Silage production from fermentable forages

A method for the production of silage from a fermentable forage substrate by admixing Lactobacillus plantarum 2B bacteria with a fermentable forage substrate. The bacteria is added in an amount effective to lower the pH of the forage substrate to a pH at which the fermentable forage is stabilized and rendered substantially free of butyric acid producing bacteria.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for the production of a silage 
from a fermentable forage by anaerobic fermentation in the presence of a 
Lactobacillus bacterium. 
2. Description of the Prior Art 
A silage is the product of anaerobic preservation of a moist forage crop or 
crop residue by acidification caused by fermentation. Although the exact 
chemical and biochemical reactions responsible for the production of a 
stable silage are unknown, the silage fermentation process can be 
explained by considering only the principal reaction, i.e., the conversion 
of carbohydrates into organic acids, thereby lowering the pH and 
preserving the ensiled materials. However, it must be recognized that this 
is a simplification. The actual process taking place includes many of the 
known biochemical and microbiological changes which typically occur in 
fermentation. 
The principal aim of preparing silages is the production of a material 
useful for feeding animals which can be preserved for long periods of time 
with a minimum loss of nutrients. For some time it has been recognized 
that silage production is benefited by maintaining anaerobic conditions 
and by inhibiting clostridia bacteria. Anaerobic conditions are needed in 
order to inhibit aerobic microorganisms which otherwise would waste the 
nutrient resources of the feedstuff through oxidative activities. 
Furthermore, clostridia are known to cause protein destruction under 
anaerobic conditions, and their activity must be reduced if maximum 
retention of nutrient value is to occur. 
Furthermore, it has also been known that silage fermentations are benefited 
by the presence of lactate-producing bacteria. Under ideal fermentation 
condition, the primary product produced from carbohydrates in the forage 
material is lactic acid. There are two general pathways that lead to the 
production of lactic acid from carbohydrates by bacteria. The 
homofermentative pathway involves the conversion of glucose into two 
molecules of lactate. The heterofermentative pathway involves the 
conversion of one molecule of glucose into one molecule each of lactate, 
ethanol, and carbon dioxide. The homofermentative pathway is especially 
preferred in silage fermentations since all dry matter is preserved for 
use as a nutrient (i.e., there is no carbon dioxide production) and energy 
loss is also minimized. In view of these advantages, rapid production of 
lactic acid by a homolactic pathway as the primary means for acidifying 
the silage is preferred. 
As previously indicated, it is also desirable to minimize the activity of 
clostridia during the ensiling process. Although clostridial fermentation 
also produces acids and may eventually result in formation of a silage, 
nutrient loss is much greater than for lactate ensiling processes. For 
example, lactic acid itself is converted by clostridia into butyric acid, 
two carbon dioxide molecules, and two hydrogen molecules (using two lactic 
acid molecules as starting material). This results in a dry matter loss of 
more than 50%. Other clostridial pathways result in the degradation of 
proteins. For example, amino acids are de-aminated or oxidized to produce 
ammonia and carbon dioxide. In addition to the obvious destruction of 
nutrients, the production of basic components like ammonia raises the pH 
of the resulting silage and prevents acid-forming bacteria from reducing 
the pH to the level required for long-term storage. 
Because of the desirability of producing rapid lactic acid production, 
various publications have suggested inoculating silage feedstuffs with 
additional latate-producing bacteria. For example, M. E. McCullough, 
Feedstuffs, 49, 49-52 (1977), suggests desirable characteristics for a 
potential organism that would be satisfactory for use in silage 
production. Typical characteristics include the following: (1) the 
organism should have a high growth rate and be able to compete with and 
dominate other organisms likely to occur in silage; (2) the organism 
should be homofermentative; (3) the organism should be acid-tolerant and 
produce a final pH of 4.0 rapidly; (4) the organism should be able to 
ferment glucose, fructose, and sucrose, and preferably be able to ferment 
fructosans and pentosans; and (5) the organism should not react further 
with organic acids. However, as pointed out in McCullough's article, no 
organism having all of these desirable characteristics was known. 
Accordingly, there remains a need for a lactate-producing organism 
suitable for improving the production of silage from fermentable forage 
materials. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a method for producing 
silage from a fermentable forage substrate suitable for use as an animal 
feedstuff. 
It is a further object of this invention to provide a method for the 
production of silage which results in rapid acidification of silage, 
thereby inhibiting the growth of coliform and gram negative butyric acid 
producing anaerobes and the growth of yeasts and molds. 
It is yet another object of this invention to provide a method for the 
production of silage which produces a silage having a maximal nutrient and 
energy value. 
These and other objects of the invention as will hereinafter become more 
readily apparent have been accomplished by providing a method for the 
production of silage from a fermentable forage or forage-like substrate 
suitable for use as an animal feedstuff, which comprises the steps of 
admixing Lactobacillus plantarum 2B bacteria with a fermentable forage 
substrate, said bacteria being inoculated in an amount effective to lower 
the pH of said substrate to a pH at which said substrate is stabilized and 
rendered substantially free of butyric acid producing bacteria, and 
allowing fermentation to proceed under anaerobic conditions until a silage 
stable to anaerobic storage is obtained.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Microorganisms suitable for the procedures described herein are exemplified 
by cultures now on deposit with the American Type Culture Collection, 
12301 Parklawn Drive, Rockville, Md. 20852, USA. These cultures were 
deposited on Jan. 18, 1983, and are identified by ATCC No. 39268. Prior to 
this deposit, L. plantarum 2B was not available outside the laboratory of 
the inventors. 
