CN104161165A - Feed for monogastric animals - Google Patents

Abstract

The present invention relates to improved feed additive or food supplement formulations obtained from more than one strain of lactic acid bacteria. The feed additive or food supplement is used for raising monogastric animals such as poultry and poultry. In addition, the feed can provide higher growth rate and feed utilization efficiency for monogastric animals, and the animal feed can also be used to control the food intake of the animals.

Description

monogastric animal feed

This application is a divisional application of the Chinese invention patent application with Chinese application number 2009801475715 (international application number: PCT/MY2009/000050) and application date: April 9, 2009.

technical field

The invention relates to a microbial feed additive, which is used in feeds for monogastric animals such as poultry, pigs and turkeys, in particular to an animal feed produced by microorganisms (preferably lactic acid bacteria).

Background technique

In recent years, it has been proposed to use various feed additives to achieve rapid growth of livestock. In this regard, antibiotic additives stand out the most. Growth-promoting antibiotics are the most common of these feed additives, primarily to have a positive effect on animal growth and feed conversion, and to reduce the incidence of certain diseases. However, the widespread use of antibiotics may lead to the development of resistance in animals to some pathogenic species (Mikkelsen and Jensen, 2000). Relatively good results have been obtained with these additives. Recent investigations have shown that such additives can lead to the development of resistant strains in livestock and the transfer of antibiotic substances to humans in small quantities. The study also found that people exposed to the feed may be at risk for allergic reactions. In modern animal husbandry, various methods have been explored to improve the health and growth of animals. These methods include strengthening livestock management, enhancing nutrition and improving the utilization rate of feed additives. Commonly used feed additives are antibiotics, probiotics, enzymes and organic acids (Bernardeau et al., 2002). Likewise, cross-resistance may develop between therapeutic antibiotics belonging to the same drug class, especially those closely related to human antimicrobial therapy. In recent years, various feed additives have been proposed to achieve rapid growth of livestock, and in this regard, antibiotic additives have attracted the most attention. It has now been found that, with the feed additive according to the invention, the above-mentioned disadvantages can be eliminated and at least the same effect can be obtained in livestock production as with an antibiotic feed additive. Therefore, the present invention can obtain ideal effects while eliminating original defects.

Some countries have moved to restrict or ban the use of antibiotics as growth promoters, which has drawn attention to possible alternatives (Wierup, 2000). In the past few years, research has mainly focused on some valuable strains of lactic acid bacteria (LAB) and their potential use as probiotics. Probiotics are considered available microbial agents that can promote the health of mammals by maintaining the natural microflora in the gut. Probiotics attach to the intestinal mucosa and multiply in the intestinal tract, thereby preventing harmful microorganisms from attaching to the intestinal tract. A prerequisite for probiotics to work is that they reach the intestinal mucosa in a suitable and feasible manner and that they are not destroyed by the low pH in the stomach. In particular, the physiology of the digestive tract of cats and dogs differs from that of the human digestive tract. For example, the average pH in the stomach of dogs and cats is 3.4 and 4.2, respectively. Lactic acid bacteria, which are probiotics, are often used as an additive instead of antibiotics. Lactic acid bacteria are widely used as starter cultures for meat and meat products, and play an important role in ensuring the safety of different food products through the production of metabolites such as bacteriocins. Bacteriocins are protein compounds that have antimicrobial properties and are able to inhibit many different bacterial species, especially pathogenic bacteria (De Vuyst and Vandamme, 1994). These compounds have attracted much attention because they are produced by bacteria that are beneficial to human health and are often used as natural food preservatives. Administration of bacteriocins has been shown to affect the bacterial ecology of the gastrointestinal tract and reduce the levels of pathogenic bacteria in different parts of the gastrointestinal tract (van Winsen et al., 2001). Gaenzale et al. confirmed in 1999 that the bacteriocin curvacin produced by Lactobacillus flexus that inhibits Escherichia coli and Listeria can be inoculated in the stomach, and at the same time confirmed that the bacteriocin produced by Lactococcus subbacteria has antibacterial properties (Mishra and Lambert, 1996). US Patent No. 5,968,569 discloses the inclusion of probiotic microorganisms for use in pet food grains, but neither this patent nor other prior art provides information on strains specifically for pet health. Therefore, there is a need for novel bacterial strains that are particularly suitable for pets and are selected for inclusion in pet food ingredients due to their high prebiotic properties beneficial to animal health.

