNaoaad 76 32 22 15 DAYS AFTER ENSILING 68 these grains had a good general appearance and their smell was not offensive and best of all treatments. From day 2 to day 32 the effects of additives, yeast and days were significant (P<.01) on butyric acid concentration of brewers' grains (Table 18). Lactic acid. Average lactic acid concentrations reached the highest level after eight days of ensiling (0.70%) and then decreased slowly until day 76. During the first 15 days, lac- tic acid was lower in the grain with 10% yeast, but after day 22 it was higher than in the grain without yeast (Figure 7). Until day 22 the difference in lactic acid content between the grains with and without yeast was not significant (Table 19). On day 32 concentrations were also similar (0.67 vs 0.65%), but on day 76 grains with yeast had about twice as much lactic acid as the grains without yeast (0.56 vs 0.25%). Statistical analysis of the changes in lactic acid con- tent of the grains on days 2, 8, 15 and 22 gave the following results: (1) The control did not differ from the grains treated with acid compounds (propionic, formic or sulfuric acid). (2) Addition of bases (ammonia or potassium carbonate) resulted in an increase when compared to the control or to the acid treated grains (P<.01). (3) Treatment with propionic acid resulted in lower lactic acid concentrations in the grains than treatment with ammonium pr0pionate or potassium carbonate plus propionic acid (P<.05). (h) Grains treated with propionic acid did not differ in lactic acid content from those treated with formic acid. (5) Lactic acid concentrations Table 18. 69 Trial 1 Analysis of variance of butyric acid content of wet brewers' grains. Source Of Variance ogegizggom Sfiizge Statistic Additives 15 0.2981 l5.87** Yeast 1 0.3496 18.60** (Additives) (Yeast) 15 0.0454 2.41* Duplicates/trt. combination 32 0.0188 Days 3 2.7092 l47.85** (Additives) (Days) 45 0.1540 8.40** (Yeast) (Days) 3 0.1519 8.29** (Additives) (Yeast) (Days) 45 0.0362 1.97** Residual error 96 0.0183 * Significant P<.05 ** Significant P<.01 70 undo» 50H mafia mcflmnm .mpozmnp pm; Aolllllov pmdwz pzonpfiz mcwmum .muozmun Pm; Acnllllov .H amass .pmdmh pSOSPfiB ho spa; Umaamcm msflmmm .mumzohp #03 mo Pampzoo Ufiom oavoma CH mwcmso .5 mpzmflm 1H9l3M 13M 1N3383d 76 32 22 15 DAYS AFT ER ENSILING 72 m5.0 m0.0 0H.H 0H.H 00.0 00.0 :0.0 A50.mv 00000H00 00000 mm.0 0:.0 0N.0 N:.0 :0.0 05.0 :0.0 0.m mm op 0000 ofiShom 00.0 00.0 00.H 00.0 00.0 05.0 00.0 0.0 :0 op 00000 mw.a 00.0 mm.m mm.H 00.H mN.N :0.N 55m.0v cwow owcowmong + 55m.av MOONM 00.H 00.0 0N.H mdeHdom 800m mm.0 00.0 0H.0 mm.0 0m.0 00.0 mm.0 Asm.av m0omm :N.0 mm.0 0m.0 ma.0 0N.0 00.0 mm.0 A5H.0v ouznowamsuowmnmm + 0.m mm op 0000 008900 0m.0 53.0 mw.0 0:.0 mm.0 0H.0 0:.0 ARH.00 ochnocamshohmnmm 00.0 NH.0 0H.0 m0.0 00.H 00.0 ma.0 Ac0wonpfla 50.00 00:0250 5:.0 HN.0 :H.o mm.0 mm.H :N.0 om.0 Afim.0v 0PMthsnomH ESHSOEE< 00.0 0H.0 NH.0 00.0 m0.0 0H.0 H0.0 Asm.0v 0002000000 ssflcoss< dm.0 :m.0 m:.0 0H.0 0m.0 NH.0 00.0 ARO.HV 0000 oHQOflmopm mm.0 0m.0 00.0 00.0 00.H 0H.0 50.0 Asm.00 0000 000000000 m:.0 no.0 mN.0 ::.0 0H.H 0:.0 mm.0 Honpfioo m 0.05 pmm mm ma 0 m 000800000 5.00 0H H0099 .mo>wpa000 pawnmmflwu upws vmawmco mcwmpw .0003099 903 CH 0809 £903 Amflmmn #03 0 so pzmohmmv zoflpmppcmocoo 0000 owvoma CH mmwcmno .0H wands 73 mmsam> 00 mo mow000>0 o 00500> 030 Mo www000>0 p mmsaw> 030% Mo 00m000>0 0 00.0 00.0 00.0 00.0 00.0 05.0 00.0 0000000 00.0 00.0 50.0 05.0 00.0 00.0 00.0 000000 000 + 000000 .0003000 002 mm.o mm.0 m0.0 0:.0 00.0 m5.0 00.0 00000» 0500003 0:0000 .0003000 003 00.0 00.0 50.0 m0.o no.0 mm.0 no.0 pmmmmo msaawo0popomg 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0000000000 «A + 00000 0000000000000 05.0 00.0 00.0 :0.0 Hm.0 :m.a Hm.0 “RH.0V opmoucmp ez0oom 00.0 00.0 :0.0 00.0 0N.0 5N.H mm.0 0&0.mv x0e noumpmnomonosm w 005 nmm mm m0 0 N #:0800009 ham A.U.Pcoov 0H wands 74 in treatments with formic acid alone or with formic acid plus paraformaldehyde were similar. (6) Addition of molasses or of a sucrose-starch mixture did not produce a change in lactic acid concentration when compared to the control or among them- selves. (7) Addition of sodium benzoate or of a Lactobacillus ‘gasgi plus L: bulgaricus culture did not change the lactic acid content of the grains when compared to control (Table 20). On day 32 grain treated with potassium carbonate plus propionic acid had the highest lactic acid concentration (2.85%). Grain in the buckets sealed with foam had 1.26% lac- tic acid. Grain to which ammonium propionate, ammonium isobu- tyrate, ammonia, potassium carbonate, formic acid or a lactic acid culture (£4.2éééi) was added had lactic acid concentra— tions from 0.07 to 0.20%. All other treatments had lactic acid concentrations higher than the control that had 0.28% at day 32. After 76 days of ensiling, grains from all treatments, except those with L;.2§§§l culture, had higher lactic acid contents than control. The highest values were found in the grains sealed with foam (0.86%) or treated with potassium car- bonate plus propionic acid (0.96%) and sodium benzoate (0.88%). The decrease in lactic acid concentration with time may be the result of secondary fermentation in the ensiled grains. Ammonia. The ammonia content of ensiled brewers' grains measured on days 2, 8, 15 and 22 after ensiling was variable, but increased markedly from day 8 to 15 and from day 32 to 76 (Table 21). Until day 15 and on day 32 the grains to which yeast was added had higher ammonia content than the grain 75 Table 20. Analysis of variance of lactic acid content of ensiled wet brewers' grains. Trial 1 Degrees Mean F Source of Variance of Freedom Square Statistic Additives 15 2-4626 35-73** Yeast 1 0.0100 0.15 (Additives) (Yeast) 15 0.2922 4.24** Duplicates/trt. combination 32 0.0689 Days 3 1.5783 28 .7o** (Additives) (Days) 45 0.0873 8.86** (Yeast) (Days) . 3 0.4017 7.30** (Additives) (Yeast) (Days) #5 0.1287 2.34** Residual error 96 0.0550 ** Significant P<.01 76 mm 000 00 0m 0 0m m A&o.mv 00000008 00000 in 0N0 :0 0m N0 00 mm 0.m mm 00 0000 008000 00 00 00 00 00 m0 0 .0.0 :0 00 00000 00 00 0 00 00 00 0 000.00 0000 000000000 + 000.00 000000 :0 om 0 00000000 800m 00 000 00 mm 00 00 00 000.00 00000 :0 :m m 0 N0 00 n ARH.0V 0000000080000000 + 0.m mm 00 0000 owShom 00 000 00 m 00 0 0 000.00 0000000000000000 an 000 0m m0 m: 0m 0m hammonpwn Rm.0v 00Sofi§< m0 mma mm mm 00 0: 0m “Rm.0v 00000059000 8500088< 00 0N0 mm 00 000 m :m 00m.0v 0000000000 8500088< mo 000 0: mm 000 m m: 0R0.Hv 0000 oonwmonm mm 0N0 mm on ma 0 0: Afim.ov 0000 owcowmopm mm 000 mm N0 00 0 00 0000000 M 900 mm mm m0 m N 000800009 HMO 00 0000s .00>000000 000000000 0003 0000000 00000m .0003000 003 00 0800 0003 000000m 003 w 000\d0m0000a wev 0000000000000 0000880 G0 0wcmno .HN 00009 77 00500> 00 00 00000000 0 00500> 030 00 000000>0 p 00500> 0500 00 000000>0 0 mm 000 0N mm 0: 00 m0 0m000>< m0 000 00 00 00 00 mm 000000 ~00 + 000000 0000000 003 00 000 00 00 mm 00 00 000000 0000000 000000 .0000000 003 mm 000 m0 mm 0 on 0 900000 0500000000000 om 000 mm :m o 00 0 0500000050 4m + 00000 0500000000000 0m mm mm mm 0 mm 00 000.0v 00000000 850000 mm 0N0 :0 :m o :0 0 0&o.mv x08 50000010000050 m 000 mm mm 00 0 0 000m 0Q080000a A.0.0800v HN 00908 78 without yeast, but at day 76, grains without yeast had only lightly more ammonia than grains with yeast (Figure 8). When formic acid plus paraformaldehyde were added to the grains, the final levels of ammonia were much lower than the average for all treatments (54 mg/100 g vs 106 mg/100 g). Formic acid or paraformaldehyde alone did not decrease the levels of ammo— niacal nitrogen from that of control. Addition of potassium carbonate plus prOpionic acid and sealing the buckets with foam resulted in ammonia levels lower than in the control after 76 days of ensiling (72 and 80 mg/100 g vs 100 mg/100 g). The highest value was found in the grains that were treated with 0.5% ammonium isobutyrate (139 mg/100 g) after 76 days of ensiling (Table 21). Analysis of variance of the ammoniacal nitrogen content of brewers' grains from day 2 to day 22 indicated that the effects of additives, yeast and days, were significant (P<.Ol) (Table 22). Ammonia is a product of protein hydrolysis caused by proteolytic bacteria. In this experiment only formic acid plus paraformaldehyde inhibited, although not totally, the growth of this kind of organisms and the formation of ammonia. Keeping the grains under anaerobic conditions or the presence of the different additives tested, did not result in ammonia levels lower than in the control. Correlations among measured constituents. In this experi- ment wet brewers' grains stored in plastic buckets had consi- derable and excessive deterioration after 32 and 76 days of 79 pmmoh §oa mSHm mflwdhw .mhozomn Po; Aolllllbv pmdmh poonpfl3-mzamsm .mhozmup pm; AOIIIIIOV .H awake .Pwmoh pzonpfla no sea; meHmCm mcflmsm .mpmzwhn pm; mo.pcmpcoo Cowonpwc awowficoeem Ca owcmno .m opsmflm nool' 80 1H9I3M 13M :10 6001/3“l 76 32 DAYS AFTER ENSIllNG 22 l5 0. 81 Table 22. Analysis of variance of ammoniacal nitrogen content of ensiled brewers' grains. Trial 1 Degrees Mean F Source of Variance of Freedom Square Statistic Additives 15 5270.75 20.