Patent Application
20080118490
The present invention generally relates to animal feeds. In particular, the present invention relates to the use of triglycerides containing medium chain fatty acids combined with exogenous active lipolytic enzymes as feed supplements for animals.
Early weaning (3 to 4 weeks of age) of piglets has become a general practice in pig husbandry systems for increasing the productivity and maintaining the profitability. Early weaning, however, burdens the piglet with a lot of stresses, mainly of environmental, nutritional and immunological origin, combined with a more or less pronounced depression of feed intake and mobilization of body reserves. Mal-digestion and mal-absorption often aggravate the situation resulting in digestive upsets due to bacterial overgrowth and/or viral infections. These phenomena greatly interfere with the profitability of the enterprise. These issues have been discussed in a vast body of literatures, e.g., VAN DER PEET (1992); and PARTRIDGE (1993).
Current solutions aim at adapting the feed to the digestive capacity of the piglet by improving the acceptability of the feed through the use of specific ingredients (e.g., milk powder and derivates, such as whey, lactose, dried blood serum, and flavors), with or without an increase of the energy content of the feed. An increase of the energy content can be obtained among others by including easily digestible or metabolizable fats, for example, medium chain triglycerides (MCTG). MCTG in this context have been well documented both in neonatal (ODLE, 1999) and in weaned piglets (CERA et al., 1989), and their utility in this aspect is determined by their specific digestive and metabolic fate (BACH & BABAYAN, 1982).
Digestive upsets are prevented and/or treated by supplementing the feed with antimicrobial substances (e.g., antibiotics or chemotherapeutics, referred to as “antibiotics” hereafter). The effects of the above interventions (prevention or treatment) mostly result in a pronounced improvement of the growth performances (referred to as “growth promotion” hereafter). This growth promotion is mainly due to, depending on the circumstances, an improved feed intake combined with a better feed conversion (i.e., kg feed/kg gain). However, there is a growing concern about the use of antibiotics for growth promotion in animal production systems; in particular, there is a well-considered fear for the risk of the emergence of cross-resistance to some last-resort antibiotics used in human medicine (CORPET, 1996; WEGENER et al., 1998). Therefore, most of those antibiotics (or so called “growth promoters”) are already or will be banned in the near future in the European Union, which justifies an urgent need for alternatives.
Because there is a general belief that digestive pathology in early weaned pig is mainly caused by Gram-negative bacteria (especially E. coli) and that Gram-positive lactic acid bacteria (e.g., Bifidobacteria, Lactobacilli) have a protective and/or antagonistic effect, the currently proposed alternatives are selected for their anti-E. coli activity. Examples of such alternatives include cupper and zinc compounds, selected organic acids such as short chain fatty acids (SCFA) (e.g., formic, acetic and propionic acids), lactic, fumaric, citric, malic and sorbic acids, probiotics (mainly lactic acid bacteria) and/or prebiotics (mainly bifidogenic oligosaccharides, or so called NDO's). Cupper and zinc compounds are effective but are not acceptable because they pollute the environment, and results obtained with probiotics and/or prebiotics are usually unpredictable and generally disappointing (CHESSON, 1994).
Similar problems exist in other animal species as well as in animals of other age groups. Only SCFA and the “classical” organic acids are the most promising alternatives at this time (ROTH et al., 1998). However, they are needed in rather high doses to be effective. That is, the utility of SCFA and “classical” organic acids is limited as there involves a high cost, a corrosive nature and an averse taste which interferes greatly with the feed intake of the animals.
The antimicrobial effects of fatty acids (FA) in general and their salts (e.g., soaps) are already known for decades. A re-evaluation of the antimicrobial effects of selected FA and derivates thereof was given in the review of KABARA (1978), wherein special attention was given to lauric acid (C12, a member of the MCFA family) and derivatives.
Relatively important contribution of MCFA has been documented in the milk-lipid of certain animal species (e.g. rabbit, goat, horse), while low or zero concentration of MCFA in sow's milk renders limited contribution (DIERICK, 1998). In most mammals, there is a more or less pronounced preduodenal (i.e., not of pancreatic origin) lipolytic activity originating from lingual or gastric secretions. The activity of those lipases is independent of the presence of colipase and bile acids, is active and stable in a broad range of pH's, and has a preference for MCFA in milk fat. The preduodenal lipase activity is high in preruminant calves and rabbits, moderate in piglets and absent in poultry (MOREAU et al., 1988). However, an excess of MCFA can have important side-effects, for example, it can be hypnotic in new born pigs (ODLE, 1999), and is a strong stimulus for cholecystokinin (CCK, an intestinal hormone with a pronounced satiating activity which could interfere with the feed intake) (LEPINE et al., 1989). In addition, a lower feed intake could also due to the strong odor (goat-like) and averse taste of free MCFA, although data in this context are scarce and non-conclusive.