It was previously known that skim milk, buttermilk and whey can be used as animal feed with the help of lactic acid bacteria. The product also contains lactic acid, vitamins, sugar and other carbohydrates, but does not contain viable microorganisms. The present invention relates to a growth-promoting feed additive comprising metabolites of natural origin produced by Lactobacillus sp. The present invention also relates to the use of metabolites obtained from Lactobacillus in an effective amount, and provides a feed additive Optimal dosage of the most potent metabolites to improve growth performance and overall bird health. The animal feed additive is combined with nutrients to obtain an animal feed. Scientifically controlled experiments on animals have shown that the economic benefits obtained by feeding animals with the animal feed mixed with the additive of the present invention are also improved due to the improved quality and value of animal products.

Contents of the invention

A preferred embodiment of the present invention relates to a biologically purified culture of a probiotic strain, wherein the strain is selected from lactic acid bacteria comprising the group Lactobacillus plantarum (preferably RI11, RG14, RS5 and RG11 strains). Lactic acid bacteria are said to be capable of producing metabolites containing bacteriocins. The present invention discloses a metabolite that can be added to animal feed as an additive and/or supplement. The animal feed comprises metabolites in an effective amount of 0.1% to 0.5% by dry weight thereof.

Thus, the animal feed comprises nutrients, bacteriocins, vitamins (preferably B vitamins), organic acids (preferably formic, acetic and lactic acids) and/or combinations thereof. In addition, the animal feed comprises a combination of bacteriocin and organic acid, or a combination of bacteriocin, B vitamins and organic acid. The proportion of bacteriocin used in the animal-specific feed is preferably between 0.05% and 0.8%.

Moreover, the present invention relates to an animal feed formulation comprising corn, meal, rice bran, wheat bran, syrup, palm oil, coconut oil crude oil, limestone, dicalcium monophosphate, calcium hydrogen phosphate, salt, lysine, Choline Chloride, Vitamins, Minerals, Threonine, Anti-Mold Compounds, Copper Sulfate, DL-Methionine and Antioxidants.

Preferably, the corn is yellow corn; the meal includes soybean meal and coconut meal; and the palm oil used includes refined palm oil and crude palm oil. Additionally, the formula includes ingredients selected from yellow corn, soybean meal, coconut meal, rice bran, molasses, crude coconut oil, limestone, salt, lysine, choline chloride, vitamins, minerals, threonine, anti-mold compounds, Any combination of copper sulfate, DL-methionine and antioxidants.

In particular, the animal feed formulation further includes metabolites such as bacteriocins in combination with B vitamins and organic acids. It is recommended that the metabolites be added in an amount of 0.5-8 kg in the total animal feed weight.

More particularly, the animal feed formulation provides a means of increasing overall feed intake by 3% to 10%, the formulation increasing animal growth rate by 5% to 7%. Accordingly, the animal feed formula can increase the feed conversion ratio by 3% to 8%.

A preferred embodiment of the present invention relates to an animal feed formula, which has the ability to reduce the number of fecal enterobacteria and increase the number of fecal lactic acid bacteria in monogastric animals or ruminants. Correspondingly, the formulation also has the ability to reduce plasma and meat cholesterol in monogastric animals or ruminants, and increase the height of small intestinal villi in their bodies.

Accordingly, the present invention relates to a process for promoting growth and/or increasing feed conversion in a monogastric animal or ruminant, wherein the process comprises feeding the animal an effective amount of bacteria mixed with B vitamins and organic acids white.

Another embodiment of the present invention relates to an animal feed comprising metabolites produced by Lactobacillus plantarum, preferably RI11, RG14, RS5 and RG11 strains.