The present invention has resulted from the discovery that a particular 
strain of bacteria, Lactobacillus plantarum 2B, is useful for the 
production of silage from a fermentable forage or forage-like substrate. 
L. plantarum 2B was isolated from a successful corn silage fermentation 
which was not inoculated with any supplemental bacteria. 
L. plantarum 2B is a Gram positive, short microaerophilic rod which is a 
catalase negative mesophile and has a temperature growth range of 
15.degree. C. to 45.degree. C. Its primary end-product of metabolism is 
lactic acid. The fermentation pattern for this organism is shown in the 
following table: 
______________________________________ 
Substrate Fermentation 
______________________________________ 
arabinose - 
fructose + 
galactose + 
glucinate + (with gas formation) 
lactose + 
maltose + 
mannitol + 
mannose + 
raffinose - 
rhamnose - 
ribose + 
sorbitol + 
glucose + 
sucrose + 
trehalose + 
xylose - 
glycerol - 
malic acid - 
citric acid - 
melibiose - 
melezitose + 
lactate - 
sorbose + 
propionate - 
______________________________________ 
L. plantarum 2B has previously been used in a model system intended to 
demonstrate the feasibility of producing animal feeds from frozen 
vegetable process waste; Moon, J. Food Sci., 44, 1460-1465 (1979). 
However, the significant difference in nutrient content of the substrate 
prevented this preliminary work from being applied directly to the 
production of silage from forage crops. Vegetable wastes contain a higher 
level of fermentable nutrients than do traditional forages. The 
freeze-thaw process that the wastes had undergone disrupted cell 
structures and provided more soluble nutrients than are available in a 
typical forage. Additionally, the freeze-thaw process destroyed many 
competing bacteria that would normally be present. The control of the 
population of naturally occurring bacteria is an important characteristic 
of the method of the present invention. 
This is particularly true in view of typical teachings in the prior art 
that certain bacteria could be used with various fermentation processes of 
traditional forages if and only if a nutrient supplement was added to the 
substrate. In the past, addition of nutrient supplements to forage crops 
has been suggested in order to increase the available carbohydrate for 
lactate production. However, carbohydrate addition is not without 
drawbacks. There is increased danger of secondary fermentation once the 
silage is uncovered or removed from the silo for feeding. The secondary 
fermentation includes growth of acid-tolerant yeast and molds which grow 
well in silages with a high level of water soluble carbohydrates. This 
results in lowering the nutrient value of the silage and possibly other 
detrimental effects. Accordingly, it was a particularly preferred goal of 
the present invention to discover a method of producing silage that did 
not require the addition of a supplemental nutrient, although, of course, 
a supplemental nutrient may be added if desired to increase the rate of 
bacterial growth. 
Lactobacillus plantarum 2B satisfies the previously cited criteria for 
organisms useful in silage fermentation: (1) it has a high growth rate and 
is able to compete with and dominate other organisms likely to occur in 
silage; (2) it is homofermentative; (3) it is acid-tolerant and produces a 
final pH of 4.0 or less quickly; (4) it is able to ferment glucose, 
fructose, sucrose and other complex sugars; (5) its reactivity with 
organic acids is negligible; and (6) it grow well from 15.degree. C. to 
45.degree. C., providing fermentation over a wide range of temperatures 
and climates. 
The present invention is carried out admixing Lactobacillus plantarum 2B 
with a fermentable forage or forage-like substrate. By forage is meant any 
plant material high in fiber suitable for consumption by domestic animals 
including cattle, sheep, goats, and the other ruminants. The forage 
material must be fermentable; i.e., it must be capable of undergoing 
fermentation in the presence of acid-producing bacteria to produce a 
silage. Suitable examples of forage materials, not intended to be 
limiting, include corn, alfalfa, wheat, rye, oats, sorghum, clover and 
grass. Many variations on the ensiling process for producing a silage from 
these fermentable forage substrates are known. Generally, ensiling 
comprises carrying out an anaerobic fermentation of the forage substrate 
using the naturally occurring bacteria present in the substrate when it is 
harvested. The present invention does not modify normal ensiling processes 
except for providing a specific species and strain of bacteria which has 
been demonstrated to produce superior results in the ensiling process. 
Superior results have been obtained for microbial counts for all forages 
used to date with the present invention. The present method, utilizing a 
particular strain of bacterium, gives unsurpassed control of other 
bacterial populations, such as coliforms and clostridia, as well as 
control of yeast and mold populations. Clostrida are capable of breaking 
down hexose and lactate to butyrate, resulting in a loss of dry matter. 
