WO2014170233A2

WO2014170233A2 - Method for improving milk quality in milk-producing animals

Info

Publication number: WO2014170233A2
Application number: PCT/EP2014/057402
Authority: WO
Inventors: Ida HINDRICHSEN; Bente Lund; Christer OHLSSON
Original assignee: Chr. Hansen A/S
Priority date: 2013-04-18
Filing date: 2014-04-11
Publication date: 2014-10-23
Also published as: AR095877A1; EP3001792A2; WO2014170233A3

Abstract

The present invention relates to a method for reducing somatic cell count and total bacterial count in milk from a milk-producing animal, the method comprising feeding the animal with a fermented feed produced by inoculating a plant material with at least one lactic acid bacterium.

Description

TITLE

Method for improving milk quality in milk-producing animals FIELD OF THE INVENTION

The present invention relates to the surprising finding that fermented plant material produced by use of at least one inoculant selected from the group consisting of lactic acid bacteria can reduce somatic cell count (SCC) and total bacterial count in milk. This reduction of somatic cell count and total bacterial count is an indication of a lower incidence of sub-acute and acute mastitis and is of considerable economic relevance both for the farmer and the dairy industry.

BACKGROUND OF THE INVENTION Mastitis is an inflammation of the mammary glands of milk-producing animals, for example dairy cows, most often caused by three major groups of pathogens which are gram-positive cocci, gram-negative bacilli and gram-positive bacilli . The most common pathogenic infection are caused by Streptococcus agalactiae, Staphylococcus aureus, Streptoccus dysgalac- tiae, Echerichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Listeria monocytogenes, En- terobacter aerogenes, Streptococcus uberis, Streptococcus bovis, Streptococcus disgalactiae, Enterococcus faecalis etc. . Pathogens enter through the teat canal of the animal and the severity is either grouped as acute, clinical, or sub-clinical mastitis. Hygiene, environmental factors and metabolic disturbances derived from high milk yield combine to create conditions favorable to the onset of mastitis. An increased somatic cell count, associated with mastitis, is positively correlated with infection and negatively correlated with mil k production.

Symptoms of mastitis includes inflammation of the mammary glands along with other symptoms like mild signs of flakes or clots in the milk and the animal may have slight swelling of infected quarter. Severe signs vary from abnormal secretion to hot and swollen quarter or udder; the cow may have fever, rapid pulse, loss of appetite, dehydration and depression ; and even death may occur.

Mastitis reduces milk yield and alters milk composition . The magnitude of these changes in individual cow varies with the severity and duration of the infection and the causative micro- organisms. Mastitis is almost always caused by pathogens. These microorganisms produce toxins that can directly damage milk-producing tissue of the mammary gland, and the presence of bacteria initiates inflammation with in the mammary tissue in an attempt to eliminate the invading microorganisms. The inflammation contributes to decreased milk produc- tion and is primarily responsible for the compositional changes observed in milk from infected quarters and cows. In general, compositional changes involve an increase in blood components present in milk and a decrease in normal milk constituents. Somatic cell count (SCC) is a measure of the number of white blood cells (leukocytes) released into the udder and indicates an increased infection. The number of somatic cells increases in response to pathogenic bacteria like Staphylococcus aureus, a cause of mastitis. Furthermore, somatic cell count is seen as an indication of the metabolic and infectious heath disorders of mammary gland, an immune parameter which increases if foreign sub- stances like pathogens and toxins (e.g. mycotoxins or enterotoxins) enter the teat canal. The SCC is quantified as cells per ml. General agreement rests on the values of less than 100,000 SCC/ml for uninfected cows.

Increased SCC causes loss both for the farmer, due to decreased milk production, lower price for the milk and higher risk for mastitis and the cost for treatment, as well as loss of holding back milk due to antibiotic treatment. Increased SCC results in additional losses, due to changes in manufacturing properties of the milk, particularly cheese manufacture (e.g. increased coagulation times, elevated cheese moisture contents, decreased cheese firmness, lower recoveries of milk solids in cheese, and reduced cheese yield and yield effi- ciency) (Auldist, 2002). Dairies therefore often choose to pay extra for milk with less than 250,000-300,000 somatic cells/ml and less than 50,000 microbial cells/ml . The level of SCC accepted by authorities is legislated by EU (Regulation (EC) No 853/2004) and is 400,000 cells/ml on average over 3 months, while microbial number should not go above 100,000 microbial cells/ml on average over 2 months.

