|1.||Process for ensilage of crop by inoculation with lactic acid bacteria, such as Lactobacillus and Pediococcus. c h a r a c t e r i z e d i n that the inoculation is performed with lactic acid bacteria being in a state of active growth/metabolism and cell division in a growth medium.|
|2.||Process according to claim 1, c h a r a c t e r i z e d i n that there is included at least one polymerproducing strain which synthesizes water binding polymer(s) .|
|3.||Process according to claim 2, c h a r a c t e r i z e d i n that the polymerproducing strain being used is Lactobacillus plantarum.|
|4.||Process according to claim 1, c h a r a c t e r i z e d i n that the dry, preferably lyophilized lactic acid bacteria are precultivated in a growth medium for approximately 24 hours and are thereafter applied to the crop in a concentrated or diluted form.|
|5.||Inoculation kit for use by the process according to any of the claims 14, c h a r a c t e r i z e d i n that it comprises an ampoule, vial or similar sealed container containing lyophilized lactic acid bacteria and a container with an appropriate volume of a growth medium.|
The present invention concerns a process for ensilage of feed plants. For many years different preservation methods for feed have been known which by their pH-decreasing effect prevent the growth of butyric acid bacteria, other spoilage bacteria and fungi. For this purpose mineral acids, formic acid or other additives which stimulate a natural lactic acid fermentation have been used.
Lactic acid fermentation is regarded as an ideal way of preservation because of the low material losses and the high nutritional value of the acids produced.
The low number of lactic acid bacteria usually present on the fresh grass has often been regarded as the most im¬ portant reason for an unsuccessful silage. Lactic acid bacteria also have a relatively long generation time compared to the aerobic and facultatively anaerobic bacteria against which they compete during the first phase of ensi¬ lage, i.e. while the supply of oxygen is good. To sti u- stimulate the lactic acid fermentation the following have therefore been attempted
a) the addition of special bacteria, preferably hσmofer- mentative lactic acid bacteria, as disclosed in Norwegian patent 74.571, US patent 3.147.121, Swiss patent 460.503 and the Swedish patent 396.275. Inoculation with a single bacterial strain has produced varying results, and today there is a tendency to choose a combination of two or more bacterial strains. A 1:1 mixture of Str. faecalis and L.piantaru is for instance more efficient in maintaining a continuous lactic acid fermentation than each of the strains alone. The reason for this is that Str. faecalis grows rapidly under aerobic conditions, and while not being particularily tolerant to lactate, it rapidly reduces pH to a level where L.piantarum is of greater advantage,
b) the addition of high numbers of lyophilized/dried lactic acid bacteria to the crop before or during the filling of the silo,
c) the addition of bacteria and bacterial nutrients immedi¬ ately before or during the filling of the silo,
d) the use of especially acid-tolerant strains of Str.- faecalis isolated by selective techniques as disclosed in Swedish patent 411.829.
The addition of molasses, glucose, cellulase or complex nutrient solutions together with the lactic acid bacteria has also proven to give a rapid fermentation and a stable product.
The commercially available inoculants are usually lyo- philized products to which a cryoprotective agent, a carrier and/or a nutrient solution have often been added. The inoculants are added to the crop either as powder or as an aqueous suspension which is sprayed onto the grass during harvesting or filling of the silo.
According to the present invention it has been developed a process for ensilage based on precultivation of selected strains of lactic acid bacteria which have been isolated from silage. The criteria for the selection of the strains are as follows:
- homofermentative fermentation pattern
- high growth rate and rapid pH-decrease
- ability to use several types of sugar present in crops (glucose, fructose, sucrose etc.)
- high osmotic tolerance - high pH-tolerance
- lack of proteolytic activity
- at least one strain shows good growth at 10°C
- at least one strain shows ability to utilize starch.
During the experiments a combination of strains belonging to the genera Pediococcus and Lactobacillus was used. Sur¬ prisingly it was found that by using 6-8 selected strains the effluent was considerably reduced. This may be due to production of water binding polymers by at least one of the strains used, e.g. Lactobacillus plantaru . It has been found that this strain is especially suited since it effectively uses the plants* polysaccharides.
Usually, the ensilage produces variable amounts of effluent depending on the dry matter content of the ensiled material, the rate of packing, the rate of cutting or grinding and the use of additives which enhance lysis (e.g. acids) . The production of effluent has been a considerable problem because of the significant loss of valuable nutritious components and because it represents a significant pollution and environmental problem. The present invention provides by reduction of the effluent a great advantage compared to the state of the art.