Meanwhile, another embodiment of the present invention relates to the use of feed for monogastric animals, in particular birds, rabbits, minks, chinchillas, dogs, rodents, pigs, calves or lambs.

Description of drawings

The drawings constitute a part of this specification and include exemplary or preferred embodiments of the invention, which can be embodied in various forms. It should be understood, however, that the disclosed preferred embodiments are exemplary of the invention only. Therefore, the drawings disclosed below are not used to limit the claims, but only serve as the basis of the claims to guide those skilled in the art.

Accompanying drawing 1 shows strains RG14, RG11, RI11 and RS5 and GenBank accession number D9210, EF536363, NC004567 and AB11208 four available sequences 16S rDNA partial sequence alignment.

Accompanying drawing 2 is the enlarged view of the bacteriocin structural gene selected from Lactobacillus plantarum strains. A) Enlarged view of the gene phytolactin EF (-450 base pairs); B) Enlarged view of the gene phytolactin W (-200 base pairs).

Detailed ways

A detailed description of preferred embodiments of the invention follows. It is to be understood, however, that the disclosed preferred embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, the following disclosures are not intended to limit the claims, but merely serve as a basis for the claims to guide those skilled in the art.

A preferred embodiment of the present invention provides a method of improving animal husbandry, particularly increasing the meat production of livestock, such as poultry. Methods that provide feed strategies can improve growth performance, carcass composition, appearance and overall animal health. For example, the methods of the invention have an improved effect on any one or combination of the following: animal product appearance (including growth performance, fecal microflora, volatile fatty acids, and intestinal villi height), plasma cholesterol, and dietary nutrient utilization.

The object of the present invention also relates to the role of metabolites produced by Lactobacillus in broiler chicken growth performance, fecal microbial count, intestinal villi height and fecal volatile fatty acids (VFA).

Another object of the present invention is to provide a means for identifying the optimal composition of Lactobacillus metabolites as feed additives or supplements for monogastric animals. Correspondingly, the metabolite composition should preferably be used in optimal amounts in animal feed additives or supplements in monogastric animal feed.

The present invention also provides a means of conferring probiotic properties to metabolites that can improve the overall health of animals, which is another object of the present invention. Animal feed additives help animals further digest food. Accordingly, the animal feed has the advantages of reducing the odor of animal manure and providing a high-quality manure for farmland.

best practice

For a given range of values, unless expressly stated otherwise in the invention, to the tenth of the unit of the lower limit, each intermediate value between the upper and lower limits of the range, and other stated values within the range Or intermediate values, should be covered within the present invention. The upper and lower limits of those narrow ranges may individually be included in the narrow range and are also included in the invention, subject to the upper and lower limits specified herein being excluded. Where the stated range includes either or both of the limits, ranges excluding either or both of the limits are still encompassed in the invention.

Unless otherwise specified, technical and scientific terms mentioned in the present invention refer to the meanings commonly known to those skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those of the present invention can be used in the practice or testing, the preferred methods and materials are now described. All publications mentioned herein should be cited to disclose and describe the methods and/or materials in connection with which the publications are cited. It must be noted that when the singular forms a, an, the are used herein or in the appended claims, these singular forms all refer to plural meanings unless the invention clearly states otherwise.

Monogastric animals such as pigs, poultry, calves, and fish are bred in large numbers to produce meat, fish, and eggs, and these animals are fed feedstuffs containing a variety of animal and/or plant materials to provide humans with energy and protein. Most of the feed used is produced commercially, but a significant portion is also produced on farms and fed directly to the animals. These feeds are usually supplemented with vitamins and minerals to meet the nutritional needs of the animals.