To avoid SCC in milk first of all high hygiene in milking parlor is advised. Nevertheless, mastitis remains the biggest economic loss for farmers in terms of cattle diseases.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to reduce somatic cell count and total bacterial count in milk from a milk-producing animal. The problem is solved by feeding the animal with a feed produced by inoculating a plant material with at least one inoculant selected from the group consisting of lactic acid bacteria (e.g. Lactobacillus, Lactococcus, Bifidobacteria) in order to produce a fermented plant material.

The examples of the present patent application demonstrate that feeding cows with silage produced by inoculating lucerne with Silo Solve® MC (Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis SR 3.54 (NCIMB 30117) and Enterococcus faecium M74 (DSM 22502) significantly reduced somatic cell count (SCC) in milk (P< 0.001) and total bacterial count (P<0.001) and a short term study indicates that a similar effect may be obtained by feeding cows with silage produced by inoculating perennial ryegrass with Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis 0224 (DSM 11037), L. buchneri Lb 1819 (DSM 22501), and optionally also Lactococcus lactis SR 3.54 (NCIMB 30117) .

It is highly surprising that a bacterial inoculum to a plant material which was incubated 90 days can have a significant effect on the hygiene of milk produced by the cows fed with the fermented plant feed, since the control fermented plant feed was also from a high quality.

The present invention is useful in the dairy industry as well as in other industries where mammalian animals are raised, i .e. the swine industry, horse breeding etc., in that the risk of transferring pathogens to the off-spring by means of the milk is reduced .

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for reducing somatic cell count in milk from a mil k-producing animal, the method comprising feeding the animal with a fermented feed produced by inoculating a plant material with at least one lactic acid bacterium. In a preferred embodiment of the invention, the total bacterial count in the milk is also reduced .

Within the concept of the present invention a fermented feed produced by inoculating a plant material with at least one lactic acid bacterium is a feed that through the growth of the lactic acid bacteria is reduced in pH, which has the effect of inhibiting growth of spoilage microorganisms. If anaerobic condition at the same time is achieved, like in silage, the fermented plant material can be conserved for a long time without losing nutrients and fed during shortage of fresh feed to animals in need thereof.

Silage, haylage and wrap are examples of fermented feed to be used in the method accord- ing to the present invention. In the present context these terms are used synonymously with fermented feed produced by inoculating a plant material with at least one lactic acid bacterium unless the context suggests otherwise. Silage, haylage and wrap are fermented crops from grasses (e.g . sorghum, maize, ryegrass, festulolium, timothy, cocksfoot, elephant grass), whole crop of oat, wheat, barley, triticale, millet, leguminous plants (e.g . lu- cerne/alfalfa, clover, peas, beans), roots (e.g . beet, cassava, taro, yam) crop residues (e.g . beet tops, vegetables and fruit stem/leaves, tree stem/leaves), by-products (e.g . brewer ^'s spent grain, vegetable pulp) or fodder trees and shrubs (e.g. Leucena, Calliandra), as well as intercrops.

The plant part from stalk, leave, kernels etc. or any combination thereof are fermented and stored in a process called ensiling or silaging. Silage can be made from many field crops, examples of which are outlined above. All of the above examples are preferred examples of fermented feed to be used in the method of the present invention.

The silage is generally produced in special feed silos or in special air-tight silage bales. The main substantiation of inoculating forages is to ensure a fast preservation of the forage to secure a high quality forage, which includes high nutrient recovery and low fungal and path- ogenic growth both when establishing the silage bunker/heap, as well as at feed out, where the silage again is exposed to air.

Some farmers are not inoculating their silage and therefore rely on that the natural microbial flora will preserve the silage efficiently enough. Many farmers, however, choose to add silage inoculants in order to obtain a more reliable effect on pH reduction, combating of fungi, mold and pathogen growth (e.g. Clostridia, Listeria, E. coli) and in order to reduce loss of nutrient.

As used herein, the term "lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconos- toc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp.

"Fermentation" in the methods and feed products of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention comprises conversion of lactose to lactic acid.

Lactic acid bacteria to be used according to the present invention may be selected from Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Propionibacterium, or any combinations thereof; preferably from Lactobacillus, Lactococcus, Enterococcus or any combinations thereof. The Lactobacillus is preferably one or more species select- ed from the group consisting of Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, and Lactobacillus buchneri. The Lactococcus is preferably a Lactococcus lactis. The Entero- coccus is preferably Enterococcus faecium.

