WO2014053977A1

WO2014053977A1 - Natural antiseptic solution to be used as teat dip (dipping) during milking for prevention and control of mastitis

Info

Publication number: WO2014053977A1
Application number: PCT/IB2013/058940
Authority: WO
Inventors: Bernardo CARRILLO LÓPEZ; Luz Haydée MOLINA CARRASCO; Juan Carlos PAREDES GALLARDO; Mariela Horzella Rademacher; Luis SALGADO GÓMEZ
Original assignee: Consorcio De Desarrollo Tecnológico Apícola S.A.
Priority date: 2012-10-02
Filing date: 2013-09-27
Publication date: 2014-04-10
Also published as: CL2012002768A1; AR092733A1

Abstract

The present invention relates to the formulation of an antiseptic solution consisting of a propolis extract and honey bee (Apis mellifera) to be used as dipping or teat dip during milking as a prophylactic measure for preventing and controlling mastitis in dairy cows, providing a natural alternative to commercially available products, which occasionally can generate residues in milk and/or cause skin irritation either in the animal or the worker who applies it. The antiseptic solution for preventing sub-clinical mastitis in dairy cows of the present invention comprises a mixture of propolis extract and honey solution in ratios ranging from 1:5 to 5:1.

Description

NATURAL ANTISEPTIC SOLUTION TO BE USED AS TEAT DIP (DIPPING) DURING MILKING FOR PREVENTION AND CONTROL OF MASTITIS

SPECIFICATION OBJECT OF THE INVENTION

The present invention relates to the formulation of an antiseptic solution consisting of a propolis extract and honey bee (Apis mellifera) to be used during milking as dipping or teat dip as a prophylactic measure for preventing and controlling mastitis in dairy cows, providing a natural alternative to commercially available products, which occasionally can generate residues in milk and/or cause skin irritation either in the animal or the worker who applies it.

BACKGROUND OF THE INVENTION

Mastitis is by definition an inflammation of the mammary gland caused mainly by infection of microorganisms and has been considered the greatest impact disease in dairy herds due to its high prevalence and the economic damage it causes. This disease occurs when a pathogenic bacteria enters into the udder, overcomes the cow's immune system, establish an infection and produces an inflammation of the glandular tissue. This inflammation is characterized by increased somatic cell concentration. Mastitis is the disease that causes more economic losses to dairy producers worldwide, with losses that account for 30% of the total cost of all diseases that affect dairy cattle. Annual global losses due to mastitis have been estimated to be 35 billion U.S. dollars. The losses caused by this disease can be grouped as follows: reduced production, discarded milk, cost of drugs, cost of veterinary services, extra labor and loss of genetic potential. Considering only milk production in Chile is estimated that annual losses ranged from 162 to 204 million liters of milk due to this disease and that reduction in milk production per lactation in cows with clinical mastitis can arise to 14%, when compared with animals that did not have the disease.

Mastitis is generally classified as clinical mastitis and sub-clinical mastitis, the former is defined as the inflammation of the udder characterized by visible abnormalities in the milk or in the udder, or both of them, alterations in the composition and appearance of the milk are present; reduced milk production, elevated body temperature of the cow, redness, warmth and swelling of infected quarter(s). While sub-clinical mastitis is a not visible inflammation of the mammary gland and requires a diagnostic test for detection. This type of mastitis is difficult to detect and cure, because the sick cow is apparently healthy without being so, the mammary gland shows no sign of disease and the produced milk has normal appearance, with no observable changes in its composition or organoleptic characteristics. It is the most dangerous form of mastitis, its frequency is 20-50 times higher than clinical mastitis because no distinct symptoms allowing opportune diagnosis are present, and it also produces alterations in milk composition and reduced production.

Among the measures that have given best results in the control of mastitis or reduction of somatic cells is the teat dip or dipping. Dipping practice consists of applying an antiseptic solution (usually iodinated) to all teats immediately after removal of the teat cups, with the aim of reducing the microorganisms remaining on the skin and teat orifice at the end of the milking process, thus preventing microorganisms for entering and growing in the teat canal, thereby reducing the risk of intramammary infections. The dipping also leaves residual disinfectant on the teat surface during milking period, during which the teat orifice remains open for at least 30 minutes, thereby preventing bacterial colonization of the teat canal and promoting recovery of lesions, sphincter, and teat canal.

After milking, teat dip with appropriate antiseptic is considered by different researchers the most effective method to prevent new infections in lactating animals. However, chemicals based on chlorine, iodine or other compounds such as chlorhexidine are used for this, its use has negative impact over the environment, and can produce some degree of irritation on the teat skin and in the hands of the milkers.

