Note: Descriptions are shown in the official language in which they were submitted.
~9S;~7
It is known to preserve various kinds of vege-table and
animal products, subs-tan-tially intended for feeding purposes, by
means of biological ensiling. This means a fermentation to lac-
tic acid of carbohydrates included in or added to the materials
by means of lactic acid forming bacteria. In the formation of
the lactic acid a lowering of the pH is obtained, which has a
preserving effect. A low redo~ potential is obtained as well,
which inhibits oxidative degradation processes such as rancidi-
fication. The biological ensiling has been used more and more
for utilizing food or feedstuffs, which otherwise would have been
cons;dered as waste and unsuitable for utilization. This is of
course of the greatest importance in these times of an i~minent
global lack of food.
In the biological ensiling a suitable growth environ-
ment for the microorganisms forming lactic acid is required. In
the processes known so far it has been necessary to have a suffi-
cient amount of carbohydrates (sugars) present, which are fermen-
table by the microorganisms, or it has been necessary to add
starch degrading enzymes or materials, such as malt, to convert ~-
the starch present to fermentable carbohydrates. Such additives
have been required in relatlvely large amounts, normally more
than 20% of starch containing material.
In ensiling it is very desirable that a sufficiently
high lactic acid content is obtained quickly i71 the ensilage so
that competing putrefactive reactions cannot proceed too far.
This requires that bacteria are available in the ensilage of a
type which can grow rapidly at the low pH-values arlsing in the
ensilage. However, in previously known ensiling processes as
above, only those bacteria have been used, which have been pre-
sent from the beginning in the starting material or the additive.However, by variations in the type of starting material and addi-
tive and also by geographical and seasonal variations of one and
~"~
the same material, variations have arisen in -the course of the
fermentation so tha-t ensilages of a varying quality have been
obtained. In order to obtain an optimal and reproducible ensil-
ing process, it would therefore be very desirable to have access
to bacteria with properties determined for ensiling. In this
way it should be possible to control the ensiling process in a
desired direction and also to adapt it to different types of star-
ting materials.
Moreover, as the fermentable sugars as well as the en-
zymes and malt are relatively expensive, it is also often desir-
able to find a biological ensiling process that can be carried out
without addition of these expensive materials. This would be
possible if bacteria forming lactic acid could be found which are
capable of converting the cheap types of starch to lactic acid.
According to the previously known literature, however, there~are
no bacteria with such properties.
This invention is now based on the discovery that it
has become possible by selective culture to produce a bacteria
strain which is capable of fermenting sugars to lactic acid at a
low pH, as well as a bacteria strain which is capable of degra-
ding starch to fermentable sugars~ According to the invention,
the first-mentioned of these strains or both are added to the
starting material to be ensiled.
In the cases when fermentable sugars in the starting
material or the additive are present in a sufficient quantity,
it is sufficient to add only the bacteria strain, which is ac-
tive at a low pHo A conversion to lactic acid is then obtained,
which is more rapid and more far-reaching than what has been the
case in previously known processes.
In the cases when fermentable sugars are not present at
all or only in an insufficient amount in the starting rnaterial or
the additive, it is necessary also to add bacteria of the starch
L7
degrading ~train, a9 an addition of the strain only active a-t a
low pH would result in a fermentation which is too slow~ It goes
without saying that starch-containing carbohydrate materials must
be present in this case,
In the ~election of the two types of bacteria, two sam-
ples are taken from a previous biological ensilacJe, and one of
them i~ cultuxed repeatedly in a substrate with a p~ decreasing to
4-4.7, preferably 4.5, and the other is cultured repeatedly in a
~ub~trate con~aining starch as the sole carbon source. The
~0 strains selected in this way can then be cultured on a larger
scale on a corresponding substrate and be recovered, e.g. by
freeze-drying, to be mixed with a carrier ~ubstance, e.g. grain
in the form of grits, to a concentrate, which after additional
dilution is added to the starting material for the ensiling pro-
cess.
In accordance with the invention, there i~ provided
a process for biological ensiling o vegetable and/or animal
materials with the use of lactic acid-forming bacteria wherein
carbohydrate materials which can ~e converted to lactic acid
and lactic acid-forming bacteria are added to the starting
material~ to be ensiledO The process consists of adding to the
starting materials:
~ a) lactic acid-forming acid-tolerant bacteria,
which have been selected fro~ naturally-occurring bacteria of
the species Streptococcus faecalis by repeated cultivation in a
medium having a pH value of 4 to 4~7, and
(b~ ~exmentable carbohydrates~
According to the above, carbohydrates are added to the
starting material for the ensiling process, which carhohydrates
can be metabolized to lactic acicd by the bacteria added. The~e
carbohydrates can consist of various starch containincJ materials,
preferably grain products, e~g. grain in the form of grits, which
~3-
. .