#6** Yeast 1 7821.19 30.36** (Additives) (Yeast) 15 lh35.82 5.57** Duplicates/trt. combination 32 257.57 Days 3 9969.66 #2.76** (Additives) (Days) 45 3109.07 13.33** (Yeast) (Days) 3 1205.14 5.17** (Additives) (Yeast) (Days) 45 522.79 2.24** Residual error 96 233.17 ** Significant P<.Ol 82 ensiling. Only the grains kept under anaerobic conditions had a low proportion of spoiled material. Come improvement was no- ticed when propionic acid, formic acid plus paraformaldehyde, a sucrose-starch mix or a lactic culture were added to the grains. All pH values were generally low (about 4.2) but were not related to the spoilage percent (Table 24). After 32 and 76 days of ensiling the grains with the most spoilage (treated with potassium carbonate) also had the highest pH values (Table 23). Grains sealed with a cover of foam showed some spoilage in only one of the four replications and had the lowest pH. Nevertheless, other treatments that had low pH values had a high amount of spoilage. There was a positive correlation between temperature and spoilage (r = .68). Aerobic fermentation increased the tem- perature in the silage. Range of average temperatures was rather narrow, from 76.20F in the grains sealed with foam to 80.10F in the grains treated with ammonium propionate. The extent of spoilage was negatively correlated with ethanol concentration (r = -.7l), but correlations between spoilage percentage and other measurements were not signifi— cantly different from zero (Table 24). The pH was positively correlated to acetic acid (r = .56) and to butyric acid (r = .79) concentration. High pH values were probably favorable for the growth of microorganisms that degraded organic matter to acetic and butyric acid. Average temperature was positively related to acetic acid (r = .49) and to butyric acid (r = .65). on.: mn.o on.o oa.o an.o ma.o om.o m.m~ «.mn m.ma 5.55 mm.n .n.:m o.n :9 o» caom oaksmasw oo.n oa.H mu.o No.0 mn.o m~.o mw.o mnmm ~.wn ¢.H~ m.mu nm.¢ n.mm Akn.ov vwom cacowqoua msaq momma ma.n oo.H w:.o ma.o :~.o no.o mm.o n.:~ ~.mn m.H~ «.mm om.n m.n mpcmauom emom oa.m oo.o ~m.a mm.o om.o :m.o oo.H n.m~ :.mn n.a~ o.au mm.¢ o.on Amn.av ovMCODumo adammMuom q; H:.~ :n.o em.o 00.0 o~.o ma.o w~.H o.n~ n.nn ~.n~ n.ms us.n n.:~ «H.o. 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Correlation coefficients among measured constituents of ensiled wet brewers' grains. Trial 18. x Y rb spoilage pH 0.35 temperature O.68** dry matter -0.0l protein 0.08 acid detergent fiber 0.04 acid detergent nitrogen -0.22 ethanol -0.7l** acetic acid 0.34 propionic acid 0.39 butyric acid 0.17 lactic acid -0.07 ammoniacal nitrogen 0.31 pH temperature 0.36 dry matter -0.23 protein 0.31 acid detergent fiber 0.16 acid detergent nitrogen -0.18 ethanol -0.l3 acetic acid 0.56* propionic acid 0.28 butyric acid O.79** lactic acid -0.03 ammoniacal nitrogen 0.44 temperature dry matter -0.l4 protein 0.18 acid detergent fiber -0.03 acid detergent nitrogen -0.33 ethanol -0.32 acetic acid 0.49* propionic acid 0.65** butyric acid -0.08 lactic acid -0.30 ammoniacal nitrogen 0.46 dry matter protein —0.75** acid detergent fiber 0.11 acid detergent nitrogen -0.l3 ethanol 0.21 acetic acid 0.11 propionic acid 0.03 butyric acid -0.03 lactic acid 0.06 ammoniacal nitrogen -0.17 Table 24 (cont'd.) 86 x Y rb protein acid detergent fiber 0.12 acid detergent nitrogen 0.04 ethanol -0.l4 acetic acid 0.13 propionic acid -0.03 butyric acid 0.22 lactic acid —0.28 ammoniacal nitrogen 0.29 acid detergent fiber acid detergent nitrogen 0.04 ethanol 0.17 acetic acid 0.34 propionic acid —o.o3 butyric acid 0.08 lactic acid -0.12 ammoniacal nitrogen 0.16 acid detergent nitrogen ethanol -0.35 acetic acid —o.48 propionic acid -o.31 butyric acid _o.o3 lactic acid —0.11 ammoniacal nitrogen -0.40 ethanol acetic acid -o.05 propionic acid 0.03 butyric acid —o.13 lactic acid 0.26 ammoniacal nitrogen -o.1o acetic acid propionic acid 0.21 butyric acid 0.28 lactic acid -o.33 ammoniacal nitrogen 0.44 propionic acid butyric acid -0.09 lactic acid —0.03 ammoniacal nitrogen 0.34 butyric acid lactic acid -0.11 ammoniacal nitrogen 0.40 lactic acid ammoniacal nitrogen —0.59* a correlation coefficients determined on values from Table 23 b critical values for a .05 = 0.482, for a .01 = 0.606 * significantly different from 0 (P<.05) ** significantly different from 0 (P<.Ol) 87 Dry matter was negatively correlated to protein content of the dry matter (r = .75), but not to the other fractions or measurements of the silage. The correlation coefficient between lactic acid and ammonia content of the silage was negative (r = -.59). Prob- ably when lactic acid reached a certain concentration, unfavorable conditions existed for proteolytic bacteria growth. Addition of autolysed yeast increased the conservation of wet brewers' grains under the conditions of this experi- ment. When 10% yeast was added to the grains several changes were noticed, such as l) spoilage decreased from 32.0 to 28.9%, 2) lower pH, 3) lower storage temperatures, 4) lower values for acid detergent fiber and acid detergent insoluble nitrogen, 5) lower acetic, propionic and butyric acid concen- trations, and 6) increased ethanol and lactic acid contents. Dry matter, protein and ammonia concentrations remained about the same with or without the addition of yeast. Silage obtained in this experiment generally had high amounts of acetic, propionic and butyric acid in relation to ethanol and lactic acid contents. High amounts of ammonia indicated considerable protein breakdown. Anaerobically stored grains had very low spoilage and lactic acid content was greater than all other treatments. Yet, there was con- siderable acetic, propionic and butyric acid and ammonia formation in the anaerobically stored grains. Evidently the secondary fermentation that decomposes lactic acid and protein was inhibited more by storing the grains anaerobically than 88 with the other treatments. In general grains treated with basic compounds had more spoilage than grains with other treatments. Organic acids added to the grains decreased the proportion of spoiled material, but were not totally effec- tive in preventing spoilage at the levels used. Neither formic acid or paraformaldehyde alone increased recovery of good silage, but when both were used together, recovery of good material was greater than for other treatments. Neither sodium benzoate or ammonium salts of propionic and isobutyric acid decreased spoilage. A sucrose-starch mix improved recovery, but dried molasses did not. The proportion of spoiled material in the silage increased between day 32 and day 76 after ensiling. This change cor- responded to an increase in pH, crude protein, acid detergent fiber, acid detergent insoluble nitrogen and ammonia content. Crude protein content was 31.4% in the fresh grains. On day 32 the average for all treatments was 34.3% and 40.9% on day 76. This increase was probably due to a seepage of ammonia from the upper part of the grain that was spoiled and had a more extensive protein degradation. Even when crude protein in the silage increased with time, total recovery of protein was low. Proportion of spoiled grain that was discarded was 26% on day 32 and 35% on day 76. Thus, average crude protein loss after 32 days of storage was 19% and 15% after 76 days. Ammoniacal nitrogen was 1.24% of the total nitrogen after 32 days and 4.47% after 76 days of ensiling. Considering that ammonia is a product 89 of protein degradation, true protein recovery in the silages was even lower than the recovery of crude protein. Increases in acid detergent fiber and acid detergent insoluble nitrogen contents could be explained by the increase observed in temperature. Fresh brewers' grains had 0.92% acid detergent insoluble nitrogen content. This value is high com— pared to normal values for haylages and silages. Haylage from the middle and bottom part of a vertical silo had 0.32% acid detergent insoluble nitrogen (Thomas, 1976). The volatile fatty acids (acetic, propionic and butyric), and ethanol in the grains increased from day 32 to day 76 and the concentration of lactic acid decreased. These phenomena indicate that a secondary fermentation occurred, in which lactic acid and other constituents were degraded to organic acids and ethanol. In this trial the ensiled grains termed good after 76 days storage were judged to be of lower quality than those after 32 days. If ammoniacal nitrogen, lactic and butyric acid are used as indicators of silage quality, then storage longer than 32 days under conditions as in this trial are contraindicated. Spoilage was a direct result of exposure of the grains to air. Addition of yeast improved preservation. Changes in the protein fraction of the silage should be avoided with the use of higher levels of preservatives or by maintaining anaerobic conditions during storage. 90 Trial 2 After studying the results of trial 1, a second experiment was carried out. Larger quantities of grain were used and the silage was kept in steel barrels instead of plastic buc- kets. No samples were taken from the barrels during the storage period so as not to disturb the fermentation process. Thus, in order to follow the chemical changes during ensiling, samples of fresh wet brewers' grains were put in test tubes, kept in the laboratory (about 75°F) and frozen on days 5, 7, 10 and 13 after the beginning of the experiment. Determination of the different constituents for days 32 and 60 were made on samples taken from the silage when the barrels were emptied. In all cases, the samples were taken from the portion of the material that was not spoiled. The barrels containing the grain were exposed to ambient temperature during the months of August, September and October,. to test storage conditions during warm weather. Complete pre- servation was achieved in trial 1 by keeping the grains under anaerobic conditions. Addition of propionic acid, formic acid plus paraformaldehyde, a sucrose-starch mix or a lactic culture reduced spoilage. These treatments or modifications were used in this experiment. Furthermore, other ways to maintain anaerobic conditions such as covering the grains with different materials were tested. Recovery. Percentage of recovery of wet brewers' grains stored for 32 or 60 days is presented in Table 25. 