The present invention provides novel metabolites for use as poultry feed additives or growth promoters, which have probiotic properties, in particular an animal feed additive of natural origin comprising an effective amount of Lactobacillus metabolites. Furthermore, the invention relates to a biologically purified culture of Lactobacillus plantarum RS5, R11, RG14 and RG11 (Comb3456) or a mutant of this biologically purified culture obtained from the microorganism. The microorganism is deposited at the National Center for Genetic Engineering and Biotechnology Culture Collection (BCC), the central laboratory of the Center for Genetic Engineering and Biotechnology in Thailand (a member of the National Science and Technology Development Agency), and can also be obtained from the University of Putra, Malaysia. Acquired from the Department of Bioprocessing Technology, Institute of Processing Technology. Preferably, biologically purified cultures of Lactobacillus RS5, R11, RG14 and RG11 or mutants thereof can be used to produce effective amounts of metabolites for use in animal feed.

Preferably, the animal feed additive comprises at least about 0.2% (w/v) of the metabolite composition by dry weight thereof. The animal feed additive may be combined with nutrients to form an animal feed, which is another aspect of the invention. The invention further includes methods of feeding animals using the animal feed. The inventive subject matter provides a method of improving the health of poultry. In particular embodiments, the present invention provides methods for accelerating and/or increasing growth, improving the immunity and general health of poultry. To achieve the above objects, the present invention provides raw materials and methods for using lactobacillus metabolites for poultry. The Lactobacillus metabolites were isolated from Malaysian food.

The performance of broiler chicks was studied with four metabolite compositions produced by Lactobacillus plantarum. 432 male broiler chickens were reared from 1 to 42 days of age in thick-bedded chicken houses (12 birds per pen). The chickens were divided into 6 groups and fed different diets: (i) standard corn, soybean based diet (negative control group); (ii) standard corn, soybean based diet plus neomycin and oxytetracycline (positive control group). group); (three) standard corn, soybean basal feed plus 0.3% metabolite composition (com3456) of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains; (four) standard corn, soybean basal feed plus 0.3% The metabolite composition (Com246) of Lactobacillus plantarum TL1, RG11 and RG11 bacterial strain; (5) standard corn, soybean basal feed plus the metabolite composition (Com256) of 0.3% Lactobacillus plantarum TL1, RG14 and RG11 bacterial strain (6) Standard corn, soybean basal feed plus 0.3% metabolite composition (Com2356) of Lactobacillus plantarum TL1, RS5, RG14 and RG11 strains. The study found that the final body weight, weight gain and average daily weight gain of the four treatment groups were significantly increased, and the feed conversion rate was significantly reduced (P<0.05). Metabolite composition supplementation increased fecal lactic acid bacteria counts, small intestinal villi height, and fecal volatile fatty acids, while reducing cholesterol and fecal Enterobacteriaceae counts. The effect of different metabolite compositions of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains (Com3456) on broiler chicks was also studied. 504 male roosters were divided into 7 treatment groups and fed with different feeds: (1) standard corn, soybean based diet (negative control group); (2) standard corn, soybean based diet plus 100ppm neomycin and soil (positive control group); (three) standard corn, soybean basal feed plus 0.1% metabolite composition (Com3456) of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains; (four) standard corn, soybean basal Feed plus 0.2% Com3456; (five) standard corn, soybean basal feed plus 0.3% Com3456; (6) standard corn, soybean basal feed plus 0.4% Com3456; (7) standard corn, soybean basal feed plus Com3456 on 0.5%. Supplementation of Com3456 at different doses improved growth performance, decreased Enterobacteriaceae numbers, and increased Lactobacillus numbers, intestinal villus height, and fecal volatile fatty acid concentrations. The 0.4% and 0.2% Com3456 treatments had the best effects, especially on growth performance, feed conversion and villi height compared to other doses. However, since the 0.2% dose is similar in effect to the 0.4% dose, but at a lower concentration, the 0.2% dose is recommended. These results show that 0.2% is the optimal dose of the metabolite composition when used in place of antibiotic growth promoters in broiler chicken diets.