Butyrate is a weaker acid and the pH of the silage will rise which can 
present a favorable medium for putrefactive clostridia which cause the 
destruction of amino acids and a loss in nitrogen. Coliforms and other 
gram negative bacteria can cause loss of dry matter and a lower pH early 
in the fermentation process. Yeasts and molds cause deterioration of 
silages following the opening of the silo and exposure to air. Treatment 
of silages with L. plantarum 2B increases the stability of the silage 
after opening by decreasing the rate of heating of the silage and 
increasing stability in the feeding stage from two to four days. Thus, the 
method of the present invention provides remarkable potential for 
improving the quality of the silage product. Additionally, improved 
nutritional value for silages of the invention over control silages to 
which no L. plantarum 2B was added have been demonstrated for such widely 
varying forages as wheat, sorghum, and alfalfa. 
Furthermore, the particular strain of bacterium used in the present 
invention does not require the addition of any supplement or other 
vegetative matter containing soluble carbohydrates, such as would be 
suitable for human consumption when used with traditional forages. As 
previously mentioned, many lactate-producing bacteria require the presence 
of additional carbohydrates in order to produce a usable silage. In the 
present invention, the forage substrate can act as the sole source of and 
contains all nutritional requirements for the Lactobacillus plantarum 2B 
organism. Accordingly, the present invention can be carried out by 
admixing L. plantarum 2B with the fermentable forage substrate as the only 
source of nutritional requirements for the microorganism as well as by 
admixing L. plantarum 2B in the presence of such supplements. 
L. plantarum 2B is inoculated in an amount effective to lower the pH of the 
forage substrate to a pH at which the substrate is stabilized and rendered 
substantially free of butyric acid producing bacteria. By substantially 
free is meant that the populations of bacteria capable of producing 
butyric acid is reduced to a level at which butyric acid production is 
less than 5%, preferably less than 1%, of the total acid production. It is 
preferred that the pH be reduced to less than 4.5, preferably to less than 
4.2, and most preferably to less than 4.0 within 2 days and preferrably 
within 24 hours. The amount of inoculated bacteria required for this pH 
reduction will vary depending on the amount of naturally occurring 
lactate-producing bacteria present in the forage crop when it is 
harvested. Admixture of 10.sup.4 to 10.sup.9 viable bacteria per gram of 
plant material is generally sufficient. A ratio of from 10.sup.5 to 
10.sup.8 (and especially 10.sup.6 to 10.sup.7) bacteria per gram is 
preferred. There is no upper limit on the number of bacteria per gram 
added except that determined by cost effectiveness. 
Although particular treatment of the forage substrate is not specifically 
required, the forage substrate is generally chopped or otherwise divided 
into relatively small parts during the collecting or processing steps of 
harvesting prior to inoculation with the L. plantarum 2B bacteria. Finely 
chopped material is easier to handle, stores more compactly, provides a 
greater surface area and release of nutrients for reaction and 
fermentation, and more efficiently packs to exclude air from the interior 
of the fermenting material than is possible with a coarsely chopped or 
unchopped forage. The size of the chopped forage substrate varies 
depending on the crop, but typically the ensiled material has a length of 
no more than 2.5 centimeters, preferably 1.75 centimeters, and more 
preferably 1.25 centimeters. 
After admixing L. plantarum 2B with substrate in an otherwise known 
ensiling process, fermentation is allowed to proceed under anaerobic 
conditions until a silage stable to anaerobic storage is obtained. This 
time will vary depending on the type of forage used. A typical minimum 
time would be approximately one month, although periods of three weeks, or 
even 10 days, will be sufficient under ideal conditions. The fermentation 
process is self limiting, since the bacteria present normally or added are 
inhibited by pH reduction and their own waste products by the time 
sufficient fermentation has occurred. The resulting product, termed a 
silage, is thereafter stable to anaerobic storage. By stable is meant that 
no more than 10%, preferably 5%, loss of nutrients occurs after storage of 
one month as measured by the average nutrient loss of the individual 
nutrients. 
The present invention comprises mixing Lactobacillus plantarum 2B with the 
substrate and allowing fermentation to proceed until a suitable silage is 
formed. However, in order to place this invention into perspective, the 
following typical example which begins with harvesting of the forage crop 
and continues through feeding of the silage to an animal is given for 
purposes of illustration only. 
In this example, alfalfa will be used as the substrate. However, other 
materials would be handled in the same general manner differing only in 
well known details of harvesting and handling of the forage material. 
Plants are harvested by any typical harvester, for example, a six-knife 
forage harvester. Alfalfa would typically be harvested in the 20-40% bloom 
stage. Plants are chopped into relatively short pieces, for example, about 
1.5 cm long. The harvested crop is transported to a silo or other storage 
container. Typically, the L. plantarum 2B inoculum would be added to the 
chopped forage as it is added to the silo or other storage container. 
Various methods for accomplishing this admixing are known and may be used 
in carrying out the process of the invention. For example, an aqueous 
suspension of L. plantarum 2B may be sprayed on the forage as it is added 
to the storage bin. Alternatively, a dry preparation of the inoculum, for 
example, L. plantarum 2B with a suitable solid carrier, such as rice 
hulls, may be added similarly to the forage. The forage containing admixed 
L. plantarum 2B is covered or the storage container closed in such a way 
as to maximally exclude air from the fermenting forage. Fermentation is 
allowed to proceed for at least 10 days, preferably three weeks and more 
preferably a month. If desired, samples of forage may be withdrawn at 
intervals of time in order to determine whether or not fermentation is 
sufficient. However, a typical ensiling process carried out on a farm 
would not sample the silage at intermediate periods. At the end of the 
ensiling process, the silage is removed for feeding to a ruminant. The 
resulting silage has improved stability after opening the silo compared to 
silages prepared without admixing of L. plantarum 2B. 