Although silage to be used according to the invention may be produced by addition of only one of the above microorganisms, the silage inoculant will often comprise two, three, four or even five or more of the above microorganisms. If more than one microorganism is used as a silage inoculant it is contemplated that the proportion of each microorganism in the product will be 1 to 99%, such as 20 to 80%, e.g . 30 to 70%, more particularly 20%, 33%, 40% or 50% of the total amount of silage inoculants calculated as cfu/g product. Generally the microorganisms will be present in about equal numbers.

In order to be useful in the method of the present invention the feed product produced by inoculating a plant material with at least one lactic acid bacterium should be able to lead to a reduction of the somatic cell count to a weekly average of 100,000 somatic cells/ml or less after 6 weeks of feeding . Preferably the somatic cell count is reduced to a weekly average of 75,000 somatic cells/ml or less after 10 weeks of feeding . The somatic cell count can be measured on the milk sample as described in Laevens et al. ( 1996) using the Somascope (Delta Instruments, Drachten, the Netherlands), which is an easy to use flow cytometry and is operated by a single button push followed by data analysis using software. By "weekly average" is meant the average of measurements on milk from the animal(s) fed with the feed product during one week, e.g . the average of daily measurements.

The total bacterial count should preferably be reduced to a weekly average of 50,000 cells/ml or less. In a preferred embodiment the total bacterial count is reduced to a weekly average of 25,000 cells/ml or less after 6 weeks of feeding, such as a weekly average of 20,000 cells/ml or less after 6 weeks of feeding . The total bacterial count can be enumerated by electronic flow cytometry using the Bactocount (Bentley Instruments, Chaska, MN, USA) . Bacterial DNA and RNA is colored by adding ethidium bromide and injected into a capillary tube. The fluorescence captures by the optic system measure the total number of bacteria, as described by Cassoli et al. (2007) .

It may be preferable that the lactic acid bacterium is capable of producing bacteriocin. An example of a such strain is SR 3.54 deposited as NCIMB 30117 which produces nisinZ.

Examples of particularly preferred inoculants are Enterococcus faecium M74 deposited as DSM 22502, Lactobacillus plantarum CH6072 deposited as DSM 16568, L. buchneri Lb 1819 deposited as DSM 22501, Lactococcus lactis 0224 which is described in EP 0928333 and deposited as DSM 11037, and Lactococcus lactis SR 3.54 which is described in SE 511 828 and has been deposited as NCIMB30117; as well as mutants and variants of the above strains, and any combinations thereof.

In the present context, the term "mutant" should be understood as a strain derived by means of e.g. radiation and/or chemical treatment, and/or selection, adaptation, screening, etc. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding reduction of somatic cell count) as the mother strain. Such a mutant is a within the scope of the present invention. Especially, the term "mutant" refers to a strain obtained by subjecting a strain mentioned above to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N- nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 1000, no more than 100, no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion or a combination thereof) compared to the mother strain. In the present context, the term "variant" should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties e.g. regarding reduction of somatic cell count). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention. According to the present invention the fermented feed can be fed to all kind of livestock, but it is preferably fed to ruminants (cud-chewing animals such as cattle/bovine (cow, bull, steer, heifer, calf), sheep/ovine (ewe, ram, wether, lamb, goat, caprine), equine species such as horse, llama, camel and swine.

The examples of the present patent application demonstrate an increased milk yield as well as significant effect on milk composition (e.g. fat, protein).

Surprisingly it was also found that feeding cows with silage produced by inoculating Lucerne with Silo Solve® MC (Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis SR 3.54 (NCIMB 30117) and Enterococcus faecium M74 (DSM 22502) significantly reduced so- matic cell count (SCC) in milk (P<0.001) and total bacterial count in milk (P<0.001).

It is highly surprising that a bacterial inoculum to silage which was incubated 90 days can have an effect on the hygiene of milk produced by the silage fed cows, since the control was of a good quality as well (no Clostridia perfringens). Furthermore, the longer time the cows were fed inoculated silage the more significant was the difference between silage inoculant and control both on milk yield, somatic cell count (SCC) and total bacterial count. As appears from Figures 2 and 3, the difference in total bacterial count between silage inoculant and control is increased in particular in the period 6 to 10 weeks and the difference in somatic cell count between silage inoculant and control is increased in the period after 6 weeks.

A further study reported in Example 2 shows that a similar effect may be obtained by feed- ing cows with silage produced by inoculating perennial ryegrass with Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis 0224 (DSM 11037), L. buchneri Lb 1819 (DSM 22501), and optionally also Lactococcus lactis SR 3.54 (NCIMB 30117).