The mechanism by which SCFA, MCFA and other organic acids excert antimicrobial activities has been well documented in the literature. The belief is that undissociated or non-ionized acids (RCOOH) are lipid-permeable and can pass across the microbial cell membrane and subsequently dissociate (RCOOH→RCOO−+H+) in the more alkaline interior of the microorganism. This dissociation or ionization reaction results in an acidification of the intracellular pH, which would be below permissible levels for microbes to survive. In other words, organic acids act as protonophores that increase the inward leak of H+ so that efflux is not rapid enough to alkalinize the cytoplasm again. Physicochemical characteristics of the organic acids greatly influence their ability to act as protonophores, for example, molecular weight, pKa (dissociation constant) and solubility. In addition, physiological environment in which the organic acids are present, especially the pH in the different locations of the gastrointestinal tract, is also a very important factor. Furthermore, the type of the microbial envelope, i.e., mainly peptidoglycan in Gram-positive bacteria and lipopolysaccharide in Gram-negative bacteria, greatly influences the passage of the organic acids through the membrane as well.
The specific characteristics of MCTG being a readily available energy source have also been well documented. Their beneficial effects as an energy source are summarized in BACH & BABAYAN (1982): (1) MCTG are digested, absorbed and transported rapidly in disorders where digestion and absorption are not optimal. Such disorders of mal-digestion and mal-absorption are frequently observed in newly weaned piglets, and are attributed to a sharp drop in the activity of most of the digestive enzymes. For example, the deficiency of lipolytic enzymes shortly after weaning is very pronounced; (2) MCTG are oxidized rapidly in the organism and are a source of abundant and rapidly available energy. However, high doses of MCTG are ketogenic and can have narcotic side effects, which are certainly undesirable in piglets.
Furthermore, the depressive effect on the voluntary feed intake, by activation of CCK, is unwanted. Also, the strong unpleasant odor of the free MCFA which evaporates relatively easily is also unwanted either by the producer or the animals.
Thus, there is a long-standing need for a feed supplement for animals, especially for early weaned pigs in order to prevent and/or alleviate the existing problems. The present invention fulfills this need.
The present invention is related to a feed supplement or feed composition and its use for animal feeds, especially for early weaned pigs in order to prevent and/or alleviate existing feeding problems. Such feed supplement or feed composition can be used as part (mostly 1 to 5%) of a complete feed (or often referred to as “feed formula”).
In particular, the present invention is directed to a feed composition comprising about 0.01% to about 20% by weight of triglycerides and about 1 ppm to about 10,000 ppm of exogenous active lipolytic enzyme, wherein the triglycerides contain about 50% to about 100% of C6-C10 medium chain fatty acids (MCFA).
The present invention is also directed to a method for controlling lipolysis in the gastro-intestinal tract of an animal. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.
The present invention is further directed to a method for treating growth impairment of an animal. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.
The present invention is still further directed to a method for treating digestive upsets of an animal. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.
The foregoing and other advantages of the present invention will be apparent to one of ordinary skill in the art, in view of the following detailed description of the preferred embodiment of the present invention, taken in conjunction with the accompanying drawings.
The present invention aims at providing new feed supplements for animal feeds, particularly for early weaned pigs, which can replace the commonly used yet contested antibiotics and other growth enhancers.
The present invention is related to a feed supplement or feed composition that is a premix of feed additives (vitamins, minerals, antibiotics, among others) with a carrier for use as part (mostly 1 to 5%) of a complete feed (or often referred to as “feed formula”). The feed composition comprises a list of different feed ingredients used in the complete feed (or feed formula).
In particular, the present invention provides a feed composition comprising at least one triglyceride (TG) containing medium chain fatty acids (MCFA) (C4-C12, preferably C6-C10 MCFA) combined with at least one exogenous active lipolytic enzyme (e.g., esterase and/or lipase) as well as its use as a feed supplement for animal feeds, especially for early weaned pigs in order to prevent and/or alleviate existing feeding problems at this time. The addition of the combination of TG and exogenous active lipolytic enzymes to an animal feed, which produces a feed formula, surprisingly results in a physiological environment in the stomach which regulates and stabilizes the gastrointestinal microflora. This effect, combined with the fact that an easily digestible and metabolizable source of energy is provided by MCFA, further results in a marked improvement of the growth which is comparable with the growth promotion obtained with commonly used (yet contested) antibiotics and other growth enhancers without negative side effects to the animal, the feed industry and the consumer.
In one embodiment of the present invention, there is provided a feed composition comprising about 0.01% to about 20% by weight of triglycerides and about 1 ppm to about 10,000 ppm of exogenous active lipolytic enzyme, wherein the triglycerides contain about 50% to about 100% of C6-C10 medium chain fatty acids (MCFA).
MCFA according to the present invention include both even and odd fatty acids, such as fatty acids containing C6 (e.g., caproic acid (or hexanoic acid)), C7 (e.g., heptanoic acid), C8 (e.g., caprylic acid (or octanoic acid)), C9 (e.g., pelargonic acid), and/or C10 (e.g., capric acid (or decanoic acid)).