Lactobacillus plantarum 2B can be prepared in bulk by culturing in MRS 
broth or other suitable media at about 30.degree. C. at a pH of about 5.8. 
MRS broth is a liquid microbiological medium containing yeast extract as a 
source of vitamins, minerals and other growth factors; trypticase as an 
amino acid source; sodium acetate; ammonium citrate; mineral salts; and 
sorbitan monooleate. 
Other complete microbiological media providing similar nutrients of the 
same type can also be used. 
Suitable inocula for application to a fermentable forage can be prepared by 
any standared method, typically by freeze-drying cultures and mixing with 
a solid carrier, such as rice or peanut hulls, cornmeal, non-fat dried 
milk, lactose, and similar materials, in order to produce a solid 
inoculum. Aqueous inocula can be prepared by diluting the original culture 
with water or another suitable aqueous carrier, such as phosphate buffer 
or cheese whey. If desired, a liquid inoculum can be stored in frozen 
form. Accordingly, the phrase "aqueous" or "liquid" inoculum as used 
herein refers to both frozen and fluid forms. A liquid inoculum can also 
be prepared by dissolving or suspending a soluble solid inoculum 
containing L. plantarum 2B, such as one prepared using lyphilized cheese 
whey as a carrier. If desired, the inoculum (solid or liquid) can be 
manufactured in concentration form which can be diluted by the ultimate 
user prior to application to a forage. 
Having now generally described this invention, the same will be better 
understood by reference to certain specific examples which are included 
herein for purposes of illustration only and are not intended to be 
limiting of the invention or any embodiment thereof, unless specified. 
EXAMPLE 1: SILAGE PRODUCTION FROM ALFALFA, WHEAT CORN AND SORGHUM 
Material and Methods 
Siliage Preparation 
Plants were harvested by a six-knife forage harvester. Alfalfa was second 
cutting 20 to 40% bloom stage harvested in June. Wheat was in the early 
boot stage and was harvested in late April. Corn in the early dent stage 
was harvested in late July. Sorghum in the late dough stage was harvested 
in late August. Plants were chopped into approximately 1.5 cm long pieces 
and transported immediately after cutting to experimental silos for 
filling. Fifty-five kg of harvested forage was packed in 6-mil 
polyethylene bags and placed in 0.21 m.sup.3 steel drums. Care was taken 
to pack material to exclude air and to seal the bags. Eight drums for each 
control and each inoculated silage were prepared. Silage drums were placed 
in an unheated barn, and an average ambient temperature was measured for 
each silage (alfalfa 24.degree. C., corn 25.degree. C., sorghum 25.degree. 
C., and wheat 17.degree. C.). After 0, 1, 2, 4 (or 5), 8, 16, and 33 days 
of fermentation, one drum each for the treatment and control was opened 
for chemical and microbiological analysis. 
Preparation of Inoculant 
The L. plantarum 2B was prepared by culturing in 10 liters MRS broth in a 
New Brunswick 14-liter fermenter. This culture medium is described in 
Rogosa et al, J. Bacteriol., 62, 132 (1951), which is herein incorporated 
by reference. Conditions of fermentation were temperature 30.degree. C., 
pH 5.8, no aeration; moderate stirring was used to help maintain the 
fermentation temperature. After 48 h of culture, cells were harvested by 
centrifugation from the spent culture medium. Cells were resuspended in 
100 ml of phosphate buffer (0.03 M, pH 7.2) and stored in polyethylene 
screw cap vials at -20.degree. C. for up to 3 months. At the time of 
inoculation, cells in storage vials were thawed rapidly in multiple 
changes of 17.degree. C. water. A direct microscopic count of the 
population per milliliter of the thawed concentrated bacterial suspension 
was determined. Inoculum, 10.sup.7 L. plantarum per gram silage, was 
prepared from the culture concentrate by a suitable dilution in 700 ml of 
water and addition of 100 ml of this inoculum to each 55 kg of silage to 
be inoculated. The viable population of bacteria was determined by plating 
the inoculum immediately after preparation and after maintaining on ice at 
4.degree. C. during inoculation of silages. 
The 100 ml of inoculant was sprayed on the silage with a thin layer 
chromatography plate sprayer. The 55 kg of silage was spread out on a 
plastic sheet (4.times.4 m), and the surface of the silage sprayed with 
about one-third of the inoculum. Silage and inoculum were mixed well, 
redistributed on the plastic, and sprayed and mixed twice more. Control 
silage was treated similarly without spraying. Inoculated and control 
silages then were packed carefully in the polyethylene bags in the 0.21 
m.sup.3 drums. 