Neither has it previously been considered important to use bacterial strains which show good growth at 10°C, despite the fact that this gives a great advantage. Due to climatic conditions in Europe, the temperature in the silage will vary considerably. Immediately after the harvesting and packing of the silo, oxidation of plant material is initiated by the consu ation of the contained oxygen. A radial temperature gradient is thereby produced as the temperature in the silage on a cold autumn day may vary from 40°C in the center to 10"C in the periphery. Such a marked temperature gradient is a temporary phenomenon, and the temperature will even out when all the oxygen has been consumed. A maximum lactic acid fermentation in the entire silage may be achieved by using at least one strain with good growth at 10°C according to the present invention.
Lack of fermentable water-soluble mono- and disaccharides in
the crop may in several cases limit the production of lactate. For this reason it has been considered important during selection of bacterial strains that at least one is able to use polysaccharides as a carbon and energy source. One of the bacterial strains used, Lactobacillus plantarum, has proven its ability to utilize starch, and this accor¬ dingly leads to an increase in the total amount of ferment¬ able carbohydrates available in the feed mass.
According to the present invention the selective growth environment is created for the applied lactic acid bacteria so they will multiply and reach an active growth phase in the absence of competing microorganisms before application to the crop. This is achieved by innoculating the lyo- philized bacteria in a specially composed sterile broth, e.g. modified MRS, which is contained in a tightly closed container, e.g. a plastic bottle. The container is there¬ after incubated at room temperature, and after 24 hours it contains a turbid bacterial culture in active growth. The bacterial culture, which is stable for 1-2 weeks at 4°C, is mixed with water and thereafter sprayed onto the crop during harvesting, e.g. by using a flail harvester.
According to the present invention the following obvious advantages are achieved:
a) The bacterial culture is propagated to a high cell density before application, i.e. a large number of active bacteria is added to the crop.
b) The bacteria are converted from a resting (lyophilized) state to active metabolizing and lactate-producing cells in the absence of interfering micro-organisms and are added to the crop in active state.
The result of a) and b) is that lactic acid is rapidly produced in the silage with an accompanying rapid decrease in pH, thus inhibiting the growth of aerobic and facultative
anaerobic, coliform bacteria. Accordingly, the loss of dry matter as carbondioxide will be reduced as well as the production of bacterial metabolites with low nutritional value.
The bacteria are cultivated under conditions which are se¬ lective for lactic acid bacteria (e.g. absence of air, ca. 20°C, specially composed growth medium) , and which promote rapid growth and a ready-to-use bacterial suspension after only 12 - 24 1-rurs. The bacterial culture is also stable for 1-2 weeks ;4'C), thus giving great flexibility for the user.
The number of lyophilized bacteria of each strain contained in the preparation may theoretically be reduced as long as the cells are viable and have the ability to multiply. This may give a significant expense-saving effect during the production of the product compared to conventional inocu¬ lants for ensilage requiring large quantities of bacteria, thus entailing higher production costs and hence higher prices.
An ensilage kit consists of a vial with lyophilized lactic acid bacteria (ca. 10 ml) and a sealed container with a sterile nutrient broth (a plastic bottle with screw-cap, 2 litres) . The ensilage kit is easy to transport, requires little storage space and is stable, which is of great importance in connection with e.g. preparedness.
The inoculant may be applied to the crop by using conventio¬ nal harvesting equipment (e.g. flail harvester) .
In addition this product has the same advantages as other inoculants used for ensilage, being environmentally safe, biologically compatible, not hazardous to the health (e.g. when splashed in the eyes), not caustic (e.g. on clothes, skin etc.) and does not corrode equipment (e.g. feed harvester etc.) .
Example of cultivation.
Production of lyophilized bacteria.
7 different strains of lactic acid bacteria, 5 Lactobacillus spp and 2 Pediococcus spp were cultivated on a medium (modified MRS) with the following composition: Meat extract 10 g
Yeast extract 5 g Peptone 10 g
Glucose 20 g
Diammonium citrate 2 g
Sodium acetate 10 g
Dipotassium hydrogenphosphate 5 g Magnesium sulphate 200 mg
Manganese sulphate 50 mg
Tween 80® 1 ml
Distilled water up to 1 litre pH 6,0
Tween 80® is a registered trade mark for polyoxyethylene- (20)-sorbitanemonooleate.