, ~, . . , ., , . ., . ~ . . .
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can advantageously be added in the forrn of a diluent for the
bacteria concentrate. As indicated above, no addition of ~ermen-
table sugar or starch degrading factors is required in the case
when both types of bacteria are used, but it may be advantageous
to add such materials in minor amounts, as this will start the
fermentation to lactic acid more rapidly. This especially applies
when ensiling animal materials, whereas it is not necessary for
certain vegetable materials, which often contain fermentable
sugars from the beginning. It may also be suitable to add small
amounts of malt, as this contains both biofactors promoting the
development of the bacteria, and starch degrading enzymes. Malt
products which decompose starch can also be added, such as dia-
~'
-3a-
.
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stase, and furthermore, other starch degradlng materials rnay also
be added, such as starch degrading yeas-ts or other fungi.
Thus, in summary, the acid tolerant bacteria should
always be present to guarantee that a fermentation to lactic acid
quic~sly starts and proceeds to a low pEI value in the silage. To
provlde the material -to be fermented by the acld tolerant bacteria,
however, various possibilities exist. Thus, the material to be
ensiled may in itself contain a sufficient amount of fermentable
sugars, or such sugars may be added to the material. Further-
more, the material may contain starch or may be mixed with starch.In this case, means for degrading the starch into fermentable
sugars must be provided, and these may consist of malt, which
contains starch degrading enzymes, such as diastase, or of these
or other starch degrading enzymes in a purified form. Further-
more, other starch degrading materials may be added, such as
starch degrading yeasts or other fungi. Finally, the starch de-
grading bacteria selected in accordance with the present inven-
tion may be used. It goes without saying that one or more of
these materials may be used together. The important condition
is that a fermentation to lactic acid and a low pH value in the
silage must be established quickly, so that competing fermenta-
tions and other reactions are subdued.
If it is intended to preserve the silage for a long
time, rod-shaped bacteria of the strain Lactobacillus, such as
Lactobacillus plantarum should also be added to the star-ting
material or the additive. These bacteria grow more slowly in the
silage than the aforesaid bacteria strains, but are tolerant to a
low pH, and have therefore a favourable influence on the mainte-
nance of a high content of lactic acid during long preservation
times.
The material to be ensiled in accordance with the inven-
-tion may con~ist of an animal material, such as various kinds of
-- 4 --
~s~
meat and fish and was-te products thereof, such as blood, meat
trimmings, fish gu-t-ting~s, etc. or of vegetab.le material, such as
various green fodders, e.g. grass, clover, alfalfa, and tops of
sugar-beets, or industrial wastes, such as brewer's grains. It
is also possible to use the process of the invention for ensiling
manure from various animals, such as cattle and poultry.
In order to obtain a rapid and safe ensiling process the
different bacteria should be added to the starting material or
additive in an amount providing at least about 103 organisms of
each type of bacteria per g mixture of starting material and addi-
tive. This is preferably achieved in such a way that a concen-
trate is provided which contains each of the desired types of bac-
teria in a concentration of 107 - 10 organisms per g, after which
the user dilutes the concentrate with grain products, e.g. grains
in the form of grits in such an amount that when this mixture is
added to the starting material for the ensiling the required bac-
teria concentration is at-tained. The concentrate is in turn a
carrier substance with admixture of the freeze-dried bacteria
from the cultures, in which the bacteria concentrations are u~sual-
ly about 101 bacteria/g.
The ensiling process according to the invention is pri-
marily intended for preservation and preparation of feedstuff for
e.g. farm animals, poultry,and domestic animals. By selection of
suitable ensiling conditions and starting material, e.g. fish
fillets or raw fish pulp, it is however possible also to prepare
feedstuffs for human consumption, and this is also comprised in
the presen-t invention.
The starch containing material and/or the fermentable
sugars should be added together with the bacteria to the starting
material to be ensiled in an amount of 1 to 12, but normally 3 to
10% by weight calculated on the starting material. When ensiling
e.g~ animal material about 9% by weight is a suitable value.
: : ,
53~ ~
This amount is considerably less than the amounts of starch con-
taining materi.al and malt required in ensiling processes of the
previously known types, where at least about 20% by weiyht are
re~uired to obtain good results. Thus this, is a great advantage
of the present invention. When ensiling vegetable materlals it
is usually preferable -to add 1 to 6% by wei.ght of carbohydrates
(with admixed bacteria), especially then about 3/O by weight.
The selection of the bacteria used according to the in-
vention is carried out in the following way:
It is started from a sample, which is preferably taken
from a previous biological ensilage, but which can also be taken
from different cereals,green forage and manure of various origins.