As can be seen in Figure 4, the 4x4 Latin square study showed an increase in SCC over time, when cows were fed control silage and no Lactococcus lactis SR 3.54 (NCIMB 30117) was added additionally. The SCC did not increase over time, when the Lactococcus lactis SR 3.54 (NCIMB 30117) was supplemented to the control silage fed to the cows or the cows were fed silage inoculated with Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis 0224 (DSM 11037), and L. buchneri Lb 1819 (DSM 22501). Combining long term inoculated silage with Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis 0224 (DSM 11037), and L. buchneri Lb 1819 (DSM 22501) with short term inoculation with Lactococcus lactis SR 3.54 (NCIMB 30117) reduced the SCC over time.

The mode of action is not known. Without being bound to any theory it is contemplated that pathogens have been more effectively combatted in the fermented plant feed produced according to the present invention thus leading to fewer pathogens in the gastrointestinal tract of the milk-producing animal and thereby fewer pathogens in the environment of the milk- producing animal. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g ., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed . No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention .

Deposited strains The Lactobacillus plantarum strain CH6072 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 16568 with a deposit date of June 20, 2008 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Pur- poses of Patent Procedure.

The Lactobacillus buchneri Lbl819 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 22501 with a deposit date of April 22, 2009 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the

International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Enterococcus faecium strain M74 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 22502 with a deposit date of May 7, 2009 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

For the above-identified deposited microorganisms, the following additional indications apply :

As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

Embodiments of the present invention are described below, by way of non-limiting exam- pies.

LEGEND TO FIGURES Figure 1 shows weekly average of milk yield incl . SEM over a 13 week period .

Figure 2 shows weekly average of somatic cell count incl . SEM over a 13 week period .

Figure 3 shows weekly average of total bacterial count (in 1,000/ml) incl . SEM over a 13 week period .

Figure 4 shows change in somatic cell count (SCC) (in 1,000/ml) over a 20 day period . A=control silage, no short term inoculation; B=control silage + short term inoculation,

C=long term inoculated silage, no short term inoculation; D= long term inoculated silage, no short term inoculation.

EXAMPLES

EXAMPLE 1

Silage inoculation

Silage was produced from lucerne (Medicago sativa) . The lucerne was wilted for 24 hours and wrapped in six layers of plastic film. Half of the lucerne bales were inoculated with 1.5 *

10⁵ CFU/g silage SiloSolve® MC containing 30% Enterococcus faecium M74 (DSM 22502), 30% Lactococcus lactis SR 3.54 (NCIMB 30117), and 40% Lactobacillus plantarum CH6072 (DSM 16568) while the other half of the bales were kept as control . The silage was incubated for at least 90 days before being used for feed .

Dairy cow production trial

36 dairy Lithuanian Black and White cows 36-62 months of age/initial weight 550-600 kg in 2nd-5th lactation were selected among 150 cows in the herd and randomly assigned to two dietary treatment groups; control silage (spontaneously fermented) and inoculated lucerne silage.

Initially a three week adaptation period was performed feeding all cows' untreated silage similar to the experimental control silage. The cows were penned individually and fed silage twice a daily according to appetite. All cows were fed a fixed amount (7.1 kg/day as fed) of concentrate with a commercial mineral mixture (Ca, P and Mg), to meet nutrient requirements. The concentrate was specially manufactured for this experiment in compliance with lucerne silage chemical composition and nutrient content. The composition of the concentrate was the same in both groups of dairy cows.

Ingredients, chemical composition, and nutrient content of concentrates used in dairy cow diet are presented in Table 2. The zootechnical part of the trial was analyzed in SAS by factorial analysis of variance (GLM-ANOVA).

Results The results are summarized in the below tables and in Figures 1-3. Both control and inoculated silages were well preserved, even though nutrient recovery was significantly better (P<0.05) for the inoculated silage in terms of dry matter, crude protein, water soluble carbohydrates and total metabolizable energy, while the fiber fraction (crude fiber, neutral detergent fiber and acid detergent fiber) were significantly reduced (P<0.05) (Table 1).

Table 1. Chemical composition and calculated nutrient content of silages

DM = Dry matter; NFE = Nitrogen free extract; WSC = Water soluble carbohydrates;

ADF = Acid detergent fiber; NDF = Neutral detergent fiber The average results from a production trial with 36 dairy cows over a 13 week period is shown in Tables 2-4.

The concentrate is kept constant and therefore it does not increase. Silage DM intake however, increased significantly (P<0.001) and thereby total energy intake (Table 2).