In a preferred embodiment, the triglycerides according to the present invention, also referred as medium chain triglycerides or MCTG, contain about 60% to about 100%, preferably about 60% to about 100%, preferably about 70% to about 100%, more preferably about 80% to about 100%, more preferably about 90% to about 100%, or yet more preferably about 100% of C6-C10 MCFA. MCFA can be caproic acid, heptanoic acid, caprylic acid, pelargonic acid, capric acid, or combination thereof. MCTG can be caproic/caprylic/capric triglycerides, heptanoic/pelargonic triglycerides, pelargonic triglycerides, heptanoic triglycerides, caprylic/capric triglycerides, caproic/caprylic triglycerides, caproic/capric triglycerides, caproic triglycerides, caprylic triglycerides, capric triglycerides, or combination thereof. Preferably, MCTG can be caproic/caprylic/capric triglycerides, caprylic/capric triglycerides, caproic/caprylic triglycerides, caproic/capric triglycerides, caproic triglycerides, caprylic triglycerides, capric triglycerides, or combination thereof.
As used herein, the term “medium chain triglyceride (MCTG)” shall refer to a glyceride in which the glycerol is esterified with three medium chain fatty acids (MCFA). As used herein, the term “caproic triglyceride” or “glyceryl tricaproate” shall refer to a glyceride in which the glycerol is esterified with three caproic acids. As used herein, the term “caprylic triglyceride” or “glyceryl tricaprylate” shall refer to a glyceride in which the glycerol is esterified with three caprylic acids. As used herein, the term “capric triglyceride” or “glyceryl tricaprate” shall refer to a glyceride in which the glycerol is esterified with three capric acids.
As used herein, the term “caproic/caprylic/capric triglyceride” shall encompass a triglyceride wherein the glycerol is esterified with caproic, caprylic and/or capric fatty acids (also termed “glyceryl caproate caprylate caprate”), as well as a combination of glyceryl tricaproate, glyceryl tricaprylate, glyceryl tricaprate, glyceryl caprylate caprate, glyceryl caprylate caproate, glyceryl caproate caprate, and glyceryl caproate caprylate caprate.
As used herein, the term “caprylic/capric triglyceride” shall encompass the triglyceride wherein the glycerol is esterified with caprylic and capric fatty acids (also termed “glyceryl caprylate caprate”, as well as a combination of glyceryl tricaprylate, glyceryl tricaprate, and glycerol caprylate caprate.
As used herein, the term “caproic/caprylic triglyceride” shall encompass the triglyceride wherein the glycerol is esterified with caproic and caprylic fatty acids (also termed “glyceryl caproate caprylate”), as well as a combination of glyceryl tricaprylate, glyceryl tricaproate, and glyceryl caprylate caproate.
As used herein, the term “caproic/capric triglyceride” shall encompass the triglyceride wherein the glycerol is esterified with caproic and capric fatty acids (also termed “glyceryl caproate caprate”), as well as a combination of glyceryl tricaprate, glyceryl tricaproate, and glyceryl caprate caproate.
In a more preferred embodiment, the C6-C10 MCFA are C6 MCFA, C8 MCFA, C10 MCFA, or combination thereof. In this case, MCTG according to the present invention contain about 0% to about 100% of C6 MCFA, about 0% to about 100% of C8 MCFA and about 0% to about 100% of C10 MCFA, wherein the sum of all three fatty acids is about 50% to about 100%, preferably about 60% to about 100%, preferably about 70% to about 100%, more preferably about 80% to about 100%, more preferably about 90% to about 100%, yet more preferably about 100%.
The above concentration of MCTG can range from, for example, 0.0125% to about 20%, from about 0.015% to about 20%, from about 0.02% to about 20%, from about 0.04% to about 18%, from about 0.05% to about 15%, from about 0.08% to about 15%, from about 0.1% to about 15%, from about 0.5% to about 15%, from about 0.8% to about 15%, from about 1% to about 15%, or from about 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% to about 20%.
MCTG according to the present invention may be a naturally occurring triglyceride-containing composition. Examples include but are not limited to coconut oil, palm kernel oil, babassu oil, cohune oil, tacum oil, cuphea oil derived from plant seeds, milk of mammalian species (e.g., milk from horse, rat, goat and rabbit), and butterfat. Alternatively, MCTG may comprise one or more industrially prepared triglycerides or a mixture of naturally occurring and industrially prepared triglycerides. Industrially prepared triglycerides may be produced by esterification, such as trans- or inter-esterification of C4-C12, preferably C6-C10 fatty acids. Examples of commercial sources of chemically synthesized MCTG include but are not limited to those given in Table 10 or those exemplified in the Materials section of the Examples.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic triglycerides, and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caprylic triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic/caprylic triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caprylic/capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic/capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic/caprylic/capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.