91 H.mo o.mo m.mn oHSPHSO ewes capomq H.0m m.am m.om AROHV Shoo vcsopo 0.05 u.mm ¢.om madam one mo mop Go choc Unsono o.ms m.mm m.ms Asmv mommmaoe Uflsufiq o.mm m.mm w.mm macaw map mo do» so mommdaos chUHA :.Ns m.mw o.ms cfimnw one mo now Go mommsaoa aha 0.3m n.0m m.sw wanw map mo no» so dommo N.Hm o.wm 3.3m :Hmnm 639 Mo now Go mflopmoswq c.mc m.mc m.ms Aema ocmv a m m m.ms m.cc m.ms Asom.ov 3ommm 3.0m 5.0m m.bm ARH.ov muznmchSMOMNHMQ mafia A&3.Hv ewes oaShom m.sm o.mw m.mm ARNV Uflom camowmonm o.nm m.om m.mm Asav cwom cacowmonm m.mm 5.50 m.mm Honpcoo m nmhwc ow «made mm pcospmmne poems ooom R ampwm voom R N Hwfihe .mcwmnw .mhozopn pm: anwmco mo hhm>oomm .mm wanes 92 osam> oco n mozam> esp mo mwmno>m m 3.55 m.m5 m.mm mwwho>< m.mm c.ooa 0.55 ace so ewes; mo Haze men cfipmcaa c spa: ccamcm p.35 o.©w m.mw AROHV shoe Unconm + mQSPHSO owpomq m.a5 5.mb w.o5 A55v mommmaos + canvaso capowq mhmu om whee mm M mopym doom 5 Wayne doom 5 psosvm6se A.U.Pcoov mm wands 93 Six days after the beginning of the experiment spoilage was noticed in the grains treated with BHA, 10% ground corn and in the control with no additive. Small mold colonies were seen on the surface of the grains covered with limestone or dry molasses. After 11 days, only the grains treated with propionic, formic and sulfuric acid had not developed surface spoilage. The grains covered with molasses, calcium sulfate and ground corn smelled rotten. Flies started to grow beneath the surface of the grains with no additive and of the grain with BHA, 7% molasses, 10% ground corn and lactic acid culture. Grains treated with propionic acid (1%) and sulfuric acid started to have surface spoilage after 15 days of ensiling. At this time about one centimeter of the surface of the grain had dried in most of the treatments while spoilage continued to develOp underneath. Dry limestone put on top of the grain (about 1 cm thick) absorbed water from the grains and remained wet until day 22 after the beginning of the experiment. Calcium sulfate was layered on top of the grains as a slurry and remained soft until day 11, then started to dry and harden. After 22 days the calcium sulfate layer was hard and started to crack especially on the edges, then flies grew beneath the cracked surface. Two of the three barrels of each treatment were emptied after 32 days of ensiling, and the remaining one after 60 days. Recovery of good silage and quantity of spoilage were measured. Grain from the barrels that had a plastic bag filled with water on top did not have any spoilage and the silage 94 looked and smelled fresh. Weight of the water in the plastic bag formed a seal between the grain and the environment keep— ing the grain entirely anaerobic. Grain treated with propi- onic acid (2%) and with formic acid plus paraformaldehyde had a dry surface and no spoilage after 32 or 60 days of ensiling. Silage from these two treatments had an acid smell and a darker color than others. Grain treated with sulfuric acid was darkest of all. All other treatments had more or less surface spoilage and fly maggots growing thereon. When the spoiled material was separated and discarded all remaining portion looked and smelled about the same from all treatments, except those treated with acids. In all cases the spoiled material had a dark brown color, an offensive-rotten smell and fly maggots growing in it. Covering the surface with limestone, calcium sulfate, dried or liquid molasses, and ground corn did not prevent spoilage or the growth of flies. Plastic bags lining the barrels allowed no seepage from the storage container. Grains from the treatments covered with limestone, calcium sulfate or liquid molasses had less spoilage than did control. Covering the surface with dried molasses or ground corn did not have any effect on the recovery when compared to the control. Addition of a lactic acid culture did not improve storability. Liquid molasses or ground corn mixed with the grain did not have any effect on recovery. Propionic acid at a level of 1% allowed more spoilage than did 2% 95 propionic acid. Acidification of the mass with sulfuric acid did not increase recovery of good material. Treating the grain with butylated hydroxyanisol (BHA) decreased percent— age recovery. This additive probably did not have any effect on the fermentation, but the initial mixing allowed more contact with air which may have increased spoilage. After 32 days of ensiling the spoilage for all treatments averaged about 17%, and after 60 days spoilage increased to about 25%. After 60 days the grains with no additive, with sulfuric acid, BHA, dry molasses on top of the grain and those with a lactic culture, had about 35% spoilage. Grains treated with propionic acid (1%), ground corn on top or mixed with ground corn had an average of 30% spoilage while those covered with limestone, calcium sulfate or dry molasses had only 20% spoilage. Effectiveness in preventing spoilage was due either to airtight sealing that did not allow aerobic fermentation to occur, or to the presence of sufficient concentration of preservatives such as propionic or formic acid mixed with paraformaldehyde. Acidity per se was not a factor in pre- venting spoilage. Addition of sulfuric acid did not have a beneficial effect. Good conservation of brewers' grains silage was the result of preservative qualities of propionic acid, formic acid and paraformaldehyde. pH. Fresh brewers' grains had a pH of 5.3. This value decreased markedly at five days of ensiling when the treat- ments had an average of 3.98 (Table 26). 96 ow 500 no Hounmp oco 0:0 mm whee no maonnmn cap 809% swam» moamsmm no eonfisuopmc mosam> 9 means pmmp CH pmmx mmamsmm so confisuopop mosam> m Hm.m 00.0 00.0 5N.3 3m.3 05.0 mm.m cmcnc>< mm.m 0m.m «5.0 00.0 00.3 om.m 00.3 0030mm cm.3 05.0 05.0 00.0 05.m 00.0 00.3 Ascav choc cescnw + caspaso cflpccq mm.m m5.m mo.m om.m oa.3 om.m mm.3 A55v mommmaoe + chapaso owpomn 30.3 05.0 m5.m 0m.3 0m.3 00.3 mH.3 massage uses oflpomg 03.3 no.0 00.0 om.m mm.m 00.0 mm.3 A5030 choc 0:30am H0.m m5.m m5.m 00.0 m0.m 05.0 03.3 A550 mcmmcacs cflsweq No.0 00.0 mm.m m5.m 00.0 00.0 mm.m Aeca oomv a m m H0.N 00.0 mm.m 00.N no.0 om.m 00.m Asm.0v snow ownsmasm 0m.m 00.0 mm.m 0m.m 0m.m ma.m 03.0 Asa.00 ccsnccacencmccmm + As3.av cecc ceases 00.3 00.3 00.0 0m.3 0m.3 00.3 m0.m Asmv cecc ceccflacum 03.3 00.0 00.0 00.m 00.0 0H.3 00.3 “sac 0300 cflccflaoum am.m 00.0 mc.m mm.3 00.3 00.3 00.0 Hocpeco m 300 pmm 00H 00H 05 cm pecseccua HMO .mc>flpaccc PamnoHMflU spa; eoawmco mswmhm N HMHHB .mpoSmhn Pm; Cw mews new; ma :0 momcmso .om manna 97 On days 10 and 13 grains treated with propionic acid (1%), 10% ground corn or lactic acid culture had pH values above 5.3, while all others were below 4.25. All other treat— ments had a stable or decreasing pH value during the ensiling process. All samples taken from silages kept for 32 or 60 days in barrels had pH values of 4.0 or lower. Grains with formic or sulfuric acid added had the lowest pH values during the ensiling period. On day 60 the values were 3.30 and 3.00, respectively. At this time the grains treated with l or 2% propionic acid had a higher pH value than did control (3.9 and 4.0 vs 3.6). Addition of molasses or ground corn did not produce a silage having a pH lower than control. The pH of the grains to which a lactic culture was added were not lower than pH of similar treatments without the culture at day 32 or day 60. The pH values indicate that an active fermentation occurred during the early days of the ensiling process. Acid addition initially lowered pH of the grains, but with other treatments low pH values occurred as a result of the fermentation process. Temperature. The brewers' grains had a temperature of 140°F arrived by truck from the brewery. Thermocouple temperatures taken during storage are presented in Table 27. Ambient temperatures taken in the area where barrels were located had an average of 79°F during the time when the experiment was performed. 98 0.35 5.05 5.05 5.05 0.30 0.05 0.05 0.05 0.05 0.05 0.05 0.00 0.00 0550 00000000 000000 0.35 5.05 0.05 5.05 0.55 0.00 0.05 0.05 5.05 5.05 0.00 0.55 0.00 00000 000 00 000 so mommmaoa 003000 0.05 5.50 0.05 0.05 0.05 5.05 0.05 5.55 5.05 0.05 0.05 0.05 5.00 00000 000 00 000 do mmmmmaos 009 0.05 0.00 5.05 0.05 0.05 5.00 5.35 0.05 5.35 5.05 0.05 0.00 5.30 0000 000 0c 000 0c 0000 0.05 0.05 5.05 5.50 5.30 0.00 5.05 0.35 0.35 0.05 5.00 0.05 0.00 00000 000 MO 000 no oQopmmE00 0.05 5.35 0.05 0.35 5.35 0.05 0.00 5.00 5.00 0.05 0.05 0.05 0.00 0000 0000 0 m m 0.05 0.35 0.05 0.05 0.05 0.05 5.55 5.05 5.55 0.05 5.00 5.05 5.00 050.00 30000 0.05 0.05 0.05 5.00 5.00 0.00 5.05 0.35 0.05 0.05 5.05 5.05 0.00 050.00 0000000000000000 + 0R3.Hv 00cm owshom 0.05 0.00 0.05 0.00 5.05 0.50 5.05 0.35 0.05 0.05 0.00 0.00 5.00 0500 0000 000000000 0.05 0.00 0.00 0.55 5.05 0.55 5.00 0.05 0.55 5.35 0.05 0.05 0.00 0000 0000 000000000 0.55 0.05 5.05 0.05 0.55 0.05 5.05 0.00 5.00 0.00 0.05 0.05 0.00 0000000 m mm mm 50 mm 00 0000000 00 0 0 0 0 000000000 0N 0000B .mc000mco Ho meonpms Ho mm>0000uw 0C0000000 0003 000000 m0000m .0003009 003 00 0800 £003 Amv 00500000800 C0 mmmcmno .5m @0909 99 00500> 0500 00 000000>0 0 m.05 0.05 o.m0 0.50 0.05 0.N5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00500000800 0800080 n.05 0.05 5.35 3.05 5.05 5.05 0.05 3.05 5.55 5.M5 5.05 0.05 0.00 00000>0 5.05 0.00 0.00 0.00 0.50 0.00 0.05 5.05 5.05 0.00 0.00 5.00 0.00 000 000 80 00003 00 0050 wmp 0000000 0 0003 000000 0.55 0.05 0.05 0.05 0.05 5.05 0.05 0.05 0.05 5.35 0.05 5.05 0.00 00000 0000 005000 + 0050050 000000 0.05 0.50 5.05 n.05 