All bacterial strains were cultivated to a density of log cell count = 9-10 and harvested by sentrifugation. The strains were mixed to a slurry, cryoprotective agent (sodium glutamate, dry milk) added and dispensed sterile into vials. The bacteria were frozen to -70*C and lyophilized. The vials were sealed in a nitrogen atmosphere. Prepared in this way, the bacteria are expected to survive at 4°C for more than 10 years. The product is in the following referred to as a silage additive according to the invention. A procedure for application of the product for ensilage of crop may be as follows:
The vial containing the lyophilized bacteria is half-filled with cold tap water (or sterile nutrient broth) , closed with
the rubber stopper and shaken well.
The contents of the vial are poured into the container with sterile nutrient broth (2 litres) , the broth having room temperature. The cap is replaced and the container is left for activation at room temperature (20-25°C) for 24 hours. The contents should be used immediately after the activation period, but may be stored for 1 week at cellar temperature (5-l0°C) .
Immediately before use the contents of the container are mixed thoroughly and diluted with cold tap water to approxi¬ mately 25 litres in a plastic can. The mixture is applied to the grass via flail harvester, and is recommended for approximately 7 tons of grass.
By this method the grass is supplied with a minimum of 1.000.000 viable lactic acid bacteria per gram.
Experiments comparing metabolizing strains according to the invention and formic acid with regards to pH, lactic acid and effluent.
Experiments were performed in 6m 3 silos (diameter 1,6 m, height 3,0 m) . Samples were taken with a i m long hollow steel bore (diameter 40 mm) which was struck vertically down into the silo from the surface.
The grass material consisted of timothy (Phleu pratense) (73%) , meadow fescue (Festuca pratensis) (17%) , red clover (Trifolum pratense) (6%) and other grass species (4%) .
The dry matter content of the grass was 14-15%. The buffer capacity was 350 mekv./kg dry matter (DM) and the sugar content, expressed as water soluble carbohydrate ( SC) , was 115-120 g/kg DM.
Table 1 pH development and lactate production PARAMETER ADDITIVE Od 2d lOd 67d
Control 5,99 4,17 3,94 4,41
PH Accord.to inv. 6,00 3,94 3,75 3,89 Formic acid 4,63 4,67 3,98 4,76
Lactic Control 2,0 28,2 59,6 21,9 acid Accord.to inv. 2,4 36,2 65,5 70,5 fg/kq DM) Formic acid 0.4 5.5 45.0 12.6
Accumulated effluent and dry matter loss after 59 days of ensilage.
ADDITIVE VOLUME (1) DRY MATTER fkσ)
Control 255 8,2
Accord. o inv. 52 2,0
Formic acid 630 21.3
The metabolizing strains according to the invention show a significantly more rapid pH-decrease than both the control and the formic acid silage which to some extent is reflected in the increased lactic acid production. In this case, neither the control nor the formic acid silo is sufficiently stabilized, which leads to a secondary pH-increase parallell to the consumption of produced lactic acid. The production of effluent by using the product according to the invention is significantly reduced compared to the control and the formic acid, this also resulting in a reduced loss of dry matter.
Example 2 Experiments comparing metabolizing strains according to the invention and formic acid. Carbondioxide loss.
The experiment was performed by using 10 1 pilot silos.
Loss of carbon dioxide was measured as weight loss.
The grass material consisted mainly of timothy (Phleum pratense) and meadow fescue (Festuca pratensis) (50/50) . The dry matter content was 23%.
Table 3 Carbon dioxide loss, expressed as % of the dry matter.
ADDITIVE 0d Id 3d lOd 50d 105d
Control 0 1,9 2,3 3,5 4,0 5,3 Accord.to inv. 0 1,0 1,6 2,1 2,2 2,3 Formic acid 0 0,1 0.6 1,5 2.0 2.1
The loss of carbon dioxide is during the first couple of days somewhat larger for the control and metabolizing strains according to the invention than for formic acid. Later in the ensilage period, however, the production of carbondioxide incresases in the formic acid silages as well, so that the total loss after 50-105 days is about the same in silos ensiled with the metabolizing strains according to the invention and the formic acid.