In a preferred embodiment, the lipolytic enzyme component is added in a concentration of about 1 ppm to about 10,000 ppm to the feed composition, for example, about 1 ppm to about 50 ppm, about 10 ppm to about 10,000 ppm, about 50 ppm to about 10,000 ppm, about 100 ppm to about 10,000 ppm, about 200 ppm to about 10,000 ppm, about 250 ppm to about 10,000 ppm, about 300 ppm to about 10,000 ppm, about 500 ppm to about 10,000 ppm, about 800 ppm to about 10,000 ppm, about 900 ppm to about 10,000 ppm, or about 1,000 ppm to about 10,000 ppm. Preferably, the lipolytic enzyme is added to the feed composition in a concentration of about 10-80 mg/g of MCTG. For example, the exogenous active lipolytic enzymes are added in a concentration of about 10 mg/g, 15 mg/g, 20 mg/g, 25 mg/g, 30 mg/g, 35 mg/g, 40 mg/g, 45 mg/g, 50 mg/g, 60 mg/g, 70 mg/g or 80 mg/g of MCTG.
The lipolytic enzyme component according to the present invention may comprise a lipase or an esterase, a mixture of lipases and/or esterases. Such lipases or esterases may be naturally occurring or industrially prepared, and may be from a microbial, mammalian or plant origin.
Examples of commercially available plant lipases include but are not limited to lipases from wheat, castor bean, rape, mustard and lupin. Examples of commercially available microbial lipases include but are not limited to the lipases as given in Table 11 or those exemplified in the Materials section of the Examples.
Examples of commercially available esterases include but are not limited to pregastric esterase (PGE) from sublingual tissue of calf, kid and lamb, rennet paste from engorged abomasa of calf, kid and lamb, esterase from rabbit liver or porcine liver.
In still a preferred embodiment, the feed composition further comprises an animal feed comprising carbohydrate, protein, saccharides and a vitamin-mineral premix, for example, an animal feed comprising starch, dextrose, casein and a vitamin-mineral premix. In still another preferred embodiment, the feed composition further comprising an animal feed comprising maize, barley, wheat, dried acid whey, cassaya, herring meal, soybean, soy-flour and a vitamin-mineral premix.
A feed formula comprising 1 to 5% of the feed composition of the present invention is also provided.
The feed composition or feed formula according to the present invention is useful for feeding animals, particularly pigs, cattle, horse, sheep, rabbits and poultry, and more particularly early weaned animals. It is also particularly useful as a growth promoter, an antimicrobial agent as well as for preventing digestive upsets.
Although the feed composition or feed formula is particularly useful for early weaned animals such as piglets, it is not excluded from being used as a feed supplement for pigs of other age categories or as a feed supplement or feed formula for other species of animals.
In another embodiment of the present invention, there is provided a method for preparing a feed composition or feed formula by mixing together different MCTG and lipolytic enzyme components according to the present invention.
In still another embodiment of the present invention, there is provided a method for controlling lipolysis in the gastro-intestinal tract of an animal, e.g., an early weaned piglet. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.
In yet another embodiment of the present invention, there is provided a method for treating growth impairment of an animal, e.g., an early weaned piglet. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.
In still yet another embodiment of the present invention, there is provided a method for treating digestive upsets of an animal, e.g., an early weaned piglet. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.
In the present study, a broad range of organic acids (SCFA, MCFA and other commonly used organic acids in the feed and food industry) were first tested in vitro for their antibacterial activity against the dominant bacteria of the small intestinal microflora. Unexpectedly, SCFA and the commonly used organic acids were found to be bacteriostatic only at higher concentrations (0.02 to 0.04 M) for the Gram-negative flora (and to a lesser extent for Streptococci). However, MCFA were shown to have high bacteriostatic and bactericidal activity against both Gram-positive and Gram-negative bacteria. The antibacterial activity of MCFA was inversely related to pH, wherein MCFA had a higher antibacterial activity at a lower pH. For example, a relatively high proportion of MCFA was in the undissociated form.
In the same in vitro study, a temptative minimal bactericidal concentration of 0.005 to 0.01 M was tested. Also unexpectedly, the antibacterial spectrum of the antibiotic growth promoters used in the intensive animal production could be duplicated completely when a combination of MCFA was used.
In order to maximize the positive effects and minimize the negative characteristics of MCFA, a combination of a MCFA-containing triglyceride and a lipolytic enzyme is used as a feed supplement in the present invention, with the intention that sufficient MCFA should be released in the stomach to have a sterilizing effect, which would result in a lesser bacterial load in the intestinal tract thereby preventing digestive upsets. This sterilizing effect, combined with the characteristics of MCFA being a readily available energy source and the supplementation of the natural lipase activity in the stomach and upper intestine by the exogenous active lipolytic enzyme(s), was shown to have unexpectedly resulted in a growth promotion without the use of antibiotics. It appears that the gradual release and absorption of the free MCFA unexpectedly avoided the unwanted side effects of MCFA.