Chemical Composition 
At each sampling period the total weight of material recovered was measured 
and used to assess percent recovery of nutrients. Subsamples were 
oven-dried at 50.degree. C. to assess percentage of moisture and were 
ground in a Wiley Mill to pass a 2-mm screen. Proximate analysis included 
fat, protein, ash, and nitrogen-free extract using standard methods of 
analysis approved by the American Association of Analytical Chemists. Acid 
detergent fiber (ADF), neutral detergent fiber (NDF), and permanganate 
lignin analyses were carried out according to methods described in Van 
Soest, J. Assoc Offic. Anal. Chem., 46, 829 (1963); Van Soest et al, 
ibid., 50, 50 (1967); and Van Soest et al ibid., 51, 780 (1978), 
respectively. Water soluble carbohydrate (WSC) was determined by the 
method of Smith, Agric. Food Chem., 20, 238 (1972). Measures of pH were on 
water extracts (10 g sample+90 ml H.sub.2 O, blended 1 min high speed 
Waring blender) by an electromark pH meter (Del Mar, CA). Volatile and 
nonvolatile fermentation acids were determined by gas chromatographic 
procedures as described in Moon et al, J. Dairy Sci., 64, 807 (1981). 
Analyses were on days 0, 2, 4, 8, and 33. All chemical analyses were in 
duplicate on duplicate subsamples from each drum. 
Microbiological Analysis 
Two 100-g samples of silage were removed from the center of the silage bag, 
one from the upper half and one from the lower half. Samples were placed 
in sterile whirl pack bags, air was removed by compression, bags were 
sealed and moved promptly to the laboratory for microbiological analysis. 
A 10-g subsample of this silage was weighed aseptically into a sterile 
200-ml blender jar. Ninety milliliters of 0.03 M 
dihydrogen-phosphate-buffered distilled water (pH 7.2) were added to the 
sample and blended in a Waring blender at high speed for 1 min. Serial 
dilutions were in the same phosphate buffered distilled water, and pour 
plates were prepared according to procedures outlined in the Standard 
Methods for the Examination of Dairy Products. 
Microflora in the fermented material were evaluated in a manner similar to 
those of others who have defined bacterial populations in silages and to 
efforts to identify predominant microflora recovered in selective 
laboratory media in the inventor's laboratory published in Moon et al, J. 
Dairy Sci., 64, 807 (1981). 
Samples were plated in duplicate on selective and nonselective agars. 
Trypticase soy broth plus agar 1.5% (TSB+A) (Difco, Detroit, MI) was used 
as a general plating medium to recover facultative anaerobic or 
microaerophilic bacteria. Lactobacillus selective agar (LBS, Baltimore 
Biological Laboratory, MD) was used to recover Lactobaccilli. Azide 
dextrose broth+1.5% agar (AZD) was used to recover lactic acid cocci which 
were predominately streptococci. Plates were incubated in a reduced oxygen 
(15% CO.sub.2, 85% air) atmosphere at 32.degree. C. for 1 week before 
colonies were enumerated. Yeasts and molds were enumerated on the rose 
bengal chlortetracycline agar (YM) described by Jarvis, J. Appl. 
Bacteriol., 36, 723 (1973). Coliforms were enumerated on violet red bile 
(VRB) agar. The YM was incubated aerobically at 30.degree. C. for 1 wk and 
VRB for 48 h before enumeration of colonies. 
Statistical Analysis 
Statistical comparisons were between treatments over the entire 
fermentation period. Days were not compared as only one drum per treatment 
per day was prepared. Effect of the additive on pH and chemical 
composition was evaluated statistically using standard methods of analysis 
including these regression models: chemical 
composition=additive+day+day.sup.2 +(day X additive)+(day.sup.2 X 
additive); and pH, log cells=additive+log day+day+(log day X 
additive)+(day X additive). The correlation coefficient of these models 
was generally above 0.70. Tests were for additive over the entire 
fermentation period at P&lt;0.05. Duncan's multiple range test also was used 
to test for differences between treatment means. Data were analyzed as 
total weight per treatment and as percent of dry matter. 
Results 
Chemical compositions of fresh forages are in Table 1. All forage had 
initial pH between 5.2 and 5.7. Percent recoveries for the four crops of 
control and treated silages after 33 days are in Table 2. All silages had 
high recoveries of nutrients in fresh forage. Addition of inoculum 
increased recovery of dry matter, crude protein, and acid detergent fiber 
in alfalfa silage but decreased nitrogen-free extract in wheat silage. 
Recovery of nutrients in corn and sorghum silage was not affected by 
addition of inoculum. 
Lactic acid and volatile fatty acid anaylsis of silages is in Table 3. 
Lactic acid was increased (P&lt;0.05) in alfalfa and wheat silages by 
addition of L. plantarum 2B. Succinic acid was decreased (P&lt;0.05) in 
alfalfa and wheat silages with addition of L. plantarum 2B. Wheat silage 
also showed increased acetic acid (P&lt;0.05) with addition of L. plantarum 
2B. There were no significant differences in any of the fermentation acids 
in corn or sorghum silages. Only in alfalfa control silage was any butyric 
acid produced (P&lt;0.05). 
The change in pH over time for corn and sorghum silage (FIG. 1) was not 
significantly affected by addition of L. plantarum 2B (Table 4). The pH 
dropped rapidly to approximately 3.7 by 1 day and remained this low 
throughout the remainder of the fermentation period. Addition of L. 
plantarum 2B lowered pH (P&lt;0.05, Table 4) of alfalfa and wheat silages 
(FIG. 2). The difference was seen by day 2 and remained through the 
fermentation period. 