For this reason arise the need for alternatives, such as the antiseptic solution of the present invention that is made from natural occurring ingredients and composed by propolis and honey, which by its natural characteristics and antibacterial properties can replace chemicals in preventing and controlling mastitis. The antiseptic solution described in this invention has the following advantages: does not damage the cows teats, has no negative effects on the hands of the operator, and does not harm the environment, it is also highly effective in preventing and treating sub-clinical mastitis , thereby preventing for cows to become ill, so is an effective prophylactic measure. On the other hand, by being a dipping solution protects the teat for a longer time avoiding infection while the teat orifice remains open, which is achieved by the adhesion of the antiseptic solution to the teat surface, given its composition.

Prior art U.S. Patent Application U.S. 2010322976 discloses a composition including honey and aloe vera useful as a tonic or food supplement for the treatment of mastitis, especially in immunocompromised animals. In contrast to what is disclosed in said document, the present invention relates to an antiseptic solution to be used during milking as teat dip or dipping as a prophylactic measure in preventing and controlling mastitis in dairy cows and not for animal consumption as food supplement or tonic.

The European Patent Application EP1230927 discloses a disinfectant composition for veterinary use in the treatment of mastitis by applying the product in gel form. The composition comprises propolis and plant extracts dissolved in water in certain specific percentages. In contrast to the present invention, said application uses only propolis as active ingredient and a number of essential oils obtained from various plants and applies the product in gel form for the treatment of mastitis and not as dipping for preventing and controlling mastitis. The present invention uses a combination of honey and propolis that allows obtaining an effective antiseptic solution for the prevention and/or control of sub-clinical mastitis, applying the product as dipping.

The International Application WO 8906955 discloses a composition for treating mastitis. The active ingredient is propolis (soft propolis) to be mixed with one or more emulsifiers and other additives. In contrast with the present invention, said application uses only propolis as the active ingredient and it is used to treat mastitis and not as dipping for preventing and controlling mastitis. The present invention uses a combination of honey and propolis that allows obtaining an effective antiseptic solution for the prevention and/or control of sub-clinical mastitis.

The UA 74087 application discloses a composition based on beekeeping products and plant extracts. The composition comprises propolis, turpentine, honey, oil carotene, pollen pellets. Other ingredients such as excipients (animal fat, beeswax, ethanol, ascorbic acid) are incorporated. Its use is mentioned for treating skin lesions and mastitis in cattle. In contrast with what is disclosed in said document, the present invention relates to an antiseptic solution to be used during milking as teat dip or dipping as a prophylactic measure for preventing and controlling mastitis in dairy cattle, and not as treatment for the disease. The advantages of the antiseptic solution described herein over other chemicals used as dipping solutions to control mastitis, are of great importance in economic terms, but even more important are the physical and chemical properties that would allow to preserve the hygienic quality in addition to avoiding the presence of unwanted residues in the milk. From the technical standpoint, the antiseptic solution based on propolis and honey is more efficient than the chemical solutions currently in use and besides it is based on naturally occurring ingredients. This invention has been shown to prevent and control sub-clinical mastitis, which is the most difficult to eradicate. Furthermore, prevention of mastitis avoids the use of antibiotics and losses in milk production.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A shows the results of field trials in which the antiseptic solution of the present invention was applied as dipping to cows at farm 1 . Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

Figure 1 B shows the results of field tests in which the commercially available composition, whose active ingredient is iodine, is applied as dipping to cows at farm

1 . Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

Figure 2A shows the results of field trials in which the antiseptic solution of the present invention was applied as dipping to cows at farm 2. Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

Figure 2B shows the results of field tests in which the commercially available composition, whose active ingredient is iodine, is applied as dipping to cows at farm

2. Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

Figure 3A shows the results of field trials in which the antiseptic solution of the present invention was applied as dipping to cows at farm 3. Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month. Figure 3B shows the results of field tests in which the commercially available composition, whose active ingredient is iodine, is applied as dipping to cows at farm 3. Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

Figure 4A shows the results of field trials in which the antiseptic solution of the present invention was applied as dipping to cows at farm 4. Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

Figure 4B shows the results of field tests in which shows the results of field trials in which the antiseptic solution of the present invention was applied as dipping to cows at farm 4. Each line represents control test results obtained for a particular cow. The control test was a somatic cell count carried out fortnightly or at least 1 time per month.

DETAILED DESCRIPTION OF THE INVENTION.

Antiseptic solution is prepared in equals parts or varying the ratios of 2-7% propolis extract to 15-25% (w/v) honey bee Apis mellifera between 1 :5 and 5:1 , both preferably from ulmo (Eucryphia cordifolia) as botanical origin, to be used as dipping solution during milking of dairy herds for the prevention and control of mastitis, as a prophylactic measure.