This sample is cultured on a substrate for lactic acid bacteria, :
e.g. tomato agar, at about 28C.
For culture of the acid tolerant bacteria a suitable :
number of the bacterial colonies formed is then taken out and
transferred to a weakly buffered li~uid medium having for example
the composition indicated in the following. At the beginning, the
pH of the medium is about 6-6.2, but it will decrease rapidly
during culture (within one day) to below 4.5. The culture îs left
.with this low pH for a long time (about 3 weeks) at 28C to select
the strains which best withstand the acid environment. After this
time the viable bacteria are transferred to a new medium of the -
same composition, and the culture is left again for about 3 weeks.
Also here a rapid pH decrease to below 4,5 is obtained. The bac-
teria are further transferred every third week~about 8 times, :.
after which the selected strain does not undergo any additional
considerable changes. After this the bacteria can be cultured on
a large scale and be freeze-dried to be used as starting material
for the bacteria concentrate.
The medium used has the following composition:
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Peptone 10 g
Meat extract 10 g
Yeast 5 g
Glucose 20 g
Tween 80~ 1 ml
K2HP04 2 g
Sodium acetate 5 g
Diammonium citrate 2 g
MgS04.7H20 200 mg
10. MnS04.4H20 50 mg
Distilled water up to 1 litre
Tween 80 ~ is a registered trade mark for polyoxyethyl-
ene(20)-sor'oitan monooleate. The medium has a pH-value of 6Ø
If it is intended to use malt as additive when ensiling,
it is suitable to add to the medium also 0.1% of malt extract to
stimulate the bacteria to grow better in an environment contain-
ing malt.
The bacteria selected in this way have been found to be
of the species Strepto_occus faecalis, and have the following
characteristic properties:
Gram reaction +
Catalase reaction - :
Growth at 15C
Growth at 45C -~
Growth at 50C -
NH3 from arginine +
Fermentation of:
Lactose ~~
Maltose +
Arabinose
Melezitose
Melebiose
~ ~9~3~L7
Trehalose -~
Starch
In their characteristlc properties these bacterla meet
the description in "Bergey's Manual of Determinative Bacterio-
logy" Edition 1957. page 522, with the addition that they well
withstand a pH below ~.5 and grow rapidly ~mder these conditions.
To produce the starch converting bacteria, a suitable
number of bacteria colonies is taken from the first preparative
culture and is inoculated into a medium containing starch as the
sole essential carbon source and is cultured about 28C for 2
weeks so that the bacteria are stimulated to produce starch de-
grading enzymes. The p~I of the medium is about 6-~.2. The bac-
teria obtained in this way are additionally transferred to a new
substrate with starch as the sole carbon source, and this ~rocess
is repeated until a strain is obtained which turns out to be `
strongly starch degrading in tests on blue-coloured starch. Then
the bacteria ar~ cultured on a large scale microaerophilically
under carbon dioxide and are freeze-dried to be used as starting
material for the bacteria concentrate.
In the cases when malt is included in the additive to
the ensiling, malt extract can be added to the culture medium
also for these bacteria to stimulate them to grow better in an
environment where malt is included. The culture medium used
here has the following composition:
Peptone 7 g
Meat extract S g
Yeast extract 2,5 g
Starch ~wheat flour) 14 g
Tween 80 ~ 1 ml
K2~IP04 2,1 g
Tomato juice 35 ml
Water up to 1 litre
Malt extract (optionally) 1 g
.
~3~3~7
rrtl~ q ~t~ r~ V~ . 2 .
rl~hf~ hr:lc~rir~ Lt~ 3cl il~ t~i$ Wrly h~ r~ r~d tc~ b~
t~ 3 ~J~3n~:C~ t r~ t c~ c ~ t~ oorlc st~,c, ~E~ec~ lly ~ ct
1 1" T~ r~t~rQ~ rht~y ~ v~ o~ t;~rlc~ c~ ac~t~ ,t~tic
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C~ rt~ t~Q~rl ~ -
itt:lii r~t .l5
a:~c~wt~ ~ St~
7~rm~ntati~n o~
c~lt~
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T r t~ h a l ~
S tr.~l~b `
0 ~ pe~e.i~ 0~ ~hes0 h~ct~riR c~rr~spsnd ts~ wha~ is
ic;~tt~A ir~ ~y~t~ n~ te~mi{lr~t~ B~acteri~ y~ c~
)57.~ S.~ th~ ~:0 ~ Rc~dl~io~n that~ hRy Rr0 capa~
d~ T $t~r?~ h Rnd ~ nt-~t~ la~ r~ci~l~
h~ p~:Q;s$~ h~ .tion is ~urth~:~ illus-
r~0d in ~ .li.o~ x~,le~, in ~i~h e~silin~ ~ ani~al aRd
t~rsæ~s~: r~t~cO pdiTl~; t~ irLveD~ ho~: ~ C4I~I-
`,pRl~ Vl~QU~ ?d ~o~ p~ce~s~
` ~ ~ 30
r.~ t~ ~m~t~ ~ Ilg.illtlg ~Rt~heS thRr6! ~e~are~l
C~r3l~ c~t~ ir~ 10 ~ rca I ti~ rrincl t~re~h~.r with a~ 3
S3:L7
as stated below. After mixing, the different batches were left
at 24~C for one month, after which the silages ob-tained were
analyzed. The analyses results obtained are given in the follow-
ing table I. The four ensillng batches have the following compo-
sition:
Batch A: lO kg Baltic herring + l.5 kg grits.
