HK1150781B - Mixture of citric flavonoids to improve ruminal fermentation - Google Patents

Description

Lemon flavonoid mixtures for improving rumen fermentation

Description of the invention

The present invention relates to the use of a mixture of lemon flavonoids for improving rumen fermentation.

Abbreviations

VFA volatile fatty acids

Increase in Delta Ct amplification cycles

Increase in delta. DELTA. Ct relative to amplification cycles of control samples in PCR

DGGE denaturing gradient gel electrophoresis

M. elsdenii E.megacoccus (Megasphaera elsdenii)

ns is not significant

PCR polymerase chain reaction

S. bovis Streptococcus bovis (Streptococcus bovis)

Mammals do not have suitable digestive enzymes for digesting structural carbohydrates, i.e., cellulose. Thus, and in order to access such important nutrient sources, herbivores have constructed fermentation compartments that contain symbiotic flora capable of digesting or fermenting such nutrients. These compartments are located either before (ruminants) or after (post-gastric fermented material, i.e. equines, rabbits) enzymatic digestion in the host. Microorganisms of the rumen only partially use these carbohydrates and release volatile fatty acids (acetic, propionic and butyric acids) as fermentation products, which are the most important energy sources for ruminants. Microorganisms that proliferate in the rumen are those that are able to adapt to the compartment conditions and utilize the food ingested. Different food fractions, carbohydrates and proteins, are fermented and supplemented with VFA production by excess biomass produced in the rumen (which in turn is the main source of finally absorbed proteins in the duodenum).

The cellulose fermenting bacteria (cellulolytic) grow optimally at a pH of 6.4-7 and produce more acetic acid than the starch and sugar fermenting substances (amylolytic). The optimum pH is about 5.5 and as these levels fall, there is an increase in the relative production of lactic acid.

With the enhancement of ruminant production systems, feed-based feeding has been replaced by concentrated rations of food formulated with grains, which increases growth rates and reduces operating costs. However, concentrated rations of food or the replacement of structural carbohydrates (cellulose) by fast-fermenting soluble carbohydrates (starch) have resulted in some digestive dysfunctions such as acidosis and timpanisms.

Rumenic acidosis can be defined as a digestive change produced by digestion of large amounts of foods rich in fermentable carbohydrates and is one of the most common dysfunctions in fattening calves. It can lead to death and outbreaks by changing the bacterial population from gram negative to gram positive, favoring the development of species such as Streptococcus bovis or Streptococcus maxima and altering the proportion of VFA. The ratio of acetic acid and propionic acid decreases, while butyric acid and lactic acid increase. The increase in lactic acid affects rumen motility, and produces an abdominal mass due to the inability to eliminate the gas produced. Acute symptoms are anorexia, yellowish green watery feces, lack of rumen tone, lameness, abdominal retraction and dehydration. Subacute forms include frequent abdominal pneumatosis, abnormal hair and liver abscesses found after slaughter.

timpanism and abdominal pneumatosis are defined as digestive alterations caused by fermentation overproduction and accumulation of gases (carbon dioxide and methane) causing abnormal swelling of the honeycomb stomach-rumen.

Usually, the rumen dysfunction is caused to exist in a latent state by the addition of antibiotic substances (ionophores, such as monensin). However, the gradual elimination of such substances as growth enhancers leads to the appearance of these conditions, causing a high mortality or morbidity, which has a negative impact on profitability. This effect on farm productivity is very important and alternatives to these antibiotic substances are therefore needed.

Patent US4443471 relates to different chemical derivatives of M-139603 and M-139603 ruminant growth enhancers, and its use in reducing the amount of methane produced by rumen fermentation.

Patent US5196432 relates to the use of alpha-2 adrenoceptor antagonists for the treatment of ruminants affected by lactic acidosis.

Patent US5709894 describes a food additive for ruminants containing glutamic acid and corn fermentation products to enhance rumen fermentation.

Patent application US2003165487 describes methods and compositions based on aspergillus oryzae (aspergillus oryzae) amylase to improve the efficiency of rumen fermentation and prevent detrimental increase in rumen lactate concentration, promoting the growth of beneficial rumen microorganisms.

Patent application US2004009209 covers a method for maintaining the rumen health of ruminants, consisting of a mixture of low-humidity molasses and a buffering agent.

PCT application WO9119489 describes a method of modulating ruminal pH levels by administering succinic acid and other carboxylic acid salts to ruminants feeding high energy rations.

PCT application WO9325616 relates to starch products encapsulated in a matrix derived from the maillard reaction of a soluble heterologous protein and a reducing sugar to improve the efficiency of microbial fermentation in the rumen of ruminants.