5.05 0.05 m.mm 5.50 m.00 0.05 0.05 5.05 m.mm 0050 00000008 + 0050050 000000 0.05 0.55 5.05 5.05 m.m5 5.05 m.55 5.05 5.05 0.05 n.05 5.05 5.00 0050050 0000 000000 5.05 n.05 5.05 0.55 5.05 0.35 5.05 0.05 m.00 5.05 5.05 0.05 5.00 00000 8000 085000 5.05 0.05 m.m5 n.05 m.05 5.00 5.M5 0.05 m.05 0.05 o.mm o.m5 0.00 80000 080 00 000 80 8000 085000 m mm mm 50 mm 00 0000000 00 0 0 0 0 000000000 A.U.0800v 0N 0HDMB 100 The second day after ensiling, temperature of the stored grain was above ambient. By the fifth day, temperatures had decreased below ambient. Temperatures generally remained below ambient except for day 29. From day 11 to 15 tempera- tures of the silage were higher than for other days, corre- sponding to a higher ambient temperature. Temperatures were taken about midday and internal temperature may also have been affected by ambient temperature hours previous to reading. Temperatures among treatments were significantly different (P<.Ol, Table 28). During the experimental period, average temperature of the control, no additive, was higher than average temperature of the treatment sealed with a plastic bag full of water (77.8 vs 71.7, P<.lO). Temperature for treatments with 2% propionic acid, formic acid plus paraformaldehyde, limestone on top of the grain and calcium sulfate on top of the grain were not significantly different from the treatment covered with a plastic bag full of water. The latter had the lowest temperature of all treatments. Temperatures of treatments with propionic acid (1%), sulfuric acid, dried or liquid molasses on top of the grain were statistically similar. Temperatures of the treatments with BHA, lactic acid culture plus 10% molasses and lactic acid culture plus 10% corn did not differ significantly from control when individual con- trasts were made. Grains covered with limestone, calcium sulfate, dried or liquid molasses, had lower average 101 Table 28. Analysis of variance of temperatures of ensiled wet brewers' grains. Trial 2 . Degrees Mean F Source Of Variance of Freedom Square Statistic Treatments l6 214.0537 2.6l** Barrels/treatment 3% 81.8693 Days 11 1764.6683 185.#4** (Treatments) (Days) 176 21.7014 2.28** Residual error 374 9.5163 102 temperatures than uncovered grains without additives (73.8 vs 77.8), but the differences were not significant. Temperatures appeared to be related to the degree of anaerobiosis in the stored grains. Dry matter and protein. Average dry matter of the silage was 22.5% at day 32 and 22.2% at day 60 (Table 29). Dry matter content of the grains was increased up to 25 and 27% by the addition of liquid molasses or ground corn. Dry matter content for other treatments were similar to control, and similar for covered and Open barrels. Liquid was observed draining from the truck in which the grains came from the brewery, but during the experiment no moisture was lost since the barrels were lined with imperme- able polyethylene plastic bags. Sample for dry matter was taken from several places in the central portion of the partially mixed contents and would not reflect changes near the surface. Protein content of fresh brewers grains was 31.4% (dry matter basis) and had only slight changes during the ensiling process (Table 29). Addition of 10% ground corn or 7% liquid molasses decreased the protein concentration of the grains as expected. Apart from these treatments protein ranged from 35.4% for the treatment with lactic culture to 31.0% for the treatment with formic acid plus paraformaldehyde. When the barrels were emptied and the spoiled material discarted, no bad smell was noticed from the silage, thus, protein decomposition was nil. On the other hand, the spoiled 103 :.mm 0.0m m.mm m.am m.mm m.am camps map so mop no Choc Undone m.mm 0.0m H.mm 3.3m m.mm m.mm Asav mmmmmaos eflswflq m.sm m.:m N.mm N.mm H.mm m.mm camps map mo mop Go mmwmmaoa casuaq o.am o.mm 6.0m N.mm m.am «.mm endow map mo new so wmmmmaoe hum m.:m o.mm H.3m m.am s.Hm H.mm “wow map go no» :0 ommo 5.3m m.mm o.mm o.HN s.am o.om cwmnm map 90 mop Co oaopmmEHq m.mm 5.3m H.mm N.Hm m.om m.mm “sag oomv a m m o.Hm H.mm o.mm e.mm o.mm m.mm Asm.ov sommm o.mm H.Hm m.mm o.mm m.mm m.mm Asa.ov mehgmeamsnommpma + AR:.HV Uflom anpom m.mm 0.0m 5.3m N.Hm m.mm o.om Aemv snow oncoamoom o.mm m.:m m.mm o.ma H.0N H.0H Asav snow oacoamonm o.mm s.mm m.mm m.om m.ma m.am Hoopsoo W mhmu om 969mm made mm nmffim W mhmv om nopmm mzmv mm noPmm psospmwne dWHmmp nmpvms hpcv mepoum & noppms app & am amass .mcflmpw .mhmzoun Po; Umawmcm mo pampsoo Camponm Usm hmpvms hum .am manna 104 Amwmmn hoppme hhuv ZQ< No.0 paw mm¢ RN.:N mm; cm>wmowh mm mhwmhm mnv mo howpwmomEoo 00 had so Hohhdp who chm mm had so mamhhmn oBP thm mosam> m w.mm m.mm o.mm d.mm m.mm m.mm mwmho>< m.:m m.wm m.mm N.HN m.om m.am no» cap ho hopmz mo Hasm man oapmmag w spas vaMom m.mm N.om .:.mm :.mm m.:m 3.0N AKOHV Choc chaohw + chapaso chpomq N.Nm 0.0m m.mm :.mm m.:m d.mm ARBV mommmaos + thpaso capowq s.mm 3.3m n.6m m.hm m.hm m.mm mhsphso whom chpomh m.wm m.mm p.0m H.5N m.nm o.mm A&oav Shoo thohw x” mhmc ow hmphm wasp mm hopmm on mhmu om hopmm mzmc mm hmpmm Amwmmn hoppme hhcv hmeOhQ\& 33.9: as «N phospwmha A.U.Pcoov mm wands 105 material had a strong rotten smell, a result of an active microbial degradation of the grain's organic matter. These results indicate no loss of nitrogen when brewers' grains are ensiled as in these trials. Acid detergent fiber and acid detergentginsoluble nitrogen. Acid detergent fiber of fresh brewers' grains was 24.2% (dry matter basis) and the average did not change during the ensiling process (Table 30). Grain to which liquid molasses or ground corn was added had lower acid detergent fiber than did other treatments due to dilution with low fiber addition. Addition of formic acid plus paraformaldehyde increased the fiber content of the grain after 32 days of ensiling, but after 60 days those values were similar to those of other treatments. Average acid detergent insoluble nitrogen (ADN) content of the grain was the same at day 32 and day 60 (0.87%). The percent in fresh brewers' grains was 0.92 (18% of the total nitrogen). Mixing of liquid molasses or ground corn with the grain decreased its ADN content. High values were observed for the control (1.02%) and for the grains covered with liquid molasses (0.95%) in relation to other treatments. Values for other treatments ranged from 0.82 to 0.90%. This corresponds to about 16% of the total nitrogen. ADN of the grains treated with formic acid plus para- formaldehyde was greater than any other treatment. This value was about 40% above all others and amounted to 24% of the total nitrogen. Formaldehyde binds proteins and must 106 es.o es.o ss.o e.om m.eh m.hm Aesv memmehos ehsehh mm.o mo.H mm.o 0.:m 5.:N 3.3m sawhm 039 he hop ho mommmaoa UHSUHQ em.o sm.o mm.o o.sm m.mm m.mm shehw esp he hop :0 mommmaos aha mm.o mm.o mm.o o.mm n.3m m.mm hmhm was he see :0 omso om.o ww.o mm.o 0.:m :.mm m.:m hamhw map mo mop ho ohopmmfiaa mm.o om.o mm.o 5.3m s.:m s.sm Ashe oomv s m m mm.o mw.o mm.o o.mm o.mm h.sm hem.ov eommm mm.h mh.h mm.h 0.0m o.mm o.sm Ash.ov messeehsehOhehee + Ass.hv shoe cheese ow.o mm.o mm.o m.mm :.mm m.mm ARNV whom owhoahohm am.o ma.o em.o e.sm m.sm o.em hehv shes ehsOheosm mo.h mo.h oo.h m.mm h.mm m.mm hohpsoo W made omihmpmm mzmv mm hoPHm m. mhwc om hopmm mzmu mm hopmm ~29 RV 29¢ R Asa &v mad & phospmoha mm HmHhe .thth .mhozohp Pm; umawmhw mo pseesoo Azosv semohphz ehsshomsh psemheeem shes use Ammsv henhm psemhepem shes .on wanes 107 Amhmmn hoppme hhvv zm< No.0 chm mm< Rm.dm mm; Uo>flocoh mm mhadhw map mo howpwmomEoo om amp :0 Hohhmn who chm mm had he mamhhMQ cap Sohh mosam> m mm.o nw.o mw.o m.mm m.mm m.mm mwmhm>< mm.o om.o sm.o m.sm h.mm s.sm hop esp ho hops; mo HHS“ wan owpmmam m mph; coammm os.o ms.o ms.o :.om m.mh m.om “echv shoe essohm + chapaso capomq 05.0 ms.o mm.o m.mm s.mm m.am Asmv mmmmmaos + mh:paso capomq ow.o mm.o ow.o m.:m m.:m m.:m chapaso chow capomq No.0 00.0 :0.0 H.mH H.mH o.mH ARQHV Shoo vhsoho Nw.o mm.o mm.o o.mm m.mm m.:m sawhm 039 no mop ho hhoo Uhsohw x mama om hophm wasp mm hopmm on mama om hmphm mmwc mm hopmm Asa sv zms R Cam $mm¢ R thSPMohe A.©.Phoov om mflnme 108 make them insoluble in acid detergent solution. For comparison, ADN in fresh forage amounts to 5-9% of total nitrogen and increases with extent of heating during ensiling (Yu and Thomas, 1976). Regression equations have been deve10ped to calculate nitrogen digestibility from ADN values in forages (Goering gt a1., 1973; Yu and Thomas, 1976). Ethanol. Ethanol content of the grains increased during the ensiling process, but the rate of increase was not the same for all treatments (Table 31). Grains treated with acids (propionic, formic and sulfuric) contained little ethanol. Propionic acid at 2% had a greater effect than did propionic acid at 1%. Grains to which a lactic acid culture plus 7% molasses were added had a high ethanol content (over 2%) throughout (day 5 to 60), while the treatment with 7% liquid molasses alone had only 0.05% ethanol on day 5 which increased to 1.74% on day 60. On day 32 and 60 silage from the treatment with lactic acid culture plus 10% corn had next to the highest of any treatment (1.54 to 2.07%). Some of the ground corn mixed with the grain could still be observed in the silage at the end of the experiment. Molasses, being a more soluble carbohydrate source, was probably degraded faster and more completely than ground corn by the microbiota during the fermentation process. Treatment with a lactic acid culture plus a substrate resulted in higher ethanol concentrations than treatment with the culture or the substrate alone. 