Example

Broilers and Design of Experiments

432 male broiler chickens from a local company were housed from 1 to 42 days of age in thick-litter houses. Each pen consisted of 12 animals and was distributed arbitrarily into an open room with wood shavings. After arrival, these broilers were manually inoculated with anti-infectious bronchitis vaccine (IB) and anti-Newcastle disease vaccine (ND) (produced by IB-DN Fort Dodge, USA). When rearing to 14 days of age, chickens were inoculated with anti-infectious bursal disease IBD vaccine (Malaysia MyVac, produced by UPM93). After inoculation, the chicken wings were tied up to detect individual body weight, and water and feed were freely available. The start feed and end feed were used to feed broilers aged 0-21 days and 22-42 days, respectively. Dietary treatment consisted of: (i) corn, soybean basal diet without antibiotics (-ve control group), (ii) basal diet containing 100 ppm neomycin and oxytetracycline (+ve control group), (iii) supplemented with 0.1 % of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains (Com3456) metabolite composition of the basal feed, (d) supplemented with 0.2% of the basal feed of the Com3456 metabolite composition, (five) supplemented with 0.3% of the Com3456 Basal diet of metabolite composition, (vi) basal diet supplemented with 0.4% Com3456 metabolite composition, (vii) basal diet supplemented with 0.5% Com3456 metabolite composition. These feeds can meet the needs of broilers for all nutrients according to the plan. The percentage composition of the feed at the beginning and the end of the feed is shown in Table 1 and Table 2 respectively:

Table 1: Percentage composition of the feed at the start

¹ Com3456 is a combination of four strains RS5, RI11, RG11 and RG14, Com246 is a combination of strains TL1, RI11 and RG11, Com256 is a combination of strains TL1, RI14 and RG11, Com2456 is a combination of strains TL1, RI11, RG14 and Composition of RG11.

² The mineral mixture provided per kilogram of feed includes: 100 mg iron, 110 mg manganese, 20 mg copper, 100 mg zinc, 2 mg iodine, 0.2 mg selenium, 0.6 mg cobalt.

³ The vitamin mixture provided in each kilogram of feed includes: 6667IU vitamin A, 1000IU vitamin D, 23IU vitamin E, 1.33mg vitamin K3, 0.03mg cobalamin, 0.83mg thiamine, 2mg riboflavin, 0.33mg folic acid , 0.03 mg biotin, 3.75 mg pantothenic acid, 23.3 mg niacin, 1.33 mg pyridoxine.

⁴ The concentration of oxytetracycline and neomycin combination is 100ppm (w/w).

Table 2: Percentage composition of the feed at the end

¹ Com3456 is a combination of four strains RS5, RI11, RG11 and RG14, Com246 is a combination of strains TL1, RI11 and RG11, Com256 is a combination of strains TL1, RI14 and RG11, Com2456 is a combination of strains TL1, RI11, RG14 and Composition of RG11.

² The mineral mixture provided per kilogram of feed includes: 100 mg iron, 110 mg manganese, 20 mg copper, 100 mg zinc, 2 mg iodine, 0.2 mg selenium, 0.6 mg cobalt.

³ The vitamin mixture provided per kilogram of feed includes: 6667IU vitamin A, 1000IU vitamin D, 23IU vitamin E, 1.33mg vitamin K3, 0.03mg cobalamin, 0.83mg thiamine, 2mg riboflavin, 0.33mg folic acid, 0.03 mg biotin, 3.75 mg pantothenic acid, 23.3 mg niacin, 1.33 mg pyridoxine.

⁴ The concentration of oxytetracycline and neomycin combination is 100ppm (w/w).

Data and Sample Collection

Individual body weight (BW) and pen feed intake (FI) were recorded weekly, and weight gain (WG), feed conversion ratio (FCR) and average daily gain (ADG) were calculated. After 6 weeks of feeding, 12 chickens were randomly and equitably selected from each treatment group and slaughtered for sampling, feces and small intestines for further analysis. The above process has been approved by the Research Advisory Committee of Universiti Putra Malaysia.