The antiseptic formulation consists of propolis extract and honey; raw materials that should comply with the following properties:

. PROPOLIS:

L it should have been harvested through mesh, a technique for obtaining a product without solid impurities such as wood cuttings, pieces of wood, paints and others.

2. The obtained extract should preferably be red-orange (CIE system).

3. It should have at least 10% of total flavonoids.

4. It should contain at least the following polyphenols and/or flavonoids: apigenin, chrysin, galangin, kaempferol and pinocembrin.

5. It should be antibiotic free.

6. Bioactivity testing and activity verification against major strains of microorganisms causing mastitis (Staphylococcus aureus ATCC 29213 and ATCC 25923, Streptococcus dysgalactiae ABEII50 , Streptococcus uberis ABEII- 46), should be performed.

. HONEY:

1 . Using annual season honey (fresh), preferably from ulmo (Eucryphia cord i folia).

2. Prior to preparation of the honey solution, the entire contents of the container should be homogenized, due to the likely heterogeneous active ingredient(s) distribution (flavonoids, hydrogen peroxide, sugars concentration, and pH).

3. Bioactivity testing and activity verification against Escherichia coli and at least against two of the microorganisms of highest incidence on mastitis, noted above, should be performed.

4. Using honey which has not undergone heat treatment above 40°C and determine the presence of hydroxymethylfurfural (HMF), not exceeding the provisions of Chilean Standard (40 meq/kg)

5. It should be antibiotic free.

METHOD OF PREPARATION OF THE ANTISEPTIC SOLUTION.

Antiseptic solution is prepared based on a propolis extract and a honey solution in ratios ranging from 1 :5 to 5:1 , preferably 1 :1 ratio. The form of preparation of these components is detailed below:

• Propolis extract:

Propolis raw material that may have different geographical origin is selected; those brown to red are considered more effective. The propolis is dissolved using methanol as solvent and then evaporated to dryness in a rotary evaporator under reduced pressure, in order to obtain the total extract. The extraction of the raw material is repeated three times.

Depending on total flavonoids content, a solution containing at least 0.5% of flavonoids is prepared in ethyl alcohol/ water 40:60.

This mixture is allowed to stand for a period between 5 to 24 hours, preferably 8 hours, at room temperature. After that, the mixture is filtered through appropriate means to separate the fat and waxes. The solution obtained is filtered through an appropriate mean to remove fats and waxes, to finally obtain a yellow colored solution.

• Honey solution:

A mixture of 15 to 25% (w/v), preferably 20% (w/v), of honey from ulmo (Eucryphia cordifolia) as botanical origin is prepared in distilled water.

The two solutions thus prepared (the solution of propolis extract and honey aqueous solution) are mixed at a 1 :5 to 5:1 ratio, preferably 1 :1 , and refiltered, if necessary, to remove fat traces from the mix. The product obtained is the antiseptic solution of the present invention developed and tested in the field.

Antiseptic solution must meet the following quality parameters (Table 1 ) to assure homogeneity of the physicochemical properties of the product.

Table 1. Quality parameters of the mixture obtained as antiseptic solution.

The following are exemplary applications that better illustrate and explain the invention, and are not to be taken in any way as limiting the scope and application of the present invention.

EXAMPLES EXAMPLE 1

Preparation of the antiseptic solution.

An antiseptic solution was prepared according to the present invention, as follows:

• Propolis extract:

Chilean propolis from different geographical origins were obtained, selecting those brown to red.

500 grams of the selected raw propolis was put in a glass vessel and was suspended in 1 L of ethyl alcohol.

The mixture was allowed to stand for 8 hours at room temperature and then filtered through a Whatman filter paper No. 1. The filtrate was evaporated under reduced pressure until a reddish oily concentrate free of solvent was obtained. The raw material extraction was repeated three times.

A solution of 5% concentration (w/v) was prepared with the extract, and it was resuspended in a mixture of ethyl alcohol/water 40% (w/v), this solution was filtered through cotton wool, to finally obtain a yellow colored solution.

• Honey solution:

200 g were dissolved up to one liter solution with distilled water.

The two solutions, 50 g/L propolis and 200 g/L honey were mixed in a 1 :1 ratio and filtered through cotton wool to remove residual fat from the mixture.

EXAMPLE 2

Determination of "in vitro" activity of the antiseptic solution of the invention

Antiseptic solution, prepared according to Example 1 , was tested "in vitro" against 5 strains of the mayor microorganisms responsible for mastitis: Staphylococcus aureus ATCC 29213 and ATCC 25923, Streptococcus dysgalactiae ABE 11 50, Streptococcus uteris ABEII 46 and Escherichia coli ABEII ATCC 8733.