109 Amanda #03 m Gov &H0.0 mm: vm>wmomh mm mhAth esp CH COMPMhthohoo Hohmnpo ow mac ho Hohhmn who chm mm had no mamhhmn 03» Sohh hmMMP mmamsmm ho umhwshopmc mosam> 9 meSP PmmP C...” PQOM mmHQEMm GO UwCHEmeU mmSHm> .m ms.o mh.h mo.h me.o 3:.0 Hm.o mm.o ethe>< mm.h sh.h mm.h mm.o :m.m mo.m mm.o emhmmm mo.h so.m em.h ho.h ho.o hm.m sm.o AsOHV shoe essohw + shephso ohposq m:.m mh.m sh.m s:.m hm.h mm.m m:.m Asnv mmmmshos + ehsphso ehpomq :m.o no.0 0:.H HH.H :n.o mo.H N:.o oh5PH30 whom capowq mm.o mo.h mm.o No.0 hm.o mm.m sm.o AsOHV shoe essohw ms.o ss.h mm.h mm.o mm.o sm.o mo.o Asuv memmshos ethhq mm.o hm.h mm.h sm.o so.o so.o sh.o Assn oomv s m m :0.0 mo.o mo.o No.0 oo.o oo.o no.0 Asm.ov shes ehhshhsm No.0 No.0 no.0 No.0 oo.o mo.o Ho.o Ash.ov sesameheshohshmh + Ase.hv shoe shahom mo.o om.o mm.o mo.o 00.0 «0.0 ho.o Asmv whom ohsohhohm sm.o :m.o mm.o oo.o oo.o mo.o mm.o fishy shes ohsoheohm me.o so.h sh.h mm.o mm.o sh.o oa.o hohpsoo m poo 9mm mmMmm moa mm mm #hmfipdmha m Hmwha .mm>wphuvm pamhmmmwu Spa: Umawmhw mhwmhm .mhmsmhn pm; ch asap Spas Amanda #03 m ho phmohomv COHFMhphmohoo Hocmnpm cw mmmhmno .Hm mdnwe 110 Addition of the lactic acid culture alone increased average ethanol from 0.49 to 0.84%. Source of the microbiota, presumably yeasts, that form ethanol is speculative but probably originated at the brewery, delivery truck or other sources. Acetic acid. Acetic acid content in fresh brewers' grains was 0.07% (on a wet basis). This concentration did not change during the ensiling process in control, in the grains with 2% propionic acid, sulfuric acid, 10% ground corn, and in the treatment sealed with a plastic bag full of water (Table 32). At day 32 and 60 grains from treatments with lactic acid culture and with 7% liquid molasses had a higher acetic acid content than did other treatments. Concentrations of acetic acid in butylated hydroxyanisol (BHA) treated grains started to increase on day 10, but in other treatments the increase started sooner. Grain treated with formic acid plus para- formaldehyde had more acetic acid than did grain with pr0pionic acid 2%, sulfuric acid and the control. Addition of liquid molasses of ground corn combined with the lactic acid culture had more effect on acetic acid forma- tion than ground corn, molasses or lactic acid culture individually. Acetic acid in the silage could be the product of heterolactic fermentation or the result of secondary fermentation of ethanol or lactic acid. Propionic acid. Fresh brewers' grains did not have any measurable amount of propionic acid. Propionic acid content lll Amwmwn pwz w hov $00.0 mm; 0w>wwowh mm mhwwhw th ha howpwhphwohoo whom owpwow 00 how ho Hwhhwp who chm mm hwo ho mehhwp ozp thm hwxwp mwanswm ho awhwshwpwu mwsaw> waSP pmwp ha Phwx mwahswm ho awhwshwpwv wwhaw> M sh.0 00.0 mh.0 NN.0 0m.0 mh.0 0h.0 mmehwes 0H.0 00.0 00.0 mH.0 0H.0 HH.0 0H.0 owawwm sh.0 ms.0 00.0 m0.0 m0.0 0h.0 mh.0 Asohv shoe ossohw + whophso ohpemh mm.0 mm.0 mm.0 mm.0 0H.0 ua.0 0H.0 ARBV mwmwwaoe + whsPst ohpowq 0H.0 0N.0 mm.0 ma.0 00.0 0H.0 NH.0 whhpaho owow owpown HH.0 HH.0 0H.0 00.0 ma.0 0H.0 0H.0 A&0Hv hhoo 0hhoh0 00.0 no.0 No.0 00.0 ms.h 00.0 mh.0 Asso memmshos ohsohq HN.0 mH.0 :H.0 mm.0 NH.0 m0.0 m0.o AEQQ oomv < m m 50.0 HH.0 0H.0 m0.o no.0 00.0 no.0 ARm.ov vwow owhhMHhm :H.0 NH.0 da.0 0H.0 3H.0 NH.0 ma.0 A&H.00 wuhhwcawshomwhwm + A&:.Hv chow washom 00.0 0H.0 da.0 00.0 :0.0 m0.0 m0.0 A&Nv chow owhowmohm 0h.0 sh.0 hm.0 no.0 No.0 :0.0 00.0 Ashv ohos ohsohoohm 00.0 00.0 no.0 00.0 00.0 NH.0 00.0 Hohphoo m 000 0mm 00h 00h on em psospswhh ham N Hwhha .mw>wphuuw phwhwhhhc hpws dwahmhw mhwwhw .mhwswhn pwz ha wsfip £903 Amflmwn 9w; w ho Phwohwmv howvwhphwohoo whom oflpwow ha mwmhwhu .Nm wanwe 112 increased during the ensiling process, but the final concen— tration in the silage was very low in relation to the other acids or to ethanol (Table 33). Grain treated with propionic acid kept its concentration constant during 60 days of storage. The actual concentration of propionic acid on day 32 and 60 in silage treated with this preservative was 1.56 and 3.00%. Butyric acid. Butyric acid was not measurable in most of the treatments during or after the ensiling process, when the spoiled material was separated from the good silage (Table 34). This fact indicates that under the conditions of this experiment clostridial activity was low. Lactic acid. Lactic acid content of fresh brewers' grains was 0.14%. Lactic acid formation was very evident by day 5 in most of the treatments, and its concentration increased with time from 0.65% on day 5 up to 1.84% on day 60 (Table 35). Addition of propionic acid, formic acid plus paraform- aldehyde and sulfuric acid inhibited lactic acid formation. PrOpionic acid at a level of 2% was more effective in pre- venting lactic acid formation than was 1% propionic acid. .Inhibition was most marked with formic acid plus paraformal- dehyde or with sulfuric acid. This indicates reduced microbial fermentation in these acid treatments. When a source of carbohydrates was added to the grain, lactic acid content of the silage was increased. Addition of molasses resulted in a higher lactic acid content than 113 Hwh was ow>wwowh mm mhwwhm whp hh howpwhphwohoo chow owhOHQOhQ 00 000 ho Hwhhwn who who Nn hwo ho mawhhwn cap sohh hwxwp mwamawm ho owhwshwpwo mwsaw> 9 meSP vmwp ha mex wwaheww ho owhfishwpwo mwsaw> w h0.0 00.0 00.0 h0.0 No.0 00.0 00.0 emshe>< H0.0 no.0 No.0 H0.0 00.0 H0.0 00q0 owawwm No.0 no.0 no.0 H0.0 No.0 H0.01 00.0 Asoav hhoo ohsohw + whSPH50 capowq H0.0 No.0 No.0 H0.0 H0.0 H0.0 00.0 ARBV mwmwwaos + whhpaso owpowq No.0 :0.0 No.0 H0.0 H0.0 H0.0 00.0 whSPHso chow capowq No.0 H0.0 no.0 :0.0 H0.0 H0.0 00.0 ARoav hhoo ohswho H0.0 no.0 No.0 H0.0 H0.0 Ho.0 00.0 A&mv mwmwwHoE ofihdwq No.0 no.0 no.0 00.0 no.0 No.0 00.0 Asmm oomv < m m H0.0 :0.0 00.0 H0.0 H0.0 H0.0 00.0 ARn.0v chow losmahm No.0 No.0 no.0 H0.0 :0.0 H0.0 00.0 ARH.0V wohhwoawshomwhwm + AR¢.HV whom washom 00.n 00.n 00.m :0.m 00.m mo.n mm.m ARNV chow owhoflmohm ms.h s0.h so.h 00.h 00.h 00.h m0.h Ashv ohos ohsohoohm H0.0 No.0 H0.0 H0.0 no.0 H0.0 00.0 Hohphoo w 900 pmn and woa mm 0“ phwspwwha QNQ m Hwhha .mw>wphoow phwhwhmwo spa; owawmhw mhhwhw .whwswhn sz ha wshp spa; Amhwwn pwa w ho phwohwmv howpwhphwohoo chow owhOHQOhm ha mwmhwho .nn wanwe ll4 Hhh mm; 0w>wwowh mm mhflwhw whp ha hohpmhphwohow chow owhhpsp 00 >00 ho Hwhhwp who ohm Nn zoo ho mawhhwn 039 Sohh hwxwp wwflmswm ho 0whflshwpw0 mwsaw> p wwpsp pmwp hh paw: mwamsww ho owhwshwpwo mwsaw> w H0.0 H0.0 H0.0 H0.0 H0.0 00.0 00.0 wmwhw>< 00.0 No.0 00.0 00.0 00.0 00.0 00.0 Uwwam 00.0 00.0 00.0 No.0 00.0 00.0 00.0 Asoav hhoo ohsohw + whhpaho ohpowq No.0 No.0 H0.0 no.0 :0.0 00.0 00.0 A&mv wwwmwaos + whhpaho 009009 00.0 00.0 00.0 00.0 00.0 00.0 oo.o wthHso Uwow 009009 00.0 00.0 H0.0 00.0 00.0 00.0 00.0 Asoav hhoo 0h50h0 H0.0 00.0 00.0 no.0 no.0 00.0 00.0 Afiuv wwmmwaoa vasofln 00.0 00.0 00.0 h0.0 00.0 00.0 00.0 hens oomv < m m 00.0 00.0 00.0 00.0 00.0 00.0 00.0 ARn.ov Uwow owhhhahm 00.0 00.0 00.0 00.0 H0.0 00.0 00.0 ARH.0V wohhwoawshomwhwm + AR:.HV owow owahom No.0 No.0 No.0 no.0 H0.0 No.0 H0.0 A&Nv chow ohhoHQOhm H0.0 no.0 No.0 00.0 00.0 No.0 H0.0 A&Hv owow owhoHQOhm Ho.o no.0 No.0 no.0 00.0 00.0 oo.o HohPhoo M 900 nmn and woa on 0W Phwstwha 000 m Hwhhe .mw>hphoow phwthMHo spa; owahmhw mhhwhw .mhwzwhp sz ha wshp spa; Amhmwn 9w; 0 ho Phwohwmv hohpwhphwohoo 000w whhhpsn ha wwwhwho .on wands 115 &:H.0 mm; ow>wwowh mw hwwhm whp ha howpwhphwohoo chow capowa 00 >00 ho Hwhhwn who who Nn hwo ho mehhwn oz» Schh hwxwp mwagewm ho owthhwpwo mwsaw> n mwpsP wap ha mex mwamewm ho owhhshwpwo mwsaw> w mo.H 3m.H ow.H 05.0 Nu.o Hm.o no.0 ww0hw>¢ n:.H HN.N #m.H wH.H wH.H 0:.H @o.H dewwm 00.0 Nn.m 0m.a Hm.0 00.0 0:.H :0.0 AR0HV hhoo ohsohm + whopaso capowa mm.H mn.m NH.N mm.H n0.H 0:.H 0:.H ARBV wwwwwaoe + whhpaso ohpowq N0.H mo.a om.H no.0 :0.0 05.0 00.0 whhpazo chow capowq 00.H HH.N ma.m 00.0 no.0 n:.H nm.0 Asoav hhoo ohsoho m0.m mm.m Hm.m mm.a n:.m 0:.H 00.0 ARNV mwwwwaoh ohhwwq 00.h mm.m sm.m s0.h mo.h mm.h 00.0 heme oomo 0 m m 00.0 nn.0 0m.0 00.0 H0.0 00.0 00.0 ARn.0v chow owhhhazm NH.0 :n.0 :m.0 00.0 No.0 00.0 00.0 ARH.0V wohhwUHwEhOHthm + “Rod.av chow whEhom 0n.0 00.0 00.0 00.0 no.0 NH.0 00.0 Asmv oflow oahoaaohm 00.0 mm.a 00.H 00.0 00.0 0n.0 no.0 A&Hv owow owhowmohm ss.h ms.m om.m mm.h sm.h sm.h sh.h hohesoo m 000 9mm 00h 00h mm mm] pswspmwha hwo N Hwhhe .mw>thoow Phwhwmmwo spas owafimhw mhwwhw .whwzwhp pwz hh wshp shag Amhmwp 9w; 0 ho pthhwmv howpwhphwohoo chow capowa hw wwwhwho .mn wanna 116 the addition of corn. The use of a lactic starter, alone or in combination with the addition of a substrate, did not result in an increase in lactic acid concentration of the final product. On day 32 and 60 the control had higher concentrations of lactic acid (2.30 and 2.72%) than any other treatment, except that with 7% molasses (2.81 and 2.72%). In general, none of the treatments increased lactic acid concentration above that of control. Marked inhibition of lactic acid formation was due to the addition of acids. Ammonia nitrogen. Ammonia nitrogen was not detected in fresh brewers' grains. The pattern of ammonia formation during the ensiling process was irregular but levels in all treatments were generally low. Grains from several treatments had little or no ammonia on day 30 or 60. Treatments allowing little ammonia forma— tion were control, propionic acid, corn mixed and plastic bag on top. The highest value was 50 mg/100 g for the grain without additive after 13 days storage in test tubes (Table 36). Addition of liquid molasses, with or without a lactic culture resulted in ammonia levels higher than in other treatments on day 32 or 60. No ammonia was found on day 5. Higher ammonia concentration were found in days 7, 10 and 13 of test tubes storage than after 32 or 60 days of barrel storage. Evidently there is much more proteolysis in small quantities of grains stored in test tubes than larger quantities stored in barrels (Table 36). 