Experiments comparing metabolizing strains according to the invention, formic acid, and two commercial inoculants containing lactic acid bacteria (Prep. 1 and Prep. 2) with regard to pH and lactic acid production.
The experiments were performed in 2 kg silos. The grass material consisted mainly of timothy (Phleum pratense) with a dry matter content of 20,9% and WSC of 129 g/kg DM.
Table 4 pH-development and lactic acid production.
PARAMETER ADDITIVE Od 3d 8d 60d 120d
Control 6,1 5,4 4,6 4,3 4,2
Formic acid 5,3 5,2 4,7 4,4 4,2 pH Accord. o inv. 6,1 5,2 4,3 3,9 3,9
Pre . 1 6,1 5,4 4,6 4,1 4,1
Prep. 2 6,1 5,4 4,8 4,1 4,1
Control 0 2,7 6,0 7,8 9,4
Lactic Formic acid 0 0,7 0,8 3,2 7,3 acid Accord.to inv. 0 4,0 7,8 12,8 11,8
(% of DM) Pre . 1 0 2,8 7,3 12,2 11,3
Prep. 2 0 1,1 7,7 12.0 22.2
The pH development in silage with the metabolizing strains show a significantly more rapid reduction compared to the control and the formic acid silo, and showed a slightly faster reduction compared to the two commercial inoculants. This pH development is also reflected in the production of lactic acid.
Utilization of starch
Medium 1: MRS - glucose + bromocresole purple Medium 2: MRS - glucose + 1% starch + bromocresol purple Incubation temperature 28 β C Incubation time 24 hours.
STRAIN MEDIUM 1 MEDIUM 2 OXIDATION* GROWTH OXIDATION GROWTH
L.piantarum 2 - 5 +++
* Scale from 0 (negative reaction) to 5 (maximum positive reaction) .
1, 2, 3 and 4 indicate intermediary reactions. 3, 4 and 5 indicate positive reactions.
Comments: The strain L.piantarum is able to utilize starch as an energy- and carbon source.
Dry matter losses (accumulated values) in the effluent from pilot scale eperiments.
C - control, N - according to invention, F = Formic acid
EFFLUENT . DRY MATTER LOSSES fkcr drv matter)
Exp< eri ent 1 Expe >riment 2 Experiment 3
Days C N F C N F C N F
1 0,4 0,1 13,6 0,3 0,0 4,3 0,0 0,0 0,0
2 1,3 0,6 16,4 2,9 1,6 7,5 0,3 0,3 1,8
3 1,7 0,6 17,5 4,8 2,7 9,2 0,6 0,5 3,5
10 3,4 0,6 18,7 9,1 6,4 16,0 3,6 2,8 8,1
40 6,8 1,4 20,6 10,6 7,7 16,6 4,9 3,8 8,6
60 8.2 2.0 21.3 11.9 9,0 17.2 5,9 4,7 9,1
I'l l, () T SCΛI.R 6 ιτf
<)HB 1. cut. Timothy (Phleum pratense) swine cress ( Polygonum ay icu lat r> ) Clover (Trifolium) (6:1:3) Dry matter 30%, WSC ~" T3θ " g/k DM,
HUI I C - capacity 400 mE/kg DM,
PΛHΛMKTKK ADDITIVE _ 0 2 10 70 12!
Con t.co 1 5, 4,7 4,0 3,9 ,9
I'll Formic acid 4,4 4,5 4,4 4, 1 4,0 Λcc.to inv. 6,0 4,1 ,9 3,8 3,8
luirl i Contro 1 0 42 8 105 101 rir it) Formic acid 0 4 I 3 21 .9 (y/ktj DM) Λcc.to inv. 0 79 111 112 119
I'Uif! I . cut. Timothy (Phleum pratense) - meadow fescue (Festuca pr l.ensis)
Clover (Trifolium) (5:2:3), Dry matter 20?,, " WSC Tlθ " cj " / ' k 1H1 Muf lr-r capaci y 330 mE/kg DM.
PΛKΛMKTKK ADDITIVE 0 2 9 55
Control 5,9 4,2 3,8 3,7
I' l l Formic acid 4,2 4,3 4,0 3,9 Λcc. to inv. 5,9 3,8 3,7 3,7
Lactic Control 0 57 106 acid Formic acid 0 3 19 34 (g/kg DM) Λcc.to inv. 0 90 106 112