CA1095317A - Process for biological ensiling of vegetable and/or animal materials - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Animal and/or vegetable materials are ensiled biologic-ally with the aid of lactic acid forming bacteria and carbo-hydrate materials which can be converted to lactic acid. To facilitate the ensiling process. selected acid tolerant lactic acid forming bacteria of the species Streptococcus faecalis are added to the starting materials, together with fermentable carbo-hydrates, materials which degrade starch into fermentable carbo-hydrates, and/or selected lactic acid forming starch degrading bacterial of the genera Streptococcus and Leuconostoc.

Description

~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 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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~S~7 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-.

S3~7 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 ~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.

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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:

~53~L7 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 .

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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.

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 ..

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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.

Claims (17)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. In a process for biological ensiling of vegetable and/or animal materials with the use of lactic acid-forming bacteria, wherein carbohydrate materials which can be converted to lactic acid and lactic acid-forming bacteria are added to the starting materials to be ensiled, the improvement consisting of adding to said starting materials;
a) lactic acid-forming acid-tolerant bacteria, which have been selected from 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) fermentable carbohydrates.

2 The process as claimed in claim 1 wherein rod-shaped bacteria of the species Lactobacillus plantarum are further added to the starting materials.

3. The process as claimed in claim 1 wherein the lactic acid-forming acid-tolerant bacteria (a) have been selected by repeated cultivation in a medium having a pH value below 4.5.

4. The process as claimed in claim 1 wherein animal materials are ensiled.

5. In a process for biological ensiling of vegetable and/or animal materials with the use of lactic acid-forming bacteria, wherein carbohydrate materials which can be converted to lactic acid and lactic acid-forming bacteria are added to the starting materials to be ensiled, the improvement consisting of adding to said starting materials:
(a) lactic acid-forming acid-tolerant bacteria, which have been selected from naturally-occuring bacteria of the species Streptococcus faecalis by repeated cultivation in a medium having a pH value of 4 to 4.7, and (b) materials which degrade starch into fermentable carbohydrates, such materials selected from the group consisting of malt, at least one starch degrading enzyme from malt, at least one starch degrading yeast, at least one other starch degrading fungus or a mixture of two or more members of the group.

6. The process as claimed in claim 5 wherein rod-shaped bacteria of the species Lactobacillus plantarum are further added to the starting materials.

7. The process as claimed in claim 6 wherein each of said bacteria species is added to the starting material in an amount that provides at least 103 bacteria per gram of the total start-ing material and the added materials combined.

8. The process as claimed in claim 5 wherein the selected bacteria after cultivation and freeze-drying are mixed with grain products to form a concentrate, which after dilution is added to the starting materials for the ensiling process.

9. The process as claimed in claim 5 wherein the lactic acid-forming acid-tolerant bacteria (a) have been selected by repeated cultivation in a medium having a pH value below 4.5.

10. The process as claimed in claim 9 wherein the materials which degrade starch into fementable carbohydrates consist of malt, starch-degrading enzymes from malt, starch-degrading yeasts or other starch-degrading fungi.

11. The process as claimed in claim 10 wherein malt is added to the starting material, and malt extract in an amount of about 0.1 weight percent is added to the nutrient media used in the selection of the bacteria.

12. In a process for biologically ensiling of vegetable and/or animal materials with the use of lactic acid-forming bacteria, wherein carbohydrate materials which can be converted to lactic acid and lactic acid - forming bacteria are added to the starting materials to be ensiled, the improvement consisting of adding to said starting materials:
(a) lactic acid-forming acid-tolerant bacteria, which have been selected from 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) lactic acid-forming starch-degrading bacteria, which have been selected from naturally-occurring bacteria of the genera Streptococcus and Leuconostoc by repeated cultivation in a medium which contains starch as the sole carbon source.

13. The process as claimed in claim 12 wherein rod-shaped bacteria of the species Lactobacillus plantarum are further added to the starting materials.

14. The process as claimed in claim 12 wherein the lactic acid-forming acid-tolerant bacteria (a) have been selected by repeated cultivation in a medium having a pH value below 4.5.

15. The process as claimed in claim 12 wherein said lactic acid-forming starch-degrading bacteria (b) have been selected from the group of naturally-occurring bacteria consisting of Streptococcus lactic and Leuconostoc mesenteroides.

16. The process as claimed in claim 12 wherein the carbo-hydrate material which can be converted to lactic acid is predominantly starch.

17. The process as claimed in claim 16 wherein vegetable materials are ensiled.