117 HHh mm; 0w>Hwowh mm hHwhm whp hH hoprhphwohoo thoesw 00 hwo ho Hwhhwp who ohm Nn zoo ho mehhwp ozp thm hwxwp meQewm ho owhHSthwo mwhHw> mwQSP mep hH phwx mehswm ho owhHshwpwo wwSHw> M n m n 0H NH HH 0 wwwhw>< n 0 0 0 0N m 0 owwam 0 0 m 0 H 0N 0 AnoHv hhoo ohhohm + wthHso oHpowH NH 0N 0 0H 0 0N 0 Asmv mwwmeos + whthso oHPowH m m N 0H m Hn 0 whopHso whom oHpowH # o o o N mm o AROHV hhoo Uhhoho HH nn nH nH 0 o 0 ARBV wwmmeoE lovHH s 0 m NH Hm m o A500 oomv a m m 0 N 0 0 Hn n 0 Ann.0v oHow loSMHsm m 0 m 0 0H 0 0 AsH.0o mosseohsshohehmo + Ass.Ho ohow 0hshom H 0 0 0 n H 0 Ast oHow OHhloohm N 0 0 0 m H 0. AKHV oHow looHQOhm :H H 0 0n mn H 0 Hohphoo M 900 nNn wnH on mm mm ham N Htha .mw>HpHoow phwhwmmHo thz owHHmhw whHwhm .mhwzwhn pws hH waHp thz AhHwhm pwz w 00H\hwm0hPHh who hoHPwhphwohoo thosz hH wwwhwho .on anwe 118 Low ammonia levels in the silage are an indication that protein degradation was not extensive in the unspoiled portion of the silage. Microbiological examination. Estimates of microbiological numbers for grain samples on day 60 are presented in Table 37. Total counts of microorganisms range from a high of 4.0 x 106 cells/g in grains treated with dry molasses on top to a low of 1.8 x 105 cells/g in the treatment with calcium sulfate on t0p. Propionic acid did not have an inhibitory effect on the number of bacteria in grains on day 60, however, formic acid plus paraformaldehyde decreased the microbial population almost ten times when compared to the control. Treatments with sulfuric acid, calcium sulfate on top of the grain, mixed liquid molasses, ground corn on top and mixed with the grains, and the treatment sealed with a plastic bag full of water, had less microorganisms than did control. Treatments with propionic acid, butylated hydroxyanisol (BHA), dried and liquid molasses on t0p of the grain had numbers of microorganisms that were close or higher than the control. Determination of coliforms was made due to the fact that the experiment was performed near dairy barns, and the original grains were dumped on the floor of a bunker silo before ensiling. No coliform organisms were detected at the end of the ensiling period, but they may have been present at ensiling time. Usually these microorganisms are not present at the end of anaerobic microbial fermentations with mixed flora (Weise, 1969). 119 moH N N.H 30H N 0.0 = moH N m.m whthho oHow oHPowH moH N 0.0 moH N N.: = moH N 0.0 AnoHv hhoo ohhoho moH N n.: n0H N N.N : m0H N m.n hHwhm whp no how ho hhoo ohsohw 30H N 3.5 :0H N 0.0 = moH N N.n ARNV wwmwwHoS lodHH :uoH N 00nA .znoH N oonA .. o0H N m.n hHwhm whp mo mop ho wwmmeoe owstH :noH N oonA o0H N N.: = 00H N 0.: hHwhw whp mo mop ho wwmmeoh Nho hyachw oh hpZohm oh = noH N m.H hHwhw whp ho mop ho domwo d0H N 0.0 moH N N.N = moH N 0.n hHwhm whp ho mop ho whopwwsHH m0H N m.0 m0H N.m.: = 00H N m.m “sow oomv a m m 00h N 0.0 00H N 0.0 = m0H N «.0 Asm.0v o0mmm hpzohw oh moH N w.H = moH N N.N AnH.0v wothonShOHwhwm + AR:.HV oHow 0Hshom hPSOhw oh moH N H.n = o0H N N.N ARNV 0How loonohm spzohw 00 00h N m.H = 00H N m.h “Rho ohom chaohoohm 30H N N.N hp3Ohw oh hpzohm oh o0H N n.N Hohphoo mowmmmwmm hHHhompopoeh mahohhhoo wmwwm esespsehe N Htha .Athwhw .mhwawhn pr owHHmhw mo swhm hwm memthmhoohOHEv mzwo 00 thhho owHHmhw thwhw .mthwhp pwa ho wpmwp HwOHNOHOHQOhoHS .mn anwe 120 moH N N.N 0H N H.H = moH N m.m hop whp ho hwpws 0 he Hhsh mop ohpmeHo s ssh; oehmem hpzohw oh hpzohw oh : moH N w.n A&0Hv hhoo ohhohw + whthSO OHPowH 140H N m.: 30H N 0.H hpSOhm oh moH N n.m A&mv mwmwwHoE + whspHso OHNowH m as h hhoo UMPmmwww HHHHomQOPowfl wEQOHHHOU WMPOB PGwEmehB A.U.Phoov mn anwB 121 Lactobacilli p0pulation was variable among treatments. The counts ranged from no growth in the control and in the treatment with calcium sulfate on top to a maximum of 4.2 x 106 in the treatment with dried molasses on top of the grain. These values reflect the number of live organisms growing on day 60 and provide no information on microbial population during the fermentation interval up to day 60. Numbers may have been more or less previous to day 60. Lactic acid con- centrations of the silage (Table 35) were not proportional to the lactobacilli count at the end of the experiment. Treat- ments to which a lactic starter was added, did not show an increased lactobacilli count at the end of the experiment. Propionic acid treated grains had a low lactic acid concen- tration yet had relatively high numbers of lactobacilli. The original number of microorganisms in a silage has been postulated to be inversely proportional to their activity and rate of growth (Weise, 1969). Yeasts and molds determinations also gave variable numbers for the different treatments. The range was from no growth in the treatments with propionic acid, formic acid plus paraformaldehyde, calcium sulfate on top and lactic acid culture plus 7% molasses, up to a maximum of 6.6 x 105 in the treatment with 10% ground corn. Ethanol concentration in the silage (Table 31) suggests yeast activity during the storage period. Nevertheless, ethanol concentration was more related to the kind of substrate and to the presence or absence of preservatives, than to the final count of yeasts and molds. 122 Presence of ethanol, acetic and lactic acid indicates the activity of a mixed microbial p0pulation, but alcoholic and lactic fermentation predominated in this experiment. Butyric acid and ammonia were almost absent in the silage, suggesting that the activity of clostridia and proteolitic bacteria was low in the portion of the silage that was not spoiled. Correlations among measured constituents in Trial 2. Correlation coefficients among the results from trial 2 are presented in Table 39. These coefficients were calculated from averages of values from samples of two barrels on day 32 and one barrel on day 60 (Table 38). Recovery of good material was negatively correlated with temperature (r = -.76). Spoilage caused by aerobic fermenta— tion may have increased the temperature of the grains. Other correlations were not significantly different from zero between spoilage and other measurements. Correlation coef- ficients between average temperatures and other parameters measured were not statistically significant. Thomas (1976) and Goering gt 31. (1973) reported high correlations (P<.Ol) between temperatures of stored forage and its acid detergent fiber and acid detergent insoluble nitrogen content. Tempera- tures in forages ranged from 91 to 108°F, while in the present experiment average temperatures during storage were lower. The range was from 71.7°F in the treatment sealed with a plastic bag full of water to 79.0°F in the treatment to which a lactic culture plus 7% molasses was added. This .1233 o.ms 3N mu.N No.0 :0.0 nn.0 oo.H om.0 2.0m 0.0N 2.:N N.:u mn.n HRNV mommuhoa vhsuhn 00 on.~ 00.0 No.0 00.0 mm.0 no.0 0.0N 0.00 N.NN 0.00 00.0 0.00 chasm 0:» he no» ho nwmmeoE 0H=UHA no ma.~ 00.0 No.0 00.0 No.0 :0.0 0.:w 0.Hn ~.- ~.nn 00.0 0.~s shahm on» he no» no mommmHos owhho N0 00.h 00.0 No.0 0H.0 0:.H 00.0 0.m~ 0.0n 0.HN H.nn no.n 0.00 chasm 0:» he no» :0 a0mg Ho mm.H 00.0 No.0 00.0 on.H 00.0 0.:N 0.:n 0.HN n.Nn H0.n N.H0 chmhm on» no no» he wcoumwehq ho om.~ 00.0 no.0 0H.0 no.H 00.0 N.s~ 0.nn ~.H~ 0.0“ 00.0 0.00 A200 oomo a m 0 no. 0N.o 00.0 No.0 0H.o no.0 00.0 0.nN 0.Hn :.NN N.mu N0.N N.Nm ARn.0v oHow loshHsm ho 0~.0 00.0 00.0 nH.0 «0.0 -.H 0.0m 0.~n 0.n~ “.mm 0~.n 0.00 ARH.00 oohsoohmshohmuaa man ARO.HV whom oHBhom 00 No.0 No.0 00.n NH.0 nN.0 0000 n.mN 0.Nn. N.HN 0.Nm 00.n 0.50 ARNV oHuw oHCOHQOhm 00 as.h No.0. on.H o~.0 am.0 00.0 0.:N 0.mn 0.0H 0.00 0n.0 0.5N hhho shes sacchooaa 00 Hm.~ No.0 No.0 00.0 0H.H N0.H N.mN o.nn n.0N 0.55 :0.n N.mu HohNCoo hwwothh whpowH ohhhpsp OHhOHhohn 0prou Handgun zo< modV .hHwNOhA so ohhuahonawv ma th>oowh cws oh HmomHCoesw R R R R R R R R R om R a pm a a~ hmhaa .mhwo 00 who \r Nn hon coHHmho thahw .mhozwhp no: we COthwonsoo ohd ohspwthEw» .xa .hhw>oowh wmaho>< .mm prme 124 .hmhmsp peso ohos oasohoesn R . M :Hahu not w 00H\z wav somehvwh Hwothoaad .HmHmun peso uHom OHpouH R . whoop pozv uhow chhhpsn R mewp perv chow 0Hvooa R .AmHmwp 903v Hohdzvo R .AmHmwn hoppua hhoo ho ohuhh oHpsHowhH pcwwhwpoo chow u 294 .Amhmwn hwvvwa hhoo .hoth pthhwpou owed a ho< .HmHmwn hvama hhoo havahn R .hwvvms hho u so .HmHmmn pogo hhw>oowh R n . 00 adv ho Hohhwn who who Nn adv ho mehhwn oz» aohh meham ho uwhHshwpwv mwus> no mwNth>w a 00 0H.o 00.0 00.0 50.0 H0.0 No.0 N.:N 0.Hn m.m~ 00.m mchmhw .mhwawhn nmwhm :0 mm.H H0.0 0N.0 0H.0 :0.H no.0 n.nN 0.Nn a.NN n.nm n0.n 3.00 mehw>< 00 00.h H0.0 no.0 nh.o on.h no.0 0.0N 0.00 ~.H~ 0.HR no.0 n.00 no» as» ho hwvm: no HHsu mun . oHumqu a nah; cwwam N0 00.N 00.0 :0.0 mn.o 00.H 05.0 3.0N n.0N a.nN N.nn mm.n 0.05 ARoHv hhoo chaohm + whopHso ohvomn nH mN.N H0.o No.0 Hn.0 ooNN 0m.o n.NN N.Nn :.nN 0.0m Hahn N.HR ARNV mwmmmHoa + whopHso oHpomH N0 00.H 00.0 no.0 0N.0 00.H 00.0 0.0N :.mn .0.HN N.om Nn.n H.0o wthHso chow whpomq 00 :H.N 00.0 No.0 0H.0 NH.H No.0 H.0H N.0N H.0N 0.05 No.n H.0m HRNV hhoo 0h50ho oh oo.