THE RELATION OF LACTIC ACID BACTERIA TO CORN SILAGE.

Transcription

1 THE RELATION OF LACTIC ACID BACTERIA TO CORN SILAGE. BY E. B. FRED, W. H. PETERSON, AND J. A. ANDERSON. (From the Departments of Agricultural Bacteriology and Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, February 4, 1921.) Recently there have appeared many reports concerning the effect of inoculation on corn silage (1). In general, these reports claim that the seeding of corn fodder with certain bact eria causes the formation of a higher grade product. According to some of the statements inoculation with selected lactic acid bacteria prevents spoilage to a large degree, reduces alcohol formation, and improves flavor and palatability. Although much has been written concerning the effect of bacteria on ensiled corn a review of the reports shows but little data to support most of the statements. No doubt the seeding of corn fodder wit,h certain of the lactic acid bacteria will hasten fermentation processes, but that this is of any advantage to the farmer is not yet established. As a rule, properly ensiled corn keeps well and is ready to feed within about 2 weeks. Spoiling of corn silage except in the upper layer exposed to the air almost never occurs. The loss in this way is small and depends to a large degree on the moisture of t,he silage and on how well it is packed. From the results of preliminary tests Gorini concluded that the seeding of corn forage with lactic acid bacteria improves the keeping qualities of the silage and that the different types act differently upon silage. He emphasized the importance of these bacteria in the making of good silage, and suggested the use of selected lactic acid bacteria for certain types of forage. * This work was in part supported by a grant from the special research fund of the University of Wisconsin. Published with the permission of the Director of the Wisconsin Agricultural Experiment Station. 319

2 320 Lactic Acid Bacteria and Corn Silage The experiments described in this paper represent an attempt to determine the effect of inoculation on the composition of silage together with a study of the normal flora of green corn and of silage. Concerning the mechanism of silage fermentations there are at present many different views (2). Some investigators look upon the plant cell or enzymes as the chief agents, while others attribute to bacteria the principal role in the formation of silage. It may be safely stated that accumulating data tend more and more to indicate the dominant influence of bacteria in the production of silage. EXPERIMENTAL. The silage was made from corn grown on the University Farm and though the ears were well matured the leaves and stalks were st,ill green. The corn was cut by a large power cutter into small pieces and packed tightly into the silo by continuous tramping. For each ton of green fodder 5 liters of a 48 hour culture were used. These cultures were made by growing the desired organism in yeast water, and were diluted, and sprinkled over the green fodder as it was packed in the silo. The lactic acidproducing bacteria used were in one case the organisms of the Lactobacillus pentoaceticus group, and in another, a mixed culture of Bacillus la&s acidi and Bacillus bulgaricus. The first series of experiments was set up as follows: one large silo was filled with uninoculated corn fodder as a control, and two tanks about 8 feet high and 4 feet in diameter were filled with inoculated corn fodder. Although the conditions in the tanks were not exactly comparable with those in the large silo, it was found that the silage from these tanks had the general appearance, odor, and taste of normal silage. Samples of 10 to 20 pounds were removed for analysis at intervals of 12 to 65 days after inoculation. To obtain representative samples the first silage for analysis was taken at a total depth of about 3 feet, or at least more than 1 foot below the zone of spoiled silage at the top. Just as soon as the sample was obtained the top silage that had been removed was returned to its proper container and t,ightly repacked. In this way it was hoped to prevent as much as possible the passage of air into the fermenting