Facultative anaerobic bacteria were increased (P&lt;0.05) by day 32 for 
alfalfa, corn, sorghum, and wheat silages with addition of L. plantarum 2B 
(FIGS. 3, 4, Table 4). Populations in corn and sorghum silages reached a 
maximum after 1 day and then declined. Populations in alfalfa and wheat 
silages reached maximum later in fermentation (FIG. 4). Total lactobacilli 
counts were increased (P&lt;0.05) by addition of L. plantarum 2B in alfalfa, 
wheat, and sorghum silages but not in corn silage (FIGS. 5, 6, Table 4). 
Initial populations of lactobacilli were much lower (10.sup.3 to 10.sup.5 
/g) in control silages of alfalfa and wheat (FIG. 6) than inoculated 
silages. Initial populations of lactobacilli were 10.sup.6 to 10.sup.7 /g 
in corn and sorghum silages which was similar to inoculum. Populations of 
lactic acid cocci recovered on azide dextrose agar were similar in all 
silages (FIGS. 7, 8, Table 4). Yeast and mold counts were lowered (P&lt;0.05) 
by addition of L. plantarum 2B in alfalfa and wheat silages (FIGS. 9, 10, 
Table 4), but it did not have a significant effect on corn or sorghum 
silages. 
TABLE 1 
______________________________________ 
Chemical composition of alfalfa, corn, sorghum, and wheat forages 
Alfalfa 
Corn Sorghum Wheat 
______________________________________ 
pH 5.74 5.24 5.40 5.79 
Dry matter, % 32.4 35.0 27.5 25.0 
Crude protein.sup.a 
16.7 7.0 6.1 10.2 
Soluble carbohydrate.sup.a 
11.1 42.7 64.4 42.4 
Crude fat.sup.a 
2.2 1.6 1.4 1.6 
Crude fiber.sup.a 
20.5 19.4 23.2 33.0 
Nitrogen free extract.sup.a 
49.8 59.8 60.1 43.3 
Neutral detergent fiber.sup.a 
50.3 42.1 52.9 58.0 
Acid detergent fiber.sup.a 
33.9 20.0 20.5 38.4 
______________________________________ 
.sup.a Percent of dry matter 
TABLE 2 
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Effect of addition of Lactobacillus plantarum to silage on percent 
recovery or nutrients at the end of the fermentation period. 
Treatment 
Lactobacillus 
Component.sup.a 
Silage Control plantarum 
______________________________________ 
Dry matter Alfalfa 89.6 93.7.sup.b 
Crude protein 82.6 95.6.sup.b 
Soluble carbohydrate 56.5 60.8 
Crude fat 100.2 100.2 
Crude fiber 100.0 100.0 
Nitrogen free extract 65.0 66.0 
Neutral detergent fiber 99.0 100.3 
Acid detergent fiber 90.7 100.1.sup.b 
Dry matter Corn 97.5 99.5 
Crude protein 100.0 100.0 
Soluble carbohydrates 33.1 24.4 
Crude fat 100.0 100.0 
Crude fiber 100.0 100.0 
Nitrogen free extract 98.0 94.0 
Neutral detergent fiber 90.5 94.4 
Acid detergent fiber 100.0 100.0 
Dry matter Sorghum 100.0 100.6 
Crude protein 100.5 100.5 
Soluble carbohydrate 12.5 14.1 
Crude fat 100.0 100.0 
Crude fiber 100.0 100.0 
Nitrogen free extract 100.0 99.0 
Neutral detergent fiber 100.0 100.0 
Acid detergent fiber 67.6 63.8 
Dry matter Wheat 100.0 100.0 
Crude protein 100.0 100.0 
Soluble carbohydrates 25.5 23.4 
Crude fat 100.0 100.0 
Crude fiber 100.0 100.0 
Nitrogen free extract 99.2 97.0.sup.b 
Neutral detergent fiber 100.0 100.6 
Acid detergent fiber 90.0 100.0 
______________________________________ 
.sup.a Recovery of nutrients is calculated as a percentage of the fresh 
forage at day 0 vs 33 
.sup.b Percents followed by b differed (P &lt; .05) from the control over th 
entire fermentation period 
TABLE 3 
______________________________________ 
Fermentation acids (mM/g silage wet wt) produced during ensiling 
Days of ensiling 
Silage 
Additive Acid 0 2 4 8 33 
______________________________________ 
Alfalfa 
None Acetic 1.07 21.49 
40.74 
33.86 
39.46 
Propionic .sup. . . ..sup.a 
. . . 
. . . 
. . . 
.61 
Lactic . . . 
14.29 
18.51 
23.83 
29.86 
Butyric . . . 
. . . 
. . . 
.51 17.40 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic .30 2.73 
3.95 
4.72 
4.36 
L. Acetic 1.59 11.19 
20.69 
43.21 
57.29 
planta- Propionic . . . 
. . . 
. . . 
. . . 
.sup. . . ..sup.b 
rum Lactic . . . 
24.89 
32.69 
29.75 
.sup. 36.00.sup.b 
Butyric . . . 
. . . 
. . . 
. . . 
.sup. . . ..sup.b 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
.sup. . . ..sup.b 
Succinic .33 .86 1.13 
.91 .sup. 1.06.sup.b 
Corn Control Acetic . . . 