The following examples and drawings merely serve to illustrate the present invention and are not meant to be limiting in any manner.
Two naturally occurring substances which are rich in medium chain fatty acid-containing triglycerides, i.e., butterfat and coconut oil, and two commercially available sources of MCTG, i.e., MCTG1 (Aldo MCT Kosher Food Grade, commercialized by LONZA Inc. (Fair Lawn, N.J. 070410, USA)) and MCTG2 (Stabilox-860, commercialized by LODERS-CROKLAAN BV (NL-1521 AZ Wormerveer)) were used in the present study. Also, lipolytic enzymes L1 (Lipozyme 10.000L, NOVO Nordisk A/S, 2880 Bagsvaerd, Denmark); L2 (Lipase 10.000P, Biocatalysts Ltd., CF37 5UT Pontypridd, Wales, UK); L3 (TP 516P, Biocatalysts Ltd., CF37 5UT Pontypridd, Wales, UK); L4 (LIPOMOD 224P, Biocatalysts Ltd., CF37 5UT Pontypridd, Wales, UK); L5 (Lipase SAIKEN, NAGASE & Co, Chuo-ku, 103 Tokyo, Japan); and L6 (Lipase ITALASE C, SBI, Systems Bio-Industries, Inc., WI 53187-1609 Waukesha, USA) were used in the present study.
The selection of above triglycerides and lipolytic enzymes does not exclude potential usefulness of other triglycerides and lipolytic enzymes and combinations thereof for the purposes of the present invention.
A lipid extraction procedure was used, wherein hexane/iso-propanol (3/2, v/v) was used to avoid any solvent evaporation thereby preventing any loss of MCFA due to their great volatility.
Next, H2SO4 was used to catalyze esterification of MCFA in the same extraction medium, wherein isopropyl esters (FAIPE) was formed without loss of shorter esters or alteration of polyunsaturated higher MCFA. FAIPE appeared in the upper hexane phase.
For the calculation of the concentration, quantitative capillary column (DB-225, 30 m, ID 0.25 mm, Film 0.25 μm) GLC chromatography of individual FAIPE was conducted, wherein two internal standards were used, i.e., C9 for C4-C12 and C17 for C14 to C18:3. Coefficients of variation on the response factors amounted to 0.94% for C9 and 2.51% for C17.
Individual free fatty acids were extracted using the above-described lipid extraction procedure with a strong anion exchange resin Amberlyst 26 before esterification in the same medium, and then analyzed by capillary GLC mean recovery of added free fatty acids amounted to 101.9%.
Lipolytic enzymes L1-L6 were selected based on their commercial availability and cost. MCTG were selected based on their specific MCFA content in the fat as specified in Table 1 below.
The in vitro incubations were done for 180 min at 37° C. in a shaking water bath in buffered circumstances at different pH's: a glycine buffer was used for incubations at pH 2 and pH 3; and an acetate buffer was used for incubations at pH 4 and pH 5. The parameters used for the incubations were chosen in order to simulate as closely as possible the in vivo conditions in gastric contents. The medium used for the incubations was made up of the following ingredients: 0.250 g of the selected TG+2.250 g of a synthetic feed (based on starch, dextrose, casein and a vitamin-mineral premix)+10 ml buffer solution+0.5 ml pepsine solution (50 mg in 100 ml aqua dest)+10,000 mg/kg fat (i.e., 10,000 ppm) of the selected commercial lipolytic enzyme preparation. The fat could be molten when needed; otherwise there were no special preparations (e.g., dispersion or emulgation) of the fat.
The results of the incubations are demonstrated in
Three female pigs (Belgian Landrace and stress free) with an initial weight of about 8.5 kg were prepared with a gastric cannula using the technique of DECUYPERE et al. (1977). The cannulae were placed midway the curvatura major in the fundic region.
Three TG (coconut oil, MCTG1 and butterfat) and two lipases (L2 and L5) were selected for this experiment. Nine feeds were prepared using 95% of a commercial feed for piglets and 5% of the selected (eventually molten) TG with or with supplementation of the selected lipases (see Table 2 for the V codes used hereafter). The fats were simply poured on the meal and thoroughly mixed in a horizontal mixer. The concentration of the lipases was 1,000 ppm of the commercial preparation in the feed.
The composition of the piglet feed was based on maize, barley, dried acid whey, cassaye, herringmeal, and soybean oil, and was supplemented with a vitamin-mineral premix. The feed contained no growth promoting supplements. The proximate analysis of the feeds (V1, V4 and V7) in % of as given was: DM: 90.6, 90.7 and 90.8; total ash: 7.8, 7.9 and 8.5; crude protein: 15.1, 15.4 and 14.8; crude fat: 8.5, 8.3 and 8.3.
The feed was given dry in three equal meals (9, 13 and 17 h) at 85% of the ad libitum intake of pigs with a comparable weight. The experiment had a 3×3 Latin square design. There were no health problems or feed refusals. Statistics were done using ANOVA (1997), differences were at p<0.01 to p<0.05 (**) or p<0.1 (*).