3 Fred, Peterson, and Anderson 321 silage. Samples for subsequent analysis were drawn from greater depths, always well below the level at which the previous sample had been taken. The silage was ground in a meat chopper, a uniform sample of the ground tissue was taken for the moisture determination, and the juice was expressed from the remainder with a strong hand press. When 400 cc. of this juice were expressed, a portion of it was diluted with sterilized water, and plated with glucoseyeast water agar. Attempts to estimate the bacterial flora by direct microscopic count failed to give satisfactory results. The remaining juice was used in the determination of volatile acid, nonvolatile acid, and alcohol. The treatment of the corn fodder and the results of the chemical and bacteriological analyses at different times are given in Table I. An examination of this table shows that inoculation of corn fodder with lactic acidproducing bacteria regardless of types of bacteria results in a slightly lowered production of volatile acid. This decrease in volatile acid as compared with that of t,he control is not,ed with the lactic acid bacteria in all three analyses. It is in the horizontal columns of this table, Nos. 4, 5, and 6, nonvolatile acid, that the effect of inoculation is most clearly shown. The culture of Lactobacillus pentoaceticus retards the production of nonvolatile acid while the mixed culture of lactic acid organisms favors this process. The analyses after 12, 28, and 54 days show a high lactic acid content of the silage inoculated with the mixed culture as compared with that of the control. Except in the very early stages of fermentation, the silage inoculated with Lactobacillus pentoaceticus is lower in total nonvolatile acid than the control. It will be seen from Nos. 7,8, and 9 that inoculation brings about a change in the alcohol content of silage. The Lactobacillus pentoaceticus organisms favor alcohol production, while the mixed cultures of lactobacilli tend to retard alcohol formation. Although the moisture content of the green corn was somewhat lower than is commonly found in silage, the fermentation processes proceeded rapidly. The chief point of interest in these determinations is the gradual increase in moisture as the silage becomes older.

4 mculated with 3. lactis acidi and B bulgaricus. r EL: w g c) w ti $ g g m 12 81,400,000 81,400, ,000,OOO % 28 8,160,OOO 97,000, ,400, ,080,000 86,660,OOO 161,500,OOO 9 No. Products TABLE I. Effect of Inoculation on the Composition of Silage from Large Containers. Volatile acid as acetic, per cent of dry silage... I I I... I I I <... Nonvolatile acid as lactic, per cent OJ dry silage... ( ed from by guest on April 8, 2018 rime after Untreated wxulsted with il mculation control. pantoaceticus du?js Alcohol as ethyl, per cent of dry silage... I I... I I I (... Moisture, per cent of moist silage... I... I... Bacteria in 1 gm. of dry silage....)

5 Fred, Peterson, and Anderson 323 For additional work with inoculated silage, barrels of 50 gallon capacity were filled with some of the same corn fodder as was used in the other experiments. To make the barrels airtight and also to prevent any absorption of the juice by the wooden st.aves they were coated on the inside with paraffin. In these miniature silos it was planned to study the types of fermentation and to compare the silage in the barrels with that in the large silo. The cut corn was inoculated uniformly and packed into the barrels. The tops of the barrels were sealed with a layer of paraffin 2 inches thick, and, except for lack of pressure, the conditions in these miniature silos were similar to those in the large silo. The barrel series was treated as indicated below: 1. Control, no inoculation. 2. Inoculated with Lactobacillus pentoaceticus. 3. Inoculated with Racillus lactis acidi and Bacillus bulgaricus. 4. Inoculated with Lactobacillus pentoaceticus, Bacillus lactis acidi, and Bacillus bulgaricus. In appearance and odor this silage resembled that of the large silo. The inoculated silage gave a more pleasant aroma than the uninoculated. The samples of silage were analyzed as previously described. The results of the chemical and bacteriological analyses are given in Table II. The silage in these barrels on analysis showed approximately the same products as those found in the larger containers. Apparently the lack of pressure did not result in a great change in the chemical composition of the silage. From the figures of the table it is clear that the bacteria are present in great numbers as late as 68 days. The seeding of silage with selected bacteria of the lactic acid group influences the silage in many ways. Except in one case that of silage with Lactobacillus pentoaceticus 21 days, the inoculated silage showed a greater number of bacteria than the control. The results of the chemical analyses agree in general with those reported in Table I. There is a slight increase in volatile acid in the corn inoculated with Lacdobacillus pentoaceticus; with lactobacilli, and lactobacilli plus Lactobacillus pentoaceticus, there is little if any difference. There is no well defined difference in the amount of the nonvolatile acid in the treated and untreated corn. In the corn inoculated with the mixed culture of lacto