growth performance

See Table 3 for growth performance. In all treatment groups, the individual body weight, total weight gain and average daily weight gain of broiler chickens at the age of 42 in the -ve control group were the lowest (P<0.05). Broilers in the treatment group supplemented with 0.4% Com3456 consisting of 4 Lactobacillus plantarum strains had the highest (P<0.05) individual body weight, gain and average daily gain at 6 weeks of age, followed by the +ve control group , ranked third was the treatment group supplemented with 0.2% Com3456. However, there was no significant difference in individual body weight and weight gain between the +ve control group and the broiler group supplemented with Com3456 (P>0.05), except for the group supplemented with 0.5% Com3456, the results of which were the same as those of all treatment groups the lowest. There was no significant difference (P > 0.05) in the mean feed intake among the treatment groups. The feed conversion ratio (FCR) of the treatment group supplemented with 0.4% Com3456 was the lowest compared with the other treatment groups (P<0.05). The data showed that the +ve control group and the 5 treatment groups supplemented with different doses of Com3456 outperformed the broilers in the -ve control group fed corn, soybean based diet in terms of individual body weight, total weight gain and average weight gain. The metabolite composition of four Lactobacillus plantarum (Com3456) at different dosage levels can partially replace the antibiotic growth promoter. According to the growth performance, the optimal dosage is 0.4% Com3456, at this time the growth performance is the best, followed by 0.2% Com3456. Broilers supplemented with different doses of the metabolite composition had 4-12% higher body weight at 6 weeks than broilers in the -ve control group, based on percent performance improvement compared to the -ve control group.

These metabolite compositions have antimicrobial effects and can be used as potential alternatives to antibiotic growth promoters (AGPs). Antibiotics have bacteriostatic and bactericidal effects, so they can inhibit and kill pathogenic bacteria present in the intestinal microbial flora, and their main effect is to increase the growth rate. The antibacterial activity of antibiotic growth promoters has been demonstrated in germ-free animals.

Table 3: Growth performance at 6 weeks of treatment groups supplemented with different metabolite compositions

^{a, b} There is no significant difference in mean ± SEM rows with the same superscript.

¹ Com3456 is a combination of four strains RS5, RI11, RG11 and RG14, Com246 is a combination of strains TL1, RI11 and RG11, Com256 is a combination of strains TL1, RI14 and RG11, Com2456 is a combination of strains TL1, RI11, RG14 and Composition of RG11.

Fecal lactic acid bacteria and Enterobacteriaceae (ENT) counts

Fecal Lactobacillus and Enterobacteriaceae flora were detected by the method described by Foo et al. (2003b). Fecal samples were diluted 10% (w/v) in sterile peptone water, left at room temperature for one hour, and then further serially diluted to 10-fold volume (v/v). Lactic acid bacteria were counted on MRS-agar (Lactobacillus agar medium, ROGOSA and SHAPE) (manufactured by Merck, Darmstadt). The plates were incubated in an anaerobic jar at 30°C for 48 hours. Enterobacteriaceae were dispersed and counted according to EMB-agar (Eosin Methylene Blue Agar Lactose Sucrose Agar) (manufactured by Merck, Darmstadt), followed by aerobic incubation at 37°C for 24 hours. The number of colony forming units is expressed in logCFU/g. Repeat the above operation three times for all samples. The numbers of fecal lactic acid bacteria and Enterobacteriaceae are shown in Table 4. The -ve control group had the lowest number of fecal lactic acid bacteria (p<0.05), while no significant difference was found in the other treatment groups. The number of Enterobacteriaceae at 6 weeks was highest in broilers of -ve control group and +ve control group (p<0.05). In comparison, the number of Enterobacteriaceae was lowest (p<0.05) for the treatment groups supplemented with 0.2% Com3456 and 0.5% Com3456. For the treatment groups supplemented with 0.1% Com3456, 0.3% Com3456 and 0.4% Com3456, the number of Enterobacteriaceae was also significantly lower (p<0.05) than that of the -ve control group and the +ve control group.