For "in vitro" tests trypticase soy agar (TSS) was used as culture medium, which was coated on a 14 cm petri dish, finally obtaining a 10⁵ CFU/mL lawn for each of the strains under study. After the lawn was solidified, holes were punched with a diameter of 7.58 mm each one, forming wells over which 50 L of sample was put.

Subsequently, inhibition halos formed around each well were measured with a digital caliper, the average results appear in Table 2.

The results presented in Table 2 clearly show the inhibitory effect of the antiseptic solution against the strains used, compared to the iodine solution (commercial solution).

Table 2. Average results of antibacterial activity inhibition halos of antiseptic solution and iodine solution over causing mastitis strains.

According to the results shown in Table 2, it can be concluded that the antiseptic solution has a broad spectrum of activity against certified strains, even against £. co//, strain responsible for environmental mastitis and which exhibits higher inhibitory activity compared with iodine solution. Example 3

Determination of the activity in the field for antiseptic solution of the present invention in dairy cows We conducted a field trial between April 201 1 and April 2012, in Chile, in which the antiseptic solution was applied as a teat dip (dipping) in 4 groups of cows randomly selected, located in 4 dairy farms. To compare the effect of the results, the commercial solution (iodine) was applied to 4 groups of cows (randomly selected) in parallel, which is commonly used in dairy farms and is applied to the cows teats at the at the end of milking process, to prevent mastitis.

In each farm, the total number of cows was different and each group was divided into two groups, one of them as a control, using the iodine solution as a teat dip. In the second group the antiseptic solution of the present invention was used as a teat dip.

To measure the effect of the batches of cows under study on the prevention and control of mastitis, biweekly or monthly counting of somatic cells in milk from each cow was used as a parameter for evaluating and comparing.

The results showed that the values of somatic cell count registered in the cows under study in all farms allow us to assure that with the antiseptic solution of the present invention are obtained better results than with the iodine solution, keeping count levels below which have been established for cows as free of sub-clinical mastitis in literature, <200,000 cells/mL. An example of these results is shown by comparing the evolution and behavior of somatic cell counts from cows that were subjected to the antiseptic solution v/s cows subjected to iodine solution in the same field (Figures 1-4 A and B). To confirm the above mentioned effect, according to the results of average somatic cell counts per cow, we proceeded to the statistical analysis of the data, which was performed by the "t-student" test for two samples, assuming unequal variances, for all farms in the trial. The total analysis of the cows results in statistically significant differences in the application of dipping solutions for sub-clinical mastitis treatment, with the antiseptic solution application showing better results (Table 3). Furthermore, the antiseptic solution proved to have greater adherence to the teat surface, remaining in this for a longer period of time.

However, when using both solutions as teat dip an increase in the number of somatic cells in the 20 biweekly observations was noticed, which is explained by the normal increase in these cells with advancing lactation, milking frequency, age, nutrition, in addition to the degree of initial mastitis and stress level of the animals under study.

Therefore, it has been suggested that a mammary quarter is a healthy one when somatic cell count of composite milk (all quarters) of a cow does not exceed 100,000 cells/mL, and if it is higher than 200,000 cells/mL the animal exhibits a degree of subclinical mastitis.

Table 3. Field analysis results for all fields and cows under study.

Claims

1 . Antiseptic solutions for prevention of sub-clinical mastitis in dairy cows, CHARACTERIZED in that it comprises a mixture of propolis extract and honey solution in ratios ranging from 1 :5 to 5:1.

2. Antiseptic solution according to claim 1 , characterized in that the best mixing ratio is 1 :1 .

3. Antiseptic solution according to claim 1 , CHARACTERIZED in that the propolis extract is prepared between 2-7% (w/v), and the honey solution is prepared between 15-25% (w/v).

4. Antiseptic solution according to claim 1, CHARACTERIZED in that the propolis extract is used at 5% (w/v) and the honey solution at 20% (w/v).

5. Antiseptic solution according to claim 1 , CHARACTERIZED in that the propolis extract is obtained using ethyl alcohol as a solvent, or methyl alcohol, or isopropyl alcohol, or acetone, or ethyl acetate, aqueous solutions or mixtures of such solvents.

6. Antiseptic solution according to claim 1, CHARACTERIZED in that the solvent mixture used is preferably ethyl alcohol/water.

7. Use of antiseptic solution according to claims 1 to 6, CHARACTERIZED in that it serves as a prophylactic measure in the prevention and control of subclinical mastitis in dairy cows and is applied as a teat dip or dipping.