Table 2. Intakes of dairy cows fed untreated and SiloSolve® MC treated silages (n = 18). Control SiloSolve® MC

Daily feed intake - silage, kg 38.5 38.9

Silage, kg DM 12.25 12 72***

Concentrate, kg DM 7.13 7.13

Total, kg DM 19.38 19.85***

Silage metabolizable energy intake, MJ day 110.13 117.92***

Total metabolizable energy intake, MJ day 199.08 206.88***

Live weight, kg (end of experiment) 564.5 564.4

Live weight change kg/day + 0.178 + 0.182

The inoculation of silage had also a significant positive effect on milk yield (P<0.001), energy-correct milk (P<0.001), fat composition (P<0.001), protein composition (P<0.001), lactose (P<0.05) and urea (P<0.01). The increase in milk was more distinct than DM-intake since Feed conversion ratio (ECM/DMI) was significantly (P<0.01) improved when inoculating silage (table 3).

Table 3. Milk yields and composition, and live weights of dairy cows fed untreated and SiloSolve® MC treated silages (n = 18).

The hygiene quality of the milk incl . somatic cell count and total bacterial contamination was significantly reduced (P<0.001), while no Clostridia perfringens spores were detected.

Table 4. Hygiene quality of the milk (n = 18). Control SiloSolve® MC

Somatic cells count¹, thous./ml 105.67 80.69***

Bacterial contamination¹, thous.cfu/ml 28.04 22.35***

Content of Clostridium perfringens² , log/g < 1 < 1

Inhibitors LPT and LPT 2 tests³ not found/not not found/not found

found

Cytometer; ²⁾ ISO 7937:2004 ³⁾ 504DI14:2006

In Figure 1 it is shown that milk yield of cows fed inoculated silage is kept constant over a period of 13 weeks, while the control drops nearly 2 kg of milk. Even the average (1.0 kg) is highly significant (Table 3). The increased milk yield could be due to increased nutrient recovery (Table 1), increased silage intake (Table 2) or improved well-being (Table 4) or a combination.

In the same time span (13 weeks) the somatic cell counts (SCC) of the cows receiving inocu- lated silage were kept constant, while the control groups increased from 80,000 to 120,000 (Figure 2). 120,000 SCC is still very low, but the average SCC is significantly (P<0.001) lower (Table 4).

The total bacterial count slowly decreased over time during the 13 weeks of measurement when cows received inoculated silage, while the control was unsteady (Figure 3) . On average the total bacterial count was significantly higher in the control compared to inoculated silage (see Table 4).

While improved nutrient recovery of silage, silage intake and production parameters are very commonly observed, the increasing improvement of production has not been shown before, as well as the effect on milk hygiene (SCC and total bacterial count) which is very surprising, especially since with time the effect of silage inoculants gets more pronounced.

In conclusion, the silage inoculation with Silo Solve® MC improved significantly several pa- rameters:

Silage quality (e.g. DM recovery, crude protein content (see Table 1)

Silage intake (see Table 2)

Production parameters (e.g. milk yield and composition (see Table 3, Fig. 1)

Milk hygiene (somatic cells and total bacterial count (see Table 4, Fig. 2)

The difference between control and inoculated silage improved with time on milk yield, SCC, and total bacterial count. EXAMPLE 2

Silage inoculation

A fourth cut of perennial ryegrass (Lolium perenne) was mown and wilted to 45% dry matter over night. The wilted forage was cut to 4 cm and sprayed with either water only or a silage inoculant before entering a Krone Big Pack 1270 XC baler and bundled to square bales. Bales were immediately wrapped in the field by using a McHale 998 wrapper. Bales were wrapped with a green Silotte Eco stretch film in 8 layers with a 50% overlap. All untreated bales

(control silage) were baled before treated bales (long term inoculated silage) to avoid contamination of lactic acid bacteria from treated to untreated bales. The silage inoculant contained L piantarum CH6072 (DSM 16568), L lactis 0224 (DSM 11037), and L buchneri Lb 1819 (DSM 22501) and was sprayed on with a concentration of 1.5 x 10⁵ CFU/g forage be- fore baling . The bales were stored outside on concrete floor over the winter until fed to the dairy cows.

On the day before feeding the silage bales were opened and weighed into buckets and additional lactic acid bacteria were added (L. lactis SR 3.54 (NCIMB30117)) using a spray flask (short term inoculation) or pure water (no short term inoculation) . A total of 100 ml water with 5* 10⁷/ml of L. lactis SR 3.54 were sprayed onto the silage 16 hours before feeding . The sample was stored in the fridge until fed the next day.