PCT application WO2004009104 relates to novel strains of megacoccus aegypti (m.elsdeniii) and uses thereof, including the prevention and treatment of lactic acidosis in ruminants.

PCT application WO2005000035 relates to a method for improving rumen fermentation, in particular for reducing methane production, comprising administering a soluble alfalfa extract obtained from fresh alfalfa.

EP1323354 describes a natural feed additive based on plant material.

It is known to feed livestock with byproducts of the crop and food processing industries. For example, citrus byproduct feed can be used as an ingredient in ruminant feed systems. They contain various energy substances for rumen microorganisms (Bampidis et al, Animal Feed Science and technology, Elsevier, vol.128, 2006, 175-. JP-52028922 describes a medicament containing bitter orange peel. The medicament is fed to horses, cattle or poultry.

The inventors of the present application have found that a mixture of lemon flavonoids (especially naringin), bitter orange extract and sepiolite is able to modulate the microbial fermentation process resulting from the administration of concentrated rations, to increase the efficiency of the microbial fermentation process and to limit certain species directly involved in the rumen acidosis process, especially streptococcus bovis.

Accordingly, the present invention relates to the use of a mixture comprising naringin, bitter orange extract and sepiolite for improving rumen fermentation in ruminants.

In this application, "ruminant" includes bovine, ovine, caprine and camelid (camelide) species.

This mixture presents a series of advantages over the state of the art, making it of high value in the field of ruminant nutrition. The advantage is that all the ingredients in the mixture are products of natural origin and are readily available. On the other hand, the mixtures are easy to handle and can be prepared according to industrial formulation methods known to the expert in the field. This mixture also has the additional advantage of limiting the growth of certain species (in particular streptococcus bovis) involved in the occurrence of ruminal acidosis. Thus, in particular, the invention relates to the use of said mixture for improving ruminant fermentation, which is impaired by an imbalance of the natural bacterial flora in the rumen. The invention also relates to the use of said mixture for preventing damage to rumen fermentation, which may be caused by an imbalance of the natural bacterial flora in the rumen. It is a particular object to use the mixture of the invention to limit the growth of bacteria causing ruminal acidosis, such as Lactobacillus (Lactobacillus spp), for example Lactobacillus acidophilus (Lactobacillus acidophilus), and especially streptococcus bovis.

The mixture is active when added to the feed in solid form at a concentration of 100 to 300ppm (100 to 300 g/ton). This is based on an in vitro study in which the effect of natural mixtures was analyzed at a dose range of 100 to 300 ppm.

According to one aspect, the naringin content of the mixture of the invention is between 10% and 25% by weight, preferably between 15% and 25% by weight, more preferably about 20% by weight.

According to a second aspect, the bitter orange extract content in the mixture according to the invention is between 10% and 65% by weight, preferably between 20% and 60% by weight, more preferably about 40% by weight. It is noted that bitter orange extracts include naringin and therefore the total naringin content in the present blends will be higher than that shown above.

According to a third aspect, the naringin, bitter orange extract and sepiolite are present in an amount of 100% by weight of the mixture.

In a first embodiment, the present invention relates to the use of a mixture comprising from 10% to 25% by weight naringin, from 10% to 65% by weight bitter orange extract and a complementary amount to 100% (complement of mixture) by weight sepiolite for improving rumen fermentation in ruminants.

In the embodiments derived from the above, the mixture comprises 15% to 25% by weight naringin, 20% to 60% by weight bitter orange extract and the balance to 100% sepiolite, more particularly 20% by weight naringin, 40% by weight bitter orange extract and 40% by weight sepiolite.

The proportions (% by weight) indicated above are relative to the total weight of the mixture.

In another embodiment, the above mixture is used to regulate the microbial fermentation process resulting from dosing the concentrated food product.

In another embodiment, the above mixture is used to limit the growth of bacteria causing ruminal acidosis, in particular streptococcus bovis. Thus, the present invention also relates to the use of the above mixture for preventing or treating ruminal acidosis, including the acute and subacute forms described herein.

In another embodiment, the above mixture is used to optimize productivity in intensive fattening of ruminants, especially cattle.

In another embodiment, the use of the above mixture comprises adding it to the feed in solid form in a concentration of 10 to 2000ppm by weight, in particular 50 to 1000ppm, more preferably 100 to 300 ppm.

Flavonoids are a class of water-soluble plant pigments for which medical attention is growing. Naringin is a flavonoid, particularly a glycosylated flavanone, obtained from the peel of Citrus fruits (grapefruit (Citrus paradisi) and bitter oranges (Citrus aurantium) and responsible to a large extent for its bitter taste.