~ 00.0 no.0 00.0 00.0 «0.0 0.n~ 0.00 0.h~ 5.05 00.n 0.00 chasm on» no no» he hhoo vhsoho hwmothh 0HpowH ohhhpzp OHhOHQOhA chewed Hohwhpw zn< . mo< .8prOhQ 2o whspwthSwu rm th>oooh Phospmwha 202805 R R R R R R R R . R as R H.n.vhoov an oHDdH 125 Table 39. Correlation coefficients among measured constituents of ensiled wet brewers' grains. Trial 2a X Y r recovery pH 0.12 temperature -0.76** dry matter -0.l7 protein 0.18 acid detergent fiber 0.40 acid detergent nitrogen 0.41 ethanol -0.37 acetic acid -0.29 prOpionic acid 0.42 butyric acid 0.19 lactic acid -0.46 ammonia nitrogen -0.30 pH temperature 0.05 dry matter -0.06 protein 0.15 acid detergent fiber -0.06 acid detergent nitrogen -0.34 ethanol 0.40 acetic acid 0.34 propionic acid 0.06 butyric acid 0.46 lactic acid 0.49* ammonia nitrogen 0.03 temperature dry matter 0.31 protein -o,36 acid detergent fiber —0.47 acid detergent nitrogen -0.41 ethanol 0.41 acetic acid 0.31 propionic acid _0.19 butyric acid 0.01 lactic acid 0.36 ammonia nitrogen 0.00 dry matter protein —0.88** acid detergent fiber -0.84** acid detergent nitrogen -0.53* ethanol 0.29 acetic acid 0.32 propionic acid -0.33 butyric acid _o.30 lactic acid 0.10 ammonia nitrogen 0.28 Table 39 (cont'd.) 126 X Y r protein acid detergent fiber 0.82** acid detergent nitrogen 0.50* ethanol -0.12 acetic acid -0.21 propionic acid 0.11 butyric acid 0.06 lactic acid -0.02 ammonia nitrogen -O.26 acid detergent fiber acid detergent nitrogen 0.75** ethanol -0.47 acetic acid -0.50* propionic acid 0.22 butyric acid 0.01 lactic acid -0.25 ammonia nitrogen -0.36 acid detergent nitrogen ethanol -0.50* acetic acid -0.34 propionic acid 0.01 butyric acid -0.01 lactic acid -0.45 ammonia nitrogen -0.33 ethanol acetic acid 0.58* pr0pionic acid -0.36 butyric acid 0.07 lactic acid 0.55* ammonia nitrogen 0.28 acetic acid propionic acid 0.00 butyric acid 0.29 lactic acid 0.12 ammonia nitrogen 0.42 propionic acid butyric acid 0.58* lactic acid -0.35 ammonia nitrogen -0.21 butyric acid lactic acid 0.07 ammonia nitrogen 0.21 lactic acid ammonia nitrogen 0.36 a correlation coefficients determined on values from Table 38. * significantly different from 0 (P<.05) ** significantly different from 0 (P<.Ol) critical values: a .05 = .48, a .01 = .61 127 could explain why in the present experiment these relations were not observed. Values for pH were low for all treatments. Addition of sulfuric acid lowered the pH of the silage to a value of 2.92, but it did not prevent spoilage on the upper part of the barrels in contact with air. Spoilage of this treatment was similar to control. Lactic acid was positively correlated with pH. Addition of acids, particularly formic and sulfuric, lowered the pH of the mass to values around 3.0. Grains from these treatments also had the lowest levels of lactic acid. In other treatments, higher pH values were favorable for the formation of lactic acid. Silage dry matter was negatively correlated to protein (r = —.88), acid detergent fiber (ADF) (r = -.84) and acid detergent insoluble nitrogen (ADN) (r = -.53), while positive correlations were found between protein and ADF (r = .82), protein and ADN (r = .50) and ADF and ADN (r = .75). Rela- tively high dry matter values in the grains mixed with molasses and ground corn corresponded to low protein, ADF and ADN contents. Thus, diluting the grain with high dry matter material with low protein, ADF and ADN contents changed these values considerably and may have been partially responsible for the high correlations obtained. Acid detergent fiber was negatively correlated with acetic acid (r = -.50). Addition of molasses or corn to the grains lowered acid detergent fiber values and probably 128 served as a substrate for the formation of acetic acid. In fact, the highest acetic acid concentrations are found in these treatments. Correlation coefficient between acid detergent insoluble nitrogen and ethanol was negative (r = -.50). Addition of carbohydrate sources (molasses or corn) lowered ADN contents and served as a substrate for the formation of ethanol. High ethanol values were related to high acetic acid concentrations (r = .58) and high lactic acid contents (r = .55). Acid treatment of the grain inhibited the formation of these three compounds while the addition of carbohydrate sources increased the content of the same in the silage. Levels of prOpionic and butyric acid were low in the silage and positively correlated (r = .58). Summary of Trial 2. In this experiment, 300 lb of wet brewers' grains were stored in steel barrels lined with poly- ethylene bags. The effect of several additives or methods of ensiling on characteristics of ensiled grains was tested. Success in ensiling was obtained with the addition of 2% propionic acid, a mixture of 1.4% formic acid plus 0.1% paraformaldehyde or when the grains were sealed with a plastic bag filled with water on top of the barrel. For these treatments, there was no spoilage. Recovery of good material after 32 days of ensiling was 83% and 68% after 60 days in the control. Covering the grains with limestone, calcium sulfate or liquid molasses improved the recovery of good silage over the control, but 129 still 17% of the grain was spoiled by day 60. Dried molasses or ground corn layered on top of the grain were somewhat less effective than the aforementioned treatments in preventing spoilage. Addition of and mixing with molasses, ground corn or a lactic acid culture, individually or in combination did not increase the proportion of material recovered as good silage after the ensiling process. Spoilage in the grains treated with sulfuric acid was similar to that of the control. Fresh brewers' grains had a pH of 5.3. In general for all treatments the pH decreased to a value below 4.0 at day 5 and remained constant until the end of the experiment on day 32 or day 60. No change was observed in dry matter, protein, acid detergent fiber and acid detergent insoluble nitrogen when comparing fresh to ensiled grains. The two main products of fermentation were ethanol and lactic acid. Concentration of acetic acid in the silage was low while propionic and butyric acids were barely detectable. Ethanol and lactic acid formation were inhibited by the presence of acids. Addition of carbohydrate sources increased the concentration of ethanol and lactic acid in the silage. Silage from the treatments with a lactic acid culture had similar composition to the grains having no additive. Ammonia was not formed during the ensiling process in most of the treatments. This, and the fact that protein content did not change with time indicates that there was not protein change or degradation. Temperature of the ensiled grains was positively correlated with spoilage. 130 Differences in the chemical components of the silage considered good were not proportional to the magnitude of spoilage. Low levels of ethanol and lactic acid were found in good preserved grains when propionic or formic acid were added. Grain to which carbohydrate sources were added had high levels of ethanol and lactic acid but still had exten- sive spoilage. Sulfuric acid lowered the pH and inhibited the formation of organic acids and ethanol, but did not pre- vent spoilage in the upper part of the silo in contact with air. Good conservation of the grains was accomplished by maintaining anaerobic conditions during storage or to the presence of preservatives such as pr0pionic acid, formic acid and paraformaldehyde. Comparison of Trial 1 with Trial 2 Two storage trials with wet brewers' grains were performed during warm weather. Different methods of ensiling and several additives were tested. In the first trial 30 lbs of grains were stored in plastic buckets. In a second experiment 300 lbs of grains were stored in steel barrels. Recovery. After 32 days of ensiling an average loss (spoiled material) of 26% was observed in the first experi— ment, but only 17% in the second experiment. After 76 days of ensiling, grains in plastic buckets had a loss of 35%. but in the second experiment spoiled material in barrels was only 24% after 60 days of ensiling. 131 Some preservatives used in the first experiment only slightly reduced spoilage but were essentially ineffective. Complete recovery was achieved in the second experiment when grains kept in barrels were mixed with 2% propionic acid or 1.4% formic acid plus 0.1% paraformaldehyde. When grains were kept under anaerobic conditions in both experiments complete preservation was also obtained. In both experiments, increases in temperature were related to the amount of spoilage. In Trial 1 grains that had more ethanol had less spoilage, but this did not occur in Trial 2. Schoch (1956) established a positive correlation between dry matter loss and butyric acid concentration in wet brewers' grains silage. This relation was not found in our experiments. There were no dry matter losses when the grains were kept under anaerobic conditions in Trial 1, but butyric acid concentration were similar to other treatments that had considerable losses. Fermentation pattern. Products of fermentation in the silage considered good varied for the two experiments. Stor- age in buckets resulted in relatively high concentrations of acetic propionic and butyric acid. Acetic acid content in the grain stored in barrels was low, and propionic and butyric acid were almost absent. There was considerable formation of ethanol and lactic acid in barrel silages while in bucket silages these two compounds were in lower concentrations. Silage from the grain stored in buckets experienced increases in nitrogen, acid detergent fiber and acid detergent 132 insoluble nitrogen, and developed large quantities of ammonia. Such changes were not observed in the second experiment, and ammonia when present was in low concentration indicating little proteolysis. Addition of 10% yeast to the grains stored in buckets resulted in less spoilage, lower pH values and temperatures during the ensiling period, less acid detergent fiber and acid detergent insoluble nitrogen, higher levels of ethanol and lactic acid and lower concentrations of acetic, propionic and butyric acid than grains without yeast. Even so, spoil— age losses for the same storage periods were higher than in grain stored in barrels. Butyric acid, lactic acid and ammonia concentration have been taken as a measure of wet brewers' grains silage quality (Allen.