Dairy cow production trial

Four primiparous lactating Holstein cows (average mil k production 27 kg ± 1.7 kg mil k/day and 112 days in mil k ± 4) were used in a 4x4 Latin square design study. The cows were chosen based on their milk performance. The cows were penned in a tie-stall and silage was fed twice daily based on the intake from the previous day. The forage concentrate ratio was fixed to 80 : 20 and adjusted based on the previous days feed intake. The cows received none or long term inoculated silage with or without additional short term inoculated silage over a period of 20 days. The amount of silage offered was adjusted based on intake of previous day.

RESULTS The results of the chemical composition of the control silage and the long term inoculation are shown in Table 5. The dry matter of the long term inoculated silage was higher and the crude protein lower. Table 5. Chemical composition and calculated nutrient content of silages

DM = Dry matter; ADF = Acid detergent fiber; NDF = Neutral detergent fiber.

Figure 4 shows an increase in SCC over time, when cows were fed control silage and no Lactococcus lactis SR 3.54 (NCIMB 30117) was added additionally. The SCC did not increase over time, when the Lactococcus lactis SR 3.54 (NCIMB 30117) was supplemented to the control silage fed to the cows or the cows were fed silage inoculated with Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis 0224 (DSM 11037), and L. buchneri Lb 1819 (DSM 22501). Combining long term inoculated silage with Lactobacillus plantarum CH6072 (DSM 16568), Lactococcus lactis 0224 (DSM 11037), and L. buchneri Lb 1819 (DSM 22501) with short term inoculation with Lactococcus lactis SR 3.54 (NCIMB 30117) reduced the SCC over time.

The reduction of somatic cell count (SCC) in milk supports example 1, where silage inocu- lants of lucerne also reduced the somatic cell count (SCC) compared to control. The meas- urement of silage inoculant in example 2 has been conducted over a shorter time period (20 days) compared to example 1 (13 weeks) and with only four cows , which have been offered all diets in different periods, so a clear effect as in example 1 was not expected.

REFERENCES

Auldist, M. (2002) : Milk Quality and Udder Health-effect on Processing Characteristics, In : Roginski H, Fuquay JW, Fox PF editor. Encyclopedia of Dairy Science. London, UK: Academic Press Inc.; 2002;p. 902-907. Cassoli, L. D., Machado, P. F., de Rodrigues, A. C, Coldebella, A. (2007). Correlation study between standard plate count and flow cytometry for determination of raw milk total bacterial count. International Journal of Dairy Technology, 60 (1), 44-48. Laevens, H., de Muelenaere, E., Ysebeart, M.-T., Deluyker, H., Vanneste, W., de Kruif, A. (1996). Comparison of somatic cell counting with Fossomatic 360 and the Somascope. Neth. Milk Dairy J. 50, 335.

Claims

A method for reducing somatic cell count and total bacterial count in milk from a milk-producing animal, the method comprising feeding the animal with a fermented feed produced by inoculating a plant material with at least one lactic acid bacterium, wherein the at least one lactic acid bacterium is a Lactobacillus, Lactococcus, Enterococcus, or any combinations thereof.

The method according to claim 1 wherein the Lactobacillus is one or more species selected from the group consisting of Lactobacillus plantarum and Lactobacillus buch- neri.

The method according to claim 1 wherein the Lactococcus is a Lactococcus lactis.

The method according to any one of claims 1 to 3 wherein the at least one microorganism is a combination of Lactobacillus plantarum CH6072 deposited as DSM 16568, Lactobacillus buchneri deposited as DSM 22501, Lactococcus lactis deposited as DSM 11037, and optionally Lactococcus lactis SR 3.54 deposited as NCIMB 30117

The method according to any one of claims 1 to 3 wherein the at least one lactic acid bacterium is a combination of a Lactobacillus, a Lactococcus, and an Enterococcus.

The method according to any one of claims 1 and 5 wherein the Enterococcus is an Enterococcus faecium.

7. The method according to any one of claims 1, 5 and 6 wherein the at least one microorganism is a combination of Enterococcus faecium M74 deposited as DSM 22502, Lactococcus lactis SR 3.54 deposited as NCIMB 30117 and Lactobacillus plantarum CH6072 deposited as DSM 16568.

8. The method according to any of claims 1-7 wherein the milk-producing animal is a bovine species such as a cow, an ovine species, an equine species or a swine.