6 3.lacti.9 acidi, tl 8. bulgarimr, p and L. patoaceticus ? ii: F * P ;z c) s ,400,000 60,000,OOO g IE CD NO I TABLE II. E.ffect of Inoculation on the Comvosition of Silage from Barrels. Products. Volatile acid as acetic, per cent of dry silage I ( C I I < I I Nonvolatile acid as lactic, per cent of dry silage I I I..... I I I I I C.... rime afte. mculatior days ed from by guest on April 8, 2018 r,. Untreated. Inoculated. control. ;. pentoacetieus. 13. ladis dar and B. bulg&cm Alcohol as ethyl, per cent of dry silage I < I I I < i < I Moisture, per cent of moist silage < I I I I I I Bacteria in 1 gm. of dry silage ( ! 56,000,OO( 10,000,OO( 13; 53,000,OO( 1: 73,500,00( B,000,00( 1 l$ 39,750,00( 72,200,00( 13: 17,750,00(

7 Fred, Peterson, and Anderson bacilli there is a somewhat greater amount of lactic acid. It is in the analyses for alcohol that the effect, of inoculation is most marked; t,he Lactobacillus pentoaceticus series shows the highest alcohol content and the mixed culture of Lactobacillus penboaceticus and lactobacilli rank next in order. It has been noted (3) that Lactobacillus pentoaceticus ferments hexose sugars with the production of considerable amounts of alcohol and acids, and the pent.ose sugars with volatile acid but no alcohol; the lactobacilli on the other hand do not ferment pentoses to any extent and do not produce appreciable amounts of alcohol or volatile acid from hexoses. Bacteriological Analyses. 1 cc. of the juice expressed as described above was diluted and plated with 1 per cent glucoseyeast water agar. After several agar media were t.ested, e.g. nutrient, litmuslactose, and NahrstoffHeyden agars, it was found that 1 per cent glucoseyeast water agar ph 6.8 gave the highest count and apparently t,he greatest number of different types of colonies. All plates were incubated at 28 C. for 1 week. Alt,hough 37 C. has been used by various investigators it was found that plates incubated at 28 C. gave greater numbers and a greater variety of colonies. To gain some idea of the fate of the organisms normally present on corn when ensiled, examinations were made of the types of bacteria on plates from the green corn and from the silage at different stages in the fermentation. The colonies on the plates were studied as to structure and form, and the different types picked off into litmus milk and into a measured amount of 1 per cent xyloseyeast water. The percentage of the various types of colonies on the plates was noted in all cases. The litmus milk cultures were examined regularly; and after 10 days at 28 C. the xyloseyeast water cultures were titrated with 0.1 N barium hydroxide. Stained mounts were made from the xyloseyeast water cultures after they were neutralized, and also from the litmus milk. Whenever an organism produced a characteristic colony on the plate and high acid in the xyloseyeast water, and showed the characteristic bacillus form in the stained mount, it was classed as one of the Lactobacillus pentoaceticus type. In