Batch B: lO kg Baltic herring + l.5 kg grits to which 104
bacteria per g ensilage of the starch degrading bacteria selected
according to the above had been added.
satch C: lO kg Baltic herring -~ 0.9 kg of a mixture of 5 parts
by weight of grits and l part by weight of malt.
Batch D: lO kg Baltic herring + 0.9 kg of a mixture of 5 parts
by weight of grits and l part by weight of malt and containing
bacteria per g of each of the acid tolerant and starch degra-
ding bacteria selected as above.
TAB1E I
Batch A B C D
.. . .. _ .. _ _ _ . . .. _ _ .. _ ~ _
Dry substance 31 31 26,4 35.6
Total nitrogen 2.70 2.772.73 2.68
Ammonia nitrogen 0.96 0.97l.05 0.31
N~I3 - N/total - N in % 35~6 35.0 38.5 ll.5
Lactic acid ~ O.l ~ O.l lo5 3.6
Butyric acid l.59 l.61 l.5 0.0
pH 7.2 7.1 6.5 4.8
In the table the concentration values are indicated in
% by weight. As a measure of the ~uality of a silage, the con-
centration of lactic acid is usually indicated, which should be
high, as well as the content of butyric acid and the ratio of
ammonia nitrogen to total nitrogen, which values should be as low
as possible. If the ratio of ammonia nitrogen -to total nitrogen
in a silage exceeds 20%, the silage can be considered as spoiled
-- 10 --
..
~95~
or on the point of being spoiled. The content oE butyric aci.d
should not exceed a few tenths of one percent.
It is evident from the table -that the silage D prepared
according to the invention ls of a considerably better quality
than the others and has a high content of :Lactic acid, whereas
the values of the butyric acid content and the ratio of ammonia
nitrogen to total nitrogen are low.
Example 2~
In this exarnple alfalfa was ensiled, which is a pasture
plant which usually is difficult to ensile. Four batches were
prepared of a composition as below and were ensiled and stored
for one month at 28C, after which the silages obtained were
analyzed. The results appear in the following table II. The
four ensiling batches had the following composition:
Batch E: Alfalfa + 5% by weight of grits
Batch F: Alfalfa -~ 5% by weight of grits + starch degrading
bacteria.
Batch G: Alfalfa -~ 5% by weight of grits + acid tolerant bacteri-
a.
Batch H: Alfalfa -~ 5% by weight of grits + starch degrading
bacteria + acid tolerant bacteria~
The various bacteria were added in each case in an a-
mount providing 10 organisms per g of the starting material for
ensiling.
_ABLE II
. . .. . .. .
Batch E F G H
. . . _ _ . _ _ . .
Dry substance 16.5 19.5 19.7 22~0
Total nitrogen 0.60 0.62 0.68 0.71
Ammonia nitrogen 0.19 0.099 0.070 0.029
N~I3-N/total-N in % 31 14 11 4
Lactic acid 0.1 0.3 1.0 1.7
Butyric acid 0.23 0.01 0.01 0.01
s3~
pEI 7.5 5.1 ~.9 ~.3
In the table all the concen-tration values have been
lndicated in % by weight.
It is evident from this table -that the batches F, G and
H all provided acceptable results, but that the very best results
are obtained with ba-tch H, where both types o-f bacteria are used.
That acceptable results have been obtained using merely the acid
tolerant bacteria is due to the fact that alfalfa normally con-
tains a small amount of sugars whlch can be fermented. This is
often the case when ensiling vegetable materials.
It is also evident from the values in the table that
the two types of bacteria strongly rein-force the influence of
each other on the fermentation process, so-called synergism.
The two new bacteria mentioned in this specification
have been deposited in the culture collection of the Royal
Agricultural College Ultuna, Sweden, with the following reference
numbers: Streptococcus faecalis 10528 (Strain F), Stre~_ococcus
lactis/Leuconostoc mensenteroides 10531-68-llSM.