36.53 
39.97 
48.07 
35.70 
Propionic . . . 
. . . 
.01 .01 .22 
Lactic . . . 
41.79 
51.32 
55.89 
42.63 
Butyric . . . 
. . . 
. . . 
. . . 
. . . 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic .22 1.65 
1.93 
1.44 
1.20 
L. Acetic .79 33.17 
47.75 
50.87 
.sup. 39.74.sup.b 
planta- Propionic . . . 
.03 . . . 
.06 .sup. .44.sup.b 
rum Lactic .18 39.75 
47.65 
47.85 
.sup. 38.95.sup.b 
Butyric . . . 
. . . 
. . . 
. . . 
. . . 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic .21 1.29 
1.23 
1.09 
.sup. 1.11.sup.b 
Sor- Control Acetic .25 48.83 
43.03 
58.30 
33.70 
ghum Propionic . . . 
. . . 
. . . 
.70 .38 
Lactic . . . 
23.65 
44.06 
42.30 
33.61 
Butyric . . . 
. . . 
. . . 
. . . 
. . . 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic .16 .11 1.17 
1.40 
1.21 
L. Acetic .69 46.18 
30.47 
42.88 
.sup. 58.45.sup.b 
planta- Propionic . . . 
. . . 
. . . 
.25 .sup. .47.sup.b 
rum Lactic . . . 
22.27 
41.73 
41.64 
34.67 
Butyric . . . 
. . . 
. . . 
. . . 
. . . 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic . . . 
1.22 
1.19 
. . . 
1.27 
Wheat Control Acetic .49 15.29 
25.10 
29.89 
35.34 
Propionic . . . 
. . . 
. . . 
. . . 
. . . 
Lactic . . . 
14.54 
26.88 
26.61 
33.21 
Butyric . . . 
. . . 
. . . 
. . . 
. . . 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic . . . 
6.49 
5.93 
6.65 
6.72 
L. Acetic . . . 
10.11 
19.33 
45.25 
.sup. 40.65.sup.b 
planta- Propionic . . . 
. . . 
. . . 
. . . 
. . . 
rum Lactic . . . 
21.29 
43.28 
36.02 
.sup. 54.40.sup.b 
Butyric . . . 
. . . 
. . . 
. . . 
. . . 
Isobutyric 
. . . 
. . . 
. . . 
. . . 
. . . 
Valeric . . . 
. . . 
. . . 
. . . 
. . . 
Isovaleric 
. . . 
. . . 
. . . 
. . . 
. . . 
Succinic .39 2.80 
3.81 
4.58 
.sup. 2.49.sup.b 
______________________________________ 
.sup.a Less than .01 mM/g silage wet weight. 
.sup.b Means followed by b differed (P &lt; .05) from the control over the 
entire fermentation period. Standard errors of means of duplicate 
determinations are acetate 2.08, propionate .02, lactate 1.92, butyrate 
.31, isobutyrate .01, valerate .01, isovalerate .02, succinate .22. 
TABLE 4 
__________________________________________________________________________ 
Effect of inoculation of alfalfa, corn, sorghum, and wheat silages with 
Lactobacillus plantarum 2B on mean pH and microbial populations 
(log.sub.10 cell 
number/g silage) recovered on four different agar media. 
Agar medium.sup.a 
Silage 
Treatment 
pH Axide dextrose 
LBS TSB + A 
YM 
__________________________________________________________________________ 
Alfalfa 
Control 
4.71 8.35 8.41 9.09 4.76 
L. plantarum 
4.36.sup.bc 
7.89 9.12.sup.c 
9.46.sup.bc 
4.25.sup.bc 
Corn Control 
3.74 8.05 8.25 8.59 5.14 
L. plantarum 
3.77 8.22 8.52 8.97.sup.bc 
5.39 
Sorghum 
Control 
3.74 7.95 7.83 8.35 5.57 
L. plantarum 
3.79 8.66.sup.bc 
8.45.sup.bc 
8.97.sup.bc 
5.39 
Wheat 
Control 
4.62 8.08 7.35 8.63 5.35 
L. plantarum 
4.14.sup.bc 
8.03 8.96.sup.bc 
9.28.sup.bc 
4.45.sup.bc 
__________________________________________________________________________ 
.sup.a Agar medium. Azide dextrose for lactic acid cocci, LBS for 
lactobacilli, TSB + A for total facultative anaerobes, YM for yeasts and 
molds. 
.sup.b,c Means followed by b differed (P &lt; .05) from control silages as 
determined by a Ducan' s tests of treatment means; when followed by c, 
means differed (P &lt; .05) from control silages using linear regression 
models determined over the 33 day fermentation period. The standared erro 
of the means of duplicate determinations was azide dextrose .288, LBS 
.189, TSB + A .288, YM .288, pH .075. 
EXAMPLE 2: STABILITY OF SILAGE AFTER OPENING SILO 
A corn silage of the invention and a corn silage control were prepred as 
describe in Example 1. The silos were then opened and the temperatures 
were measured at intervals as shown in the following Table and in FIG. 11. 