8 326 Lactic Acid Bacteria and Corn Silage glucoseyeast water agar the colony is deep, small, lanceshaped, yellowish, and opaque. The edge is usually smooth and uniform, but. sometimes has a woolly appearance. The organism is a Grampositive, bluntended rod, which does not form spores. It is a fairly large organism, 0.6 to 0.7~ wide and 1.6 to 3.Oh long under different conditions. It occurs singly or in short filaments, wit,h a characterist.ic grouping in bundles of parallel organisms. The results of the plate counts show that the corn at the time it was placed in the silo had about 86,000,OOO bacteria in 1 gm. of dry tissue, while there were 81,400,OOO in 1 gm. of dry silage after 12 days. 16 days later the total number of bacteria had decreased to 8,160,000, and after 54 days there were only 1,080,OOO. This rapid decrease in the bacterial flora is in agreement with the findings of earlier investigators. Counts made under similar conditions have shown that within 1 to 3 days after ensiling there is an enormous increase in the total number of bacteria followed by a rapid decrease. After 12 days the count in the inoculated silage had reached a figure at which it remained practically constant for 53 days instead of steadily decreasing as in the count on the uninoculated silage. The increase in numbers usually noted at first is largely due 00 the presence of a large supply of fermentable carbohydrates. After the consumpt,ion of most of this available food and after the production of acid, unfavorable conditions for the growth of many types of organisms are produced. The bacteriological examination indicates that the fermentation activity is soon narrowed down to a few aciduric types, wit.h a consequent reduction in Ootal numbers as the other types rapidly disappear. The bacterial flora of the fresh green corn was noteworthy in respect to variety of forms, but most of these organisms persisted for only a short time. There were very few organisms that produced acid from lactose; and none was found that produced high acid from xylose broth. After 12 days the Lactobacillus pentoaceticus type in particular had begun to assume a prominent place in the flora. Inoculation with Lactobacillus pentoaceticus apparently caused this type of organism to predominate earlier with a greater percentage of the type at the final examination than in the silage from the other silos. In both inoculated and

9 Fred, Peterson, and Anderson 327 uninoculated silage the final examination after 8 or 10 weeks shows that the Lactobacillus pentoaceticus type formed 50 per cent or more of the flora. Accompanying it was found a short roundended rod, often in pairs, which resembled Bacillus lack acidi, but was iridescent on a glucose agar slant. Lactobacillus pentoaceticus and this short rod both gave a negative test for catalase, indicating t(hat they belong to Ohe lactic group. SUMMARY. An examination of the bacteriological and chemical analyses of inoculated and uninoculated silage indicates: 1. That the Lactobacillus pentoaceticus type plays an important part in t.he chemical changes produced. These organisms are present throughout the fermentation and in the last stages are the predominant type. An increase in Lactobacillus pentoaceticus means an increase in alcohol and volatile acid with a decreased amount, of lactic acid. 2. That the organisms of the Bacillus lactis acidi type persist and act only during the first days of the fermentation. 3. That the production of alcohol is undoubtedly due in part to the action of the predominant pentosefermenting type of lactic organism. 4. That inoculation may hasten and intensify the production of certain products during the early stages of the fermentation, but that in the later stages both chemical and bacteriological analyses of all inoculated and uninoculated silage show approximately the same chemical composition and the same kinds of microorganisms. BIBLIOGRAPHY. 1. See Boving, P. A., J. Agric. and Hart., 1920, xxiv, suppl. 13. Gorini, C., Stazioni sper. agrar. ital., 1918, li, 199; Reale Inst. Lombard0 SC. e Lettere, 1919, lii, 193. Heinze, B., Jahresb. Ver. angew. Bot., 1913, xi, 142. Stratton, G. F., Country Gent., 1919, lxxxiv, For a review of the literature see Esten, W. M., and Mason, C. J., Connecticut Agric. Exp. Station, Bull. 70, Hunter, 0. W., and Bushnell, L. D., Kansas Agric. Exp. Station, Il'echn. Bull. 2, Lamb, A. R., Iowa Agric. Exp. Station, Research. Bull. 40, Peterson, W. H., and Fred, E. B., J. Biol. Chem., 1920, xli, 181. Sherman, J. M., J. Bact., 1916, i, Peterson. W. H.. and Fred, E. B., J. Biol. Chem., 1920, xlii, 273.

10 THE RELATION OF LACTIC ACID BACTERIA TO CORN SILAGE E. B. Fred, W. H. Peterson and J. A. Anderson J. Biol. Chem. 1921, 46: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#reflist1