§tyal., 1975). If concentrations of these compounds are taken as criteria of silage quality, then grains with or without yeast kept in buckets were inferior to the grains stored in barrels. Type of additive. Mineral and organic acids were used as additives in these experiments. Mineral acids (sulfuric acid) lowered the pH and inhibited the formation of volatile fatty acids, ethanol and lactic acid in grains kept in buckets and barrels but did not prevent spoilage. Krinstad and Ulvesli (1951) and Schoch (1957) observed a decrease in dry matter losses when mineral acids were added to brewers' grains and ensiled. This difference is probably due to the type of silo used. Krinstad and Schoch used concrete silos 133 for their experiments where more anaerobic conditions were maintained than in our experiment. Propionic acid at a level of 2% and 1.4% formic acid plus 0.1% paraformaldehyde prevented spoilage of grain kept in barrels, but were essentially ineffective at lower levels in grains kept in buckets. Formic acid mixed with paraformal- dehyde was more effective in reducing spoilage than formic acid or paraformaldehyde alone in the first experiment. Allen et a1. (1975) reported decreased spoilage when formic or pr0pionic acid, or a combination of both were added at a level of 0.40% to wet brewers' grains stored in uncovered piles. Ammonium isobutyrate increased recovery of haylage ensiled in barrel silos (Thomas, 1976). In our experiments with wet brewers' grains, addition of ammonium propionate or ammonium isobutyrate at levels of 0.5% did not have a benefi— cial effect on recovery. Increasing the concentrations of these preservatives above 0.5% might produce more desirable results. Bases such as ammonia or potassium carbonate did not reduce spoilage when added to the grains. Potassium carbonate was added alone or mixed with propionic acid to displace air by means of formation of carbon dioxide. Nevertheless, this did not reduce spoilage of the grains stored in buckets. The use of carbohydrate sources did not improve preservation in any of the trials. Similar results were reported by Allen.§t a1. (1975 a and b) for grains stored in 134 test tubes or in uncovered piles. However, Schoch (1957) found that adding 10 to 15% dried apple residue reduced dry matter losses of brewers' grains stored in silos. Brewers' grains mixed with corn have been successfully stored in air- tight silos (Anonymus, 1976). The difference is probably due to the degree of anaerobiosis reached in the silos and amount of material added. In our experiments grains mixed with carbohydrates were kept with the surface exposed to air and that was where spoilage occurred. Some increases in recovery was observed when a lactic culture was added to brewers' grains stored in buckets. How- ever, addition of a lactic acid culture to grains stored in barrels did not have any beneficial effect. Furthermore, lactic acid concentrations in the grains to which a lactic acid culture was added were not higher than in other treat— ments. Weise (1969) postulated that activity and rate of growth of microorganisms was inversely proportional to their original number in the silage. Type of silg. Differences in recovery and fermentation pattern in grains stored in buckets or in barrels are due primarily to the type of model silo used. Surface of exposure 2 and the weight to air of grains in buckets was about 850 cm of the grains was 30 lb while 300 lb of grain stored in barrels had a surface of exposure of about 2400 cm2. Thus, surface exposed to air per pound of grain was 28 cm2 in buckets and 8 cm2 in barrels. Furthermore, during the first experiment, samples were taken periodically from the buckets. 135 The surface of the grain had to be removed and air was allowed to penetrate in the mass of the grain causing more spoilage. Test tubes (100 ml) were used to follow fermentation in the second experiment. Allen and Stevenson (1975) compared test tubes and buckets as model silos for brewers' grains and observed that buckets were ineffective in simulating horizontal silo conditions. In our experiments barrels were more similar in storage conditions to test tubes than to buckets. Nevertheless, some irregular results in pH, ethanol, acetic acid, lactic acid and ammonia were observed in samples of brewers' grains from test tubes. In some samples these values were higher or lower than the concentrations in samples taken from barrels at the end of the experiment. Such differences may be due to the weight of the sample that can be stored in test tubes. A sample of less than 100 g cannot be compacted in the same way as grain stored in barrels. Furthermore, a small sample might not be representative of the large amount of grains stored in a large silo. Air is more easily excluded by the pressure created by the weight of a large mass of grain than by the weight of a small mass. A small silo, 10 ton capacity, was filled with wet brewers' grains at the same time when the storage trial in barrels was initiated. Grain was covered with a polyethylene sheet over which a layer of corn silage about 20 cm thick was placed. After 60 days of ensiling some spoilage, 2—3 inches, was observed around the edges of the silo but the 136 rest of the grain appeared well preserved. Liquid was observed draining from the bottom of the silo until day 20. Silage at day 60 appeared drier in the upper part of the silo than in the lower part. Even when no chemical analyses were made on this silage good preservation of wet brewers' grain was apparently achieved by storing it in silos, provided there is adequate sealing. Practical considerations In practice wet brewers' grains are delivered to the farms by truck from the brewery where they are placed in some type of storage facility. Grains must be utilized within one week in most storage facilities or they deteriorate and spoil. In order to prevent losses due to spoilage and decreased nutritive properties, good storage is necessary. From the experiment described above, using small model silos, wet brewers' grains were completely preserved under oxygen free storage conditions. Spoilage and formation of ammonia and butyric acid were reduced in the silage by increasing size of the silo. An extrapolation of these findings would indicate that wet brewers' grains ensiled in a large silo would have even lower concentrations of ammonia and butyric acid. Good quality silage can be expected by storage of wet brewers' grains in vertical silos if their construction per- mits the maintenance of anaerobic conditions. Silo strength and ease of automatic unloading may be a problem when wet brewers' grains are stored in large upright silos. 137 To store wet brewers' grains in bunker or pit silos, the surface area exposed to air should be minimized. This could be achieved by sealing the grains in these silos with poly— ethylene sheeting. The use of preservatives is necessary when the grains cannot be stored under anaerobic conditions. In experiments described above, mixing the grain with propionic acid at the level of 2% or with formic acid (1.4%) and paraformaldehyde (0.1%) resulted in complete preservation of wet brewers' grains silage. Care must be taken in handling these organic acids since they are corrosive and can cause burns in the skin. The preservatives could be mixed with the grains at the brewery since most of the farms do not have mixing equip- ment. Special facilities at the farm should be built in order to store the grains since existing silos would not always be available for ensiling grains. When grains to which preservatives have not been added are removed from silos to feed animals, they must be utilized in a short period of time, probably not more than two days, since contact with air will cause rapid spoilage. Grains with added preservatives would be expected to last longer. The reduction in losses and the increases in quality of ensiled and preserved wet brewers' grains would likely com- pensate for the additional costs of handling and preserving. Ensilage of wet brewers' grains will solve the problem caused by fluctuation in supply and demand. A feedable pro- duct would then be available when needed and prices would tend to stabilize. CONCLUSIONS Wet brewers' grains can be stored for long periods of time if anaerobic conditions are maintained in the silo. When anaerobic conditions cannot be maintained, the use of preservatives is indispensable. Propionic acid at the level of 2% or a mixture of formic acid (1.4%) and paraformal- dehyde (0.1%) are recommended. Addition of a source of carbohydrates, as corn or molasses, does not exert a beneficial effect on the conser- vation of wet brewers' grains if anaerobic conditions are not maintained. Principal products of fermentation of wet brewers' grains are ethanol and lactic acid in barrel silos. In smaller silos there is formation of acetic, propionic and butyric acid, and evolution of ammonia. Size and type of the silo determine the recovery and quality of ensiled brewers' grains. Small silos produce more deterioration of the silage than larger silos. 138 LITERATURE C ITED LITERATURE CITED Ademosun, A. A. 1973. Evaluation of brewers dried grains in the diets of growing chickens. British Poultry Science. 14:463. Allen, W. R. and K. R. Stevenson. 1975. Influence of additives on the ensiling process of wet brewers' grains. Can. J. Anim. Sci. 55:391. Allen, W. R., K. R. Stevenson and J. Buchanan-Smith. 1975. 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