Sampling of the gastric contents for the chemical analysis was done on 2 consecutive days, 2 times a day, and 30 min after the 9 h and 13 h meal. The pH was measured directly; thereafter the samples were stored at −20° C. till further analysis.
The sampling of the gastric contents for the bacteriological analysis was done during 1 day, and 90 min after the 9 and 13 h meal. The bacterial counts were done using the technique of VAN DER HEYDE et al. (1964). The media (all from OXOID, UK) used were RCM agar+hemin for the total count (48 h, anaerobic), Rogosa agar for the Lactobacilli (48 h, anaerobic), Slanetz & Bartley agar for the fecal Streptococci (24 h, aerobic), and EMB agar for E. coli (24 h, aerobic). All incubations were performed at 37° C. Results are expressed as log10 colony forming units (CFU)/g fresh contents.
It is shown that the pH of the stomach contents measured 30 and 90 min after feeding did not differ between the treatments (feeds) and ranged between 4.2 and 5.01. This is within the optimum range for the lipolytic activity of L2 and L5 as was found in the above experiment described in Example 1. The results of the bacteriological counts are presented in Table 3 and in
E. coli
The data show the following: (1) with coconut oil, both L2 and L5 reduced 10-fold of the total count and lactobacilli count; (2) with MCTG1, both enzymes had a very pronounced (mostly p<0.001) effect and reduced the total count and individual lactobacilli count by a factor of 100 to 1,000, and reduced individual streptococci and E. coli counts to non detectable levels; and (3) with butterfat, there was a 10-fold reduction of the total count and individual streptococci count.
It is concluded that the combination of a MCTG and a lipolytic enzyme in the feed is able to suppress the total bacterial count and individual counts of the dominant flora. This effect most likely is due to the release of free MCFA from the triglycerides used, which was confirmed by the chemical analysis of the different fat fractions in the gastric contents collected during the present study. The results of the chemical analysis are given in
There was no preferential release of specific FA (results not shown), that is, the release of individual FA is roughly proportional to their content in the triglycerides used. Results presented in
It is striking and unexpected that the release of MCFA runs parallel with the degree of suppression of the bacterial load in the stomach with the most efficient suppression observed with the combination MCTG1+L5, which caused 60.9% hydrolysis of the triglycerides in the stomach (corresponding with a concentration of about 1% of FFA and 0.6% of MCFA), followed by coconut oil+L5 (0.8% FFA and 0.3% MCFA) and butterfat+L5 (0.8% FFA and 0.06% MCFA).
This experiment was designed to check if the above-mentioned concept was applicable and suitable in commercial settings and if obtained, whether a growth promotion was comparable with the growth promotion obtained in early weaned piglets with antibiotics or a combination of commonly used organic acids with proven effectiveness.
244 freshly weaned piglets (Seghers Hybrid F1, initial weight about 6.5 kg) were divided according to litter, sex and weight in 4 groups: A: 68; B: 61; C: 60 and D: 55 piglets. The experiment was run in commercial settings in temperature controlled facilities.
The feed composition used was based on barley, wheat, maize flakes, extruded maize, extruded soybeans, soy-flour, herring meal, 2.5% TG, and a commercial premix (mainly based on milk products, vitamins+minerals) for early weaned piglets (12.5%). The treatments (A to D) differed in TG and the additives used (see Table 5). The feeds contained no growth promoting antibiotics. Feed A was a negative control, feed D a positive control containing a mix of commonly used organic acids. The calculated proximate analysis of the feeds used was equalized. The formulated contents were (% fresh): DM: 90.0 to 88.8, crude protein: 18.7 to 18.9, crude fat 6.9, total ash: 5.1-5.3. The energy content was (Nef97): 2463-2475 kcal/kg, the ileal digestible amino acids were set at: Lys: 1.07%, Met+Cys: 0.65, Thr: 0.66, Try 0.19.
The feed was prepared by a commercial feed company which used a spray-equipment for fats and other liquid supplements. The feed was offered dry, ad libitum; water was continuously available via a nipple.
The experiment lasted 3 weeks. The piglets were weighed individually at the start of the experiment and weekly thereafter; feed intake was recorded daily per two pens (joint feed hopper for two pens with about 15 piglets each). Therefore statistics only could be done for the weights. The visual health condition of the pigs per pen was checked daily and coded on a scale from 0 (extremely bad) to 10 (excellent). The zootechnical results on a weekly basis are presented in Table 6.
The visual health score ranged between 4 and 9 on treatment A; while for the other treatments, the range was 8 to 9 without marked differences.
The daily growth did not differ between treatments A and D as well as between B and C. The most pronounced differences were obtained in the first two weeks after weaning during which the best growth performance (with about 30% increase over the control) was obtained with treatments B and C. The better results obtained with the feeds B and C (MCTG2 without or with lipase) are due to an increase of the feed intake. The best feed conversion, however, was obtained with the feed containing MCTG2+lipase. The improvement of the growth using a combination of a MCTG (MCTG2) and a lipase was in the same range as obtained with quinoxalines (additives with both a Gram-positive and Gram-negative spectrum).
Two weeks after weaning, 5 barrows of each experimental group were euthanized. Because the pigs were fed ad libitum, there was no control of the feed intake. Upon dissecting the gastrointestinal tract, samples were taken from the stomach and the upper (duodenum) small intestine, and then analyzed chemically and bacteriologically in the same way as explained in the above experiments. Only the total anaerobic count was reported below.
The pH of the gastric contents was about 3.5 and about 5.7 in the duodenum; there were no differences among the treatments. The total anaerobic counts are reported in Table 7.
The results suggest that the feed containing the combination of MCTG2 and L5 caused a significant 10-fold suppression of the bacteriological load, both in the stomach and upper intestine. It is noted that this effect was somewhat lower than in the previous experiment with the gastric cannulated pigs, which could be due to the lower amount of MCTG used in the present experiment (2.5% used herein vs. 5% used previously) and/or the different feeding and sampling procedures. Nevertheless, the present experiment confirmed the results obtained in the cannulated pig reported in above Example 3. The same can be stated for the results of the analysis of the different fat fractions (g/100 g of fresh contents) and the degree of hydrolysis (g FFA/100 g of total FA) in the gastric contents, which are given in Table 8.
It can be calculated from the hydrolysis results that for Feed B (MCTG2) and Feed C (MCTG2+L5), 0.3 and 0.4% free MCFA were present in the stomach, respectively. In the previous experiment with the cannulated pigs, the highest concentrations of free MCFA (and the strongest inhibition of the bacterial load, i.e., about 100 fold) were obtained with MCTG1+L5 and coconut oil+L5, which were 0.60 and 0.30%, respectively.
The combined results of Experiments 2 and 3 clearly suggest that there is a correlation between the amount of released free MCFA and the inhibitory effect on the gastric flora.
As growth promotion is related and proportional to the inhibition of the total bacterial load in the small intestine, this in vitro experiment was designed to evaluate an optimal combination of different concentrations of MCTG with different doses of lipolytic enzymes, wherein a combination of MCTG1, MCTG2 or coconut oil and a proven effective lipase (L5) was used.
In detail, four concentrations of MCTG were used: 0, 2.5, 5 and 10%; for each of these concentrations, the lipase (L5) was incorporated at 10,000, 1,000 or 100 ppm. The medium contained also 2.5 g per incubation flask of the same synthetic feed (based on starch, dextrose, casein and a vitamin-mineral premix) as used in Experiment 1. However, in the present experiment, the triglyceride was dispersed using gum arabic and gum tragacanth before adding to the medium. The incubations were done at pH 5 using an appropriate acetate buffer. Finally the medium (15 ml) was inoculated with 1 ml of a suspension of bacteria originating from the ileal contents of two cannulated pigs fed a diet without growth promoting additives. Incubations were done for 180 min at 37° C. in a shaking water bath and were done in duplicates.
The methods for the analysis of fats and the bacterial counts were the same as used in the previous experiments with the exception that only the total anaerobic count was reported. As a relationship between the antibacterial activity and the molecular weight of the fatty acid was expected, the results for the free fatty acids were also expressed on a molar basis. The results are given in Table 9.
The above data demonstrate the following results: (1) the amount of released fatty acid is nearly proportional to the concentration of the triglyceride, while a 10-fold dose increase of the lipolytic enzyme used only doubled the concentration of the free fatty acid. For each combination of a specific % of triglyceride and a given ppm of lipolytic enzyme, the release of the fatty acid follows the order: MCTG1>MCTG2>coconut oil; (2) the higher the concentration of the free fatty acid, the more pronounced the suppression of the number of bacteria. A minimal concentration of about 0.35% of fatty acid in the medium appears necessary for a significant suppression of the flora, which concentration corresponds to 0.025 moles/liter. The order of MCTG1>MCTG2>coconut oil corresponds with an increase of the molecular weight of the quantitatively most important MCFA in the triglycerides: MCTG1=C8, MCTG2=C10, coconut oil=C12; and (3) the used in vitro protocol offers an excellent tool for the screening of the numerous combinations of MCTG and available lipolytic enzymes for their usefulness as feed supplements with a stabilizing or suppressive effect on the gastrointestinal microflora. This effect is generally accepted as the basis for obtaining a growth promotion.
(1) Source: tested products + literature compilation
Candida sp.
Candida rugosa*
Candida antartica A/B
Candida lipolytica
Candida paralipolytica
Saccharomyces lipolytica
Thermomyces lanuginosus**
Rhizomucor Miehei
Rhizopus sp.
Rhizopus delemar
Rhizopus oryzae
Rhizopus niveus
Rhizopus arrhizus
Rhizopus javanicus
Aspergillus sp.
Aspergillus niger
Aspergillus usamii
Aspergillus oryzae
Mucor sp.
Mucor javanicus
Mucor lipolyticus
Penicillium sp.
Penicillium roquefortii
Penicillium cyclopium
Penicillium simplissimum
Penicillium camembertii
Geotrichum candidum
Neurospora crassa
Ustilago maydis
Fusarium solani
Burkholderia cepacia***
Pseudomonas sp.
Pseudomonas alcaligenes
Pseudomonas mendocina
Pseudomonas fluorescens
Pseudomonas aeroginosa
Pseudomonas spp.
Chromobacterium viscosum****
Staphylococcus sp.
Staphylococcus aureus
Staphylococcus carnosus
Staphylococcus hyicus
Achromobacter lipolyticum
Acinetobacter
Propionibacterium acnes
Bacillus sp.
(1) Source: tested products + literature compilation
Salmonella Thypimurium SL 3144 cultures grown in a LB-medium overnight were brought to equal densities (5×108 CFU/ml) and diluted 1.000-fold in 300 μl fresh medium. Bacteria were grown in a 100-well bioscreenplate at 37° C., and the optical density at 600 nm (OD600) was measured automatically every 30 min during 6.5 hours in a BioscreenC apparatus. For each time point, the average optical density was calculated from three independent measurements. By plotting the measured optical density versus time, microbial growth curves were generated for the given conditions (pH, MCTG type and concentrations, as well as lipase concentrations). Each point in the growth curve was an average value of the three measurements.
The incubation medium used was LB or M9. The incubation was performed at a temperature correlating well with the animal temperature (37° C.-40° C. for pigs or poultry), and the incubation pH also corresponded to specific pH zones prevailing in the intestinal tract (ranging from 2.5 to 7.0). Test results shown herein were obtained at pH 4.5, 5.2 or 7.0.
Lipase dosage used in the present study ranged from 10 to 80 mg of lipase/g of MCTG. Lipase activity was of 200,000 LU/g lipase. The lipase activity was defined as “Lipase activity Unit” (LU) wherein 1 LU is the amount of enzyme which liberates 1 μmol of fatty acid per minute from olive oil after incubation for 30 minutes at 40° C. and at pH 7.0.
MCTG used was Aveve MCT 8/10 and Aveve MCT 6/8, the compositions of which are shown in Table 12. The concentration of MCTG in the incubation medium ranged from 0.01 to 0.04%.
The first experiment was performed at pH 4.5 using Aveve MCT 8/10. The concentrations of MCTG and lipolytic enzyme in the test compositions are shown in Table 13. The control (composition H) used in this experiment consisted of incubation medium alone at pH 4.5.
The results are shown in
The second experiment was performed at pH 7 using MCT 8/10. The concentrations of MCTG and lipolytic enzyme in the test compositions are shown in Table 14. The control (composition T) used in this experiment consisted of incubation medium alone at pH 7.
The results are shown in
The third experiment was performed at pH 4.5 using Aveve MCT 6/8. The concentrations of MCTG and lipolytic enzyme in the test compositions are shown in Table 15. The control (composition Y) used in this experiment consisted of incubation medium alone at pH 4.5.
The results are shown in
The fourth experiment was performed at pH 5.2 using Aveve MCT 6/8. The concentration of MCTG and lipolytic enzyme in the test compositions are shown in Table 16. The control (composition Y1) used in this experiment consisted of incubation medium alone at pH 5.2.
All MCTG/lipase incubations with different MCTG types and different concentrations of MCTG and lipase showed inhibition of the growth curve of the bacterium tested. The data shows that the inhibition of bacterial growth is detectable starting from a concentration of 0.01% MCTG and 10 mg lipase/g of MCTG. Growth curve reduction in the time frame of 0-6.5 h correspond to a normal digestion time in pigs and is longer than the digestion time in poultry.
The results presented herein clearly show reduced growth of the bacterium tested at different pH ranges which was dependent of the concentration of MCTG in the incubation medium. Activity of the lipase at a concentration of 10-80 mg/g of MCTG induced a reduction of the microbial growth. The above lipase concentration in the incubation medium with 0.01%-0.04% MCTG corresponds to 1 ppm to 32 ppm of lipase in the incubation medium.
The antimicrobial activity demonstrated in the present study is consistent with the in vivo bacterial reduction measurement, which is from 30% to 100% reduction of bacterial counts depending on the bacterial group tested.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 99870120.5 | Jun 1999 | EP | regional |
This application is a continuation-in-part application of U.S. patent application Ser. No. 10/009,235, filed Jun. 11, 2002, which is a 35 U.S.C. §371 national phase application of PCT/EP00/05192, filed Jun. 6, 2000, which claims the benefit of European patent application No. 99870120.5, filed Jun. 7, 1999, the entire contents of which are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10009235 | Jun 2002 | US |
| Child | 11895691 | US |