The data show the effect of the metabolite composition on reducing the number of gastrointestinal enterobacteria. Furthermore, the metabolite composition increased the number of gastrointestinal lactic acid bacteria. All dosage levels of the metabolite composition were able to increase the number of gastrointestinal lactic acid bacteria, although the degree of increase varied. All treatment groups supplemented with Com3456 had a positive effect on reducing the number of Enterobacteriaceae. The metabolite composition can reduce gastrointestinal enterobacteria, so that lactic acid bacteria can better increase the number of flora in the small intestinal microbial flora through flora exclusion.

Table 4: Fecal Lactobacillus and Enterobacteriaceae and Volatile Fatty Acid Counts in Treatment Groups Supplemented with Different Metabolite Compositions

There are no significant differences in the ^ac Means±SEM rows with the same superscript.

¹ Com3456 is a combination of strains RS5, RI11, RG11 and RG14, Com246 is a combination of strains TL1, RI11 and RG11, Com256 is a combination of strains TL1, RI14 and RG11, Com2456 is a combination of strains TL1, RI11, RG14 and RG11 combination.

small intestine morphology

This process is a modified method proposed by Hair-Bejo (1990). Remove 5–6 cm segments from the duodenum, jejunum, and ileum as follows: (1) in the middle of the duodenal loop; (2) at the end of the duodenal loop and Meckel’s diverticulum and (3) the midsection between Meckel's diverticulum and the ileocecal junction (ileum). Intestinal segments were rinsed with 10% neutral buffered formalin solution for subsequent morphological analysis. For morphological analysis, intestinal segments were fixed overnight in 10% neutral buffered formalin solution, and intestinal samples were excised, dehydrated in a tissue processor (manufactured by Leica, Japan), and embedded in paraffin. 4 micron sections were excised from each sample, mounted on slides, stained with hematoxylin and eosin, and mounted under a microscope for observation. Observations of morphological changes included: intestinal villi height (the height from the tip of the villi to the junction of villi and crypts), and crypt depth (defined as the depth of invagination of adjacent villi). Villus height and crypt depth were measured using an image analyzer. These values are the average of the best 20 villi for which crypts were assayed for each slide.

Determination of Volatile Fatty Acids (VFA)

Volatile fatty acid concentrations in feces were determined by a modified method of Jin et al. (1998). Weigh 1 gram of stool samples (stored at -20°C) from each sample into sampling tubes. 1 ml of 24% metaphosphoric acid was diluted in 1.5M sulfuric acid (produced by BHD Laboratories, Poole, UK), and then added to the sampling tube. The mixture was left overnight at room temperature, and centrifuged at a speed of 10,000 rpm for 20 minutes at 4°C, and the collected supernatant was stored in a 2 ml screw-cap sample bottle (produced by Kempel Glass Co., Ltd., USA) middle. An internal standard of 20 mM 4-methylpentanoic acid (Sigma, St. Louis, MO, USA) was added to the supernatant to bring the composition to 10 mM and stored at -20°C until gas liquid chromatography (GLC) analysis. Use Quadrex007 series (produced by Quadrex Company, New Haven, Connecticut, 06525, U.S.) bonded item fused silica capillary column (15m, 0.32mm number, film thickness 0.25 micron) to volatile fatty acid in 6890N (Pennsylvania) with flame particle detector installed produced by Hewlett-Packard Co., Avondal, Avondal) for separation. Purified nitrogen was used as carrier gas at a flow rate of 60 ml/min. The temperature of the injector and detector was 230°C, and the column temperature was set constantly at 200°C. Commercial standards of 20 mM acetic acid and 10 mM propionic, butyric, isobutyric, valeric and isovaleric acids from Sigma (Sigma Chemicals, Inc., St. Louis, MO, USA) were used as external standards to identify peaks. Fecal volatile fatty acids are mentioned in Table 5. The volatile fatty acid is mainly acetic acid, followed by propionic acid and butyric acid in lower concentrations. Treatment groups supplemented with 0.4% and 0.3% Com3456 had the highest (p<0.05) acetate and overall volatile fatty acid levels. In contrast, chickens in the -ve control group had the lowest levels (p<0.05). However, there were no significant differences in acetate and total volatile fatty acid concentrations among the other treatment groups (p > 0.05). Propionic acid and other volatile acid concentrations did not differ significantly (p > 0.05) among all treatment groups. In the broiler feed supplemented with 0.1% Com3456, the butyric acid level was significantly different from the treatment groups supplemented with 0.3%, 0.4% and 0.5% Com3456 (p<0.05). However, there was no significant difference in the other six treatment groups (p>0.05). Supplementation of a metabolite composition increases fecal volatile fatty acid levels. Volatile fatty acid content was increased by mixing certain doses of Com3456, especially with 0.3% and 0.5% Com3456 at week 3 and with 0.4% Com3456 at week 6. A major reason for the increase in volatile fatty acid levels in the treated broilers was the increased presence of lactic acid bacteria, lactic acid bacteria and other in vivo microorganisms that convert various substrates such as lactose, Biogenic amines and other allergens are fermented to short-chain fatty acids and other organic acids and gases (Gibson and Fuller, 2000).

Table 5: Intestinal villus height and crypt depth in the treatment groups supplemented with different metabolite compositions

^{a, b} There is no significant difference in mean ± SEM rows with the same superscript.

¹ Com3456 is a combination of strains RS5, RI11, RG11 and RG14; Com246 is a combination of strains TL1, RI11 and RG11; Com256 is a combination of strains TL1, RI14 and RG11; Com2456 is a combination of strains TL1, RI11, RG14 and RG11 combination.

data analysis

Complete randomized design data were analyzed using the General Linear Modeling program of the Statistical Analysis System (SAS, 1998). Duncan's multiple range test was used to compare these treatments. Data are presented as mean ± standard error of the mean (SEM). The pilot unit takes the chicken house with broiler chickens as the experimental object. The statistical model is as follows:

Yijk＝μ+τi+εij+δijk

μ = mean effect

τi = treatment effect of group i

εi j = random error

δijk = sampling error

Claims (12)

1. An animal feed formulation, wherein the formulation comprises corn, meal, rice bran, wheat fine bran, syrup, palm oil, coconut oil crude oil, limestone, dicalcium monophosphate, calcium hydrogen phosphate, salt, lysine, Choline Chloride, Vitamins, Minerals, Threonine, Anti-Mold Compound, Copper Sulfate, DL-Methionine, Antioxidant. 2. The animal feed formulation according to claim 1, wherein said corn is yellow corn; said meal is soybean meal and coconut meal; and said palm oil is refined palm oil and crude palm oil. 3. The animal feed formula according to claim 1, wherein said formula further comprises a compound selected from yellow corn, soybean meal, coconut meal, rice bran, molasses, coconut oil crude oil, limestone, table salt, lysine, choline chloride, Any combination of vitamins, minerals, threonine, anti-mold compounds, copper sulfate, DL-methionine and antioxidants. 4. The animal feed formulation according to claims 1-3, wherein the animal feed further comprises a combination of metabolites such as bacteriocins, B vitamins and organic acids. 5. The animal feed formulation according to claim 4, wherein said metabolites are added in an amount of 0.5-8 kg in the total animal feed ration. 6. The animal feed formulation of claim 1, wherein the animal feed increases overall feed intake by 3% to 10%. 7. The animal feed formulation of claim 1, wherein the animal feed can increase animal growth rate by 5%-7%. 8. The animal feed formulation of claim 1, wherein the animal feed conversion ratio is between 3% and 8%. 9. The animal feed formula according to claim 1, wherein the formula has the ability to reduce the amount of fecal enterobacteria in monogastric animals or ruminants, and increase the amount of fecal lactic acid bacteria. 10. The animal feed formulation of claim 1, wherein said formulation has the ability to lower plasma and meat cholesterol in monogastric or ruminant animals. 11. The animal feed formulation of claim 1, wherein said formulation has the ability to increase the height of small intestinal villi in monogastric or ruminant animals. 12. Animal feed according to any one of claims 1-11, comprising metabolites produced with Lactobacillus plantarum.