Several studies have shown that, like other flavonoids, naringin biosynthesis is influenced by environmental and genetic factors which determine the differences in the concentration levels of these compounds, with values estimated to be common for fresh grapefruit peel ranging from 15 to 18 g/kg. The content in the peel is also different, higher in unripe fruits than in ripe fruits.

Naringin is used in perfumery and to flavor candies, beverages and baked goods, is also used for its antioxidant and antimutagenic properties, and as an oil stabilizer.

Bitter orange extract may be obtained from ground citrus fruit (especially bitter oranges) by conventional procedures in the art such as extraction, filtration, concentration, precipitation, clarification and final drying. The extraction treatment may be carried out in a binary alkanol/water system, wherein the alkanol is selected from methanol, ethanol, propanol, etc. Methanol is preferably used. A typical bitter orange extract will comprise 31.5 to 71.5% by weight total flavonoids, for example 45 to 55% by weight (if determined by HPLC). In particular, the flavonoids include naringin, neohesperidin and poncirin. Typically, the total flavonoid content will comprise (a)17.5 to 35.1% by weight naringin, e.g., 25 to 27% by weight, (b)7.7 to 16.9% by weight neohesperidin, e.g., 11 to 13% by weight and (c)2.1 to 6.5% by weight poncirin, e.g., 3 to 5% by weight.

Sepiolite, in turn, is a natural hydrous magnesium silicate, the source of which is attributed to the calcareous deposits of marine fossils. It is a white or yellowish clay mineral. It was used as a powdered toothpaste since ancient times.

The composition of the invention is applied to ruminants suffering from or at risk of suffering from reduced rumen fermentation. In particular, these ruminants include:

1) feeding ruminants on a high concentration (cereal) diet, e.g. for improving feed conversion ratio and avoiding ketosis processes, especially if the ruminant is a ruminant

a) Animals fattening under a booster system;

b) females with high lactation yield at peak lactation when the concentrate supply reaches the highest ratio; or

c) The intake capacity of heavy pregnant ewes or goats with more than one lamb has been limited.

2) Ruminants receiving sudden dietary changes, especially such changes as

a) Change from pasture (high-roughage) to high concentration ration; or

b) Changing from low digestible starch (sorghum or corn) to high consumable (wheat or barley).

For illustrative purposes, the following examples are provided to better understand the present invention.

Example 1: effect of mixtures at doses of 100 and 300ppm in cattle

Fermentation kinetics were analyzed based on the protocol set out in Theodorou M K et al, Animal Feed Science and Technology, 48(3), p.185-197, month 8, 1994. The inoculum used was rumen fluid from commercial intensive fattening cattle. The mixture of the invention (20% by weight naringin, 40% by weight bitter orange extract and 40% by weight sepiolite) was inoculated with an inoculum and commercial cattle feed (600mg) supplemented with cereal straw in the usual ratio (80: 20 concentrate: straw). This mixture dose was incubated in triplicate in two batches (2 cattle) in a sealed bottle containing 800ml of inoculum dilution.

The test lasted 48 hours and the pressure in the bottle was recorded at 2, 4, 6, 12, 24, 36 and 48 hours. The generation of gas is established from the pressure value. After 10 hours of incubation, one bottle was separated and its contents were sampled for determination of VFA and DNA isolation for molecular genetic testing.

By passingDNA Stool Mini kit (Qiagen Ltd, Carwley, West Sussex, UK) for DNA extraction. By means of a spectrophotometerThe DNA concentration and purity (total DNA) were determined and the absorbance was measured at 260 and 280 nm.

Use of ABI by real-time PCR7000 sequence detection System bacterial and bovine streptococcal DNA were quantified using specific primers for total bacteria (Maeda H et al, FEMS Immunology and medical Microbiology, 39(1), p.81-86, month 10 2003) and bovine streptococcal (TajimaK et al, Applied and Environmental Microbiology, 67(6), p.2766-2774, month 6 2001). As a reference for calculating the total bacterial concentration in the culture medium, we used a DNA concentrate of bacterial samples obtained by differential centrifugation of the rumen fluid (500g, 10 min, followed by 20.000g, 20 min). The abundance of Streptococcus bovis was expressed relative to total bacteria using Livak K J et al, Methods (san Diego, Calif.), 25(4), p.402-408, Δ Δ Ct expression described in 12 months 2001.

Bacterial biodiversity was carried out by DGGE using specific primers (N ü bel U et al, Journal of bacteriology, 178(19), p.5636-5643, 10 months 1996). Electrophoresis used an 8% acrylamide gel with a gradient of 50% to 65% denatured urea/formamide at 80V for 16 hours. Gels were stained using the Amersham Biosciences (Sweden) kit and after scanning, the band patterns were analyzed using the UPGMA (Unweighed Pair-Group Method Arithmetric instruments) program.

The effect of the mixture on rumen fermentation was analyzed according to a 2 x 2 (mixture x dose) factor design, considering animals as experimental modules.

Results

1. -gas generation

Under the in vitro conditions described previously, inclusion of the mixture in the medium had no effect on the kinetics of VFA production or total VFA production. The experimental treatment also did not affect the molar ratio of VFA of most importance, which showed an average ratio of 60% acetic acid, 29% propionic acid and 9% butyric acid.

2. Real-time bacterial quantification by PCR

Table 1 shows the total DNA concentration obtained by spectrophotometry (260nm), and we can see how the DNA concentration in the medium was not altered by the inclusion of the mixture. However, the determination of the amount of bacterial DNA by a more specific procedure (real-time PCR) showed that the inclusion of the mixture resulted in a significant increase in bacterial DNA, indicating an increase in bacterial growth. The presence of significant differences in microbial growth due to the presence of the mixture (in gas (CO)₂+NH₃) No such difference in VFA release was found to be produced or higher) indicates that the mixture has a positive effect on the microbial synthesis efficiency.

The same table also shows the concentration of specific DNA sequence copies of S.bovis, which has been identified as one of the main producers of lactic acid and thus is involved in the conditions caused by ruminal acidosis. The addition of the mixture allows a very significant limitation of the concentration of this species in the culture medium, expressed as the reduction in the number of DNA copies corresponding to streptococcus bovis (2(Δ Ct) × 1000) relative to the total number of bacterial DNA copies or as a percentage of the copy number relative to the control medium (2(Δ Δ Ct)).

Table 1: the concentrations of the different DNA types in the medium calculated by real-time PCR; making

DNA 16S copy number in culture medium as an indicator of bacterial biodiversity

Significance of diet control mixture
	Dose (ppm) 100300 RSD dietary dose
Total DNA (. mu.g/ml) 37.339.732.36.98 ns bacterial DNA (. mu.g/ml) 8.1924.321.52.750.0018 ns Streptococcus bovis 2 (. DELTA.Ct). 10004.031.171.10.840.01 ns Streptococcus bovis 2 (. DELTA.Ct) 0.310.290.14 ns DGGE N15.5015.015.91.44 ns

When we analyzed the effect of the dose of the mixture on the above parameters, we found that there was no significant difference with the mixture at this concentration. Thus, the results obtained indicate that initial concentrations of 100 and 300ppm exceed the activity limit of the compound on the rumen fermentation process. On the other hand, although the study mixture altered the concentration of Streptococcus bovis, it did not alter the biological diversity of the population. This demonstrates the selective effect of the substance on certain microbial populations.

3. Conclusions and advantages

Supplementation of the study mix with a quantitative amount of inoculum from a concentrated diet administered under commercial intensive fattening conditions produced significant changes in the rumen fermentation process.

The presence of the mixture did not alter the gas or VFA production level. However, it has a significant effect on the different microbial populations in the inoculum. Although promoting a significant increase in the level of population synthesis, it significantly inhibited the growth of Streptococcus bovis.

Claims (8)

1. A mixture for improving ruminal fermentation in ruminants comprising 10% to 25% by weight naringin, 10% to 65% by weight citrus aurantium extract comprising 44 to 55% by weight total flavonoids and the balance to 100% by weight sepiolite, wherein the total flavonoids comprise (a)17.5 to 35.1% by weight naringin, (b)7.7 to 16.9% by weight neohesperidin and (c)2.1 to 6.5% by weight poncirin, wherein the improvement comprises limiting the growth of Streptococcus bovis (Streptococcus bovis) bacteria.

2. The mixture of claim 1 wherein the total flavonoids comprise (a)25 to 27% by weight naringin, (b)11 to 13% by weight neohesperidin and (c)3 to 5% by weight poncirin.

3. The mixture of claim 1, wherein the improvement modulates microbial fermentation processes in ruminants dosed with concentrated food.

4. The mixture of claim 1, wherein the improvement optimizes ruminant productivity in intensive fattening.

5. The mixture of claim 1, wherein the ruminant is a calf.

6. The mixture of claim 1 for addition to feed in solid form at a concentration of 10 to 2000ppm by weight.

7. The mixture of claim 6 for addition to feed in solid form at a concentration of 50 to 1000ppm by weight.

8. The mixture of claim 7 for addition to feed in solid form at a concentration of 100 to 300ppm by weight.