TABLE 5 
__________________________________________________________________________ 
Effect of inoculation of corn silage with L. plantarum 2B 
on stability of silage after opening 
Temperature (.degree.C.) at indicated time (hrs) after opening 
Silage 0 6 24 32 48 56 72 80 106 
126 
144 
__________________________________________________________________________ 
Control 
25 
26.4 
28.2 
35.2 
42.7 
36.2 
37.5 
35.9 
33.6 
37.1 
39.2 
L. plantarum 
25 
26.1 
26.3 
26.8 
28.2 
29 42.4 
46.4 
32.6 
32.4 
40.2 
(ambient 
25 
25.2 
26.0 
26.0 
26.3 
25.9 
26.2 
25.9 
27.4 
-- 26.9 
temperature) 
__________________________________________________________________________ 
Temperature is a measure of stability of the forage after opening as heat 
is generated by yeasts and molds after the silage is exposed to air. The 
nearly two day lag in reaching maximum temperature after opening indicates 
greater stability for the silage prepared according to the invention over 
a silage prepared without inoculation of L plantarum 2B. 
EXAMPLE 3: COMISON OF SILAGES OF THE INVENTION WITH SILAGES PREED 
USING A DIFFERENT STRAIN OF L. PLANTARUM 
Silages were prepared as described in Example 1. The comparision inoculum 
(designated L. plantarum H) was received from Chr. Hansen's Laboratory, 
Inc., 9015 W. Maple St., Milwaukee, Wis. 53214, on dry ice and immediately 
transferred to a -20.degree. C. freezer and stored until the day of silage 
preparation. The cans of inoculum were thawed by placing in room 
temperature (17.degree. C.) water for about 15 min. The cans were suface 
sterilized with alcohol, flamed and opened. A direct microscopic count of 
the population per ml in each can was determined. A level of inoculum of 
L. plantarum H equal to that used with L. plantarum 2B was prepared from 
the culture concentrate by adding 50 ml of the concentrate to 700 ml of 
water and adding 100 ml of this to 55 kg of silage giving an approximate 
level of inoculum of 10.sup.7 /g silge. L. plantarum 2B was treated as 
described in Example 1. Experimental procedures are also described in 
Example 1. 
TABLE 6 
______________________________________ 
Effect of inoculation of the silage with L. plantarum on the percent 
recovery.sup.a of nutrients at the end of fermentation period 
Additive.sup.b 
Component Silage Control LpH Lp2B 
______________________________________ 
Weight Alfalfa 87.45 97.87 94.43 
Dry matter 100.00 101.37 100.17 
Protein 77.88 97.87 84.11 
Soluble carbohydrate 98.88 108.32 111.29 
Fat 116.68 130.61 148.87 
Crude fiber 79.87 100.47 88.16 
Nitrogen free extract 91.11 93.48 94.69 
Neutral detergent fiber 
94.88 99.95 102.47 
Acid detergent fiber 85.08 95.23 102.09 
Weight Corn 98.67 101.87 96.74 
Dry matter 100.00 98.70 101.34 
Protein 108.87 101.26 105.63 
Soluble carbohydrate 48.26 34.08 46.02 
Fat 100.37 103.02 93.73 
Crude Fiber 69.86 80.06 66.62 
Nitrogen free extract 138.30 135.01 137.52 
Neutral detergent fiber 
86.33 98.09 90.69 
Acid detergent fiber 89.12 97.99 87.37 
Weight Sorghum 90.07 91.13 90.09 
Dry matter 100.00 93.67 96.67 
Protein 88.23 91.13 87.33 
Soluble carbohydrate 25.76 26.06 28.71 
Fat 98.38 99.19 81.30 
Crude Fiber 88.24 91.13 84.59 
Nitrogen free extract 91.27 93.36 93.89 
Neutral detergent fiber 
94.57 94.17 96.10 
Acid detergent fiber 96.50 104.15 102.96 
Weight Wheat 95.07 101.05 96.28 
Dry matter 102.88 101.32 103.95 
Protein 93.55 100.05 96.29 
Soluble carbohydrate 29.11 35.70 33.74 
Fat 234.33 197.63 219.09 
Crude fiber 107.79 88.67 101.12 
Nitrogen free extract 72.92 98.73 81.05 
Neutral detergent fiber 
85.40 98.36 88.13 
Acid detergent fiber 83.55 93.99 93.37 
______________________________________ 
.sup.a % recovery (total amount in drum Day 32)/(Total amount in drum Day 
0) .times. 100 
.sup.b LpH = Lactobacillus plantarum H, Lp2B = Lactobacillus plantarum 2B 
As can be seen from an examination of Table 6, the nutrient values present 
in the resulting silages were comparable, apparently since all silages 
were prepared under optimum conditions. However, L. plantarum 2B gave 
better control of the total population of anaerobic bacteria present 
during fermentation for all silages as shown in FIGS. 12-15. These figures 
show total microaerophilic populations in silages (as demonstrated on a 
trypticase soy broth plus agar medium described in Example 1). In all 
cases the total microaerophilic populations is higher for L. plantarum 2B 
than it is for L. plantarum H during the initial stages of fermentation, 
indicating that L. plantarum 2B is superior at controlling the total 
population of anaerobic bacteria during fermentation. This control is 
believed to be important for silages prepared under non-optimal 
conditions, as would typically occur on a farm. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein.