KR20160040790A - A feed additive composition comprising fermented Eucommia ulmoides extract and a method thereof - Google Patents

Abstract

The present invention relates to a livestock feed additive composition comprising an Eucommia ulmoides OLIV. extract fermented by using Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus plantarum, and to a manufacturing method thereof. The bioactivity of the Eucommia ulmoides OLIV. is increased by a fermentation process using L. brevis NJ42, L. curvatus NJ40, and L. plantarum NJ45 strains.

Description

[0001] The present invention relates to a composition for adding a livestock feed containing an extract of fermented mushroom and a method for preparing the same,

The present invention relates to a composition for adding a livestock feed comprising an extract of Leptobacillus brevis, Lactobacillus carvatus and lactobacillus plantarum fermented by using a plant, and a process for producing the same.

In Korea, antibiotics have been used for livestock productivity and growth, and ionophore antibiotics such as monensin, chloroform and halogenated analogues have been used as representative antibiotics (Odongo et al., 2007 Journal of Dairy Science . 90: 1781-1788; McAllister and Newbold, 2008 Animal Production Science . 48: 7-13). However, the problems of such antibiotics and chemicals have been reported and their use is limited.

Many studies have been carried out on plant extracts derived from natural products in animal husbandry, but there are insufficient results of verifying various medicinal plants.

Medicinal plants have excellent physiological activity by themselves. However, for industrial application, it must meet the criteria of economic efficiency and productivity. That is, it is important to improve the activity per unit weight, to improve the productivity of the drug substance, and to secure economical efficiency. Efforts have been made to improve the efficacy of useful substances contained in medicinal plants through bioconversion according to this purpose. One of these efforts is the entry into force.

Fermentation is a biological process that proceeds by microorganisms, and as microorganisms grow, they secrete various enzymes, and biological substances are converted by these enzymes. Of course, material conversion may occur in the direction of lowering physiological activity, but material conversion in the direction of improving physiological activity is mainly used for industrial purpose. Kim et al. (2003, Korean Journal of Food Science and Technology . 35: 272-279) reported that total phenolic content and antioxidant activity were enhanced by fermentation of plant extracts, and some plants showed high preference according to fermentation.

It is a leaf of a mulberry tree which is a deciduous tree. Diapung leaves contain glycosides, alkaloids, pectin, sugars and vitamins, and are known to have effects such as blood pressure lowering, diuretic stimulation and nourishing tonic.

[Prior Patent Literature]

Korean Patent Publication No. 10-2005-0074009

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and an object of the present invention is to provide an effective composition for adding a livestock feed.

Another object of the present invention is to provide an effective method for preparing a composition for adding a livestock feed.

In order to achieve the above object, the present invention provides a fermented product obtained by fermenting at least one strain selected from the group consisting of Lactobacillus brevis, Lactobacillus caballus, and Lactobacillus plantarum, Which comprises the steps of:

In one embodiment of the present invention, the antimicrobial composition preferably has antimicrobial activity against at least one bacterium selected from the group consisting of Staphylococcus aureus, Listeria monocytogenes, Salmonella gallinarum, and Mannheimia haemolytica, but is not limited thereto.

The present invention also provides a composition for antioxidant comprising an effective amount of a bivalent fermentation product prepared by inoculating a bacterium with Lactobacillus brevis (L. brevis).

The present invention also provides a composition for adding a livestock feed comprising, as an active ingredient, a fermented product obtained by inoculating and fusing one or more strains selected from the group consisting of Lactobacillus brevis, Lactobacillus caballus and Lactobacillus plantarum, .

The present invention also relates to a method for producing a fermented beverage comprising the step of adding to the feed a bivalent fermentation product obtained by inoculating a bivalent one or more strains selected from the group consisting of Lactobacillus brevis, Lactobacillus caballus and Lactobacillus plantarum, A method for preparing a composition for feed addition is provided.

Hereinafter, the present invention will be described.

The present invention has developed lactic acid bacteria capable of fermenting two kinds of livestock for the development of a livestock feed additive, and evaluated the physiological activity thereof to develop a fermented feed additive.

Through the present invention, it was found that the bioglasses possessed by the bioglasses themselves are excellent, and that the physiological activities of the bioglasses were improved by the fermentation process.

It is a physiological function that can replace antibiotics added to livestock feed, and it is the most commonly recognized activity, and it has antibacterial activity. Although it did not exhibit antimicrobial activity, it was found that when fermented using L. curvatus strain NJ40, which was developed through the present invention, the antibacterial activity was excellent.

Antioxidant activity is an important function for livestock producing livestock products by supplying high energy in a short period of time, and it also plays an important role in productivity improvement. This antioxidative activity was also greatly improved when the bacterium L. brevis NJ42 was fermented with the strain developed through the present invention.

In terms of ruminal fermentation, which is closely related to the productivity of ruminants, These effects were improved by fermentation using L. curvatus NJ40 and L. plantarum NJ45 which were developed through the present invention.

As can be seen from the present invention, the fermentation process using L. brevis NJ42, L. curvatus NJ40 and L. plantarum NJ45 in the development of feed additive using diapauses shows that the physiological activity I could.

Fig. 1 shows the results of growth of the lactic acid bacteria starter strains used in the bivalve fermentation. NJ28: W. confusa, NJ33: W. cibaria, NJ40: L. curvatus, NJ42: L. brevis; NJ45, L. plantarum, NJ48: L. sakei.
Fig. 2 shows the antioxidative activity of the extracts of the mungbean sprouts fermented with the mungbean extract and various lactic acid bacteria. Test: Con: non-fermented mulberry extract, NJ28: W. confusa, NJ33: W. cibaria, NJ40: L. curvatus, NJ42: L. brevis; NJ45, L. plantarum, NJ48: L. sakei.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  1. Isolation of Lactobacillus sp.

Isolation and Identification of Strain for Fermentation : Kimchi and salted seafood were used as strain isolates. Two kinds of fermented seafood (herring rosemary and hwangseok sauce) were obtained from the bears' sound of Jinseo - myeon, Buan - gun, Jeollabuk - do. The fermented seafood was used as a strain isolate. Fermented salted and fermented kimchi were milled using a household grinder, sterilized in 0.8% NaCl, and plated on MRS (Difco, USA) solid medium and cultured at 30 ° C for 24 hours. Bacterial strains cultured on MRS medium were randomly selected to evaluate the β-glucosidase enzyme activity (MUG test, 4-methylumbelliferyl-β-D-glycopyranoside reaction). Six strains of lactic acid bacteria were selected based on enzyme activity and identified using 16S rRNA gene sequence. DNA sequences were used to identify selected strains. The 16S rRNA gene sequence of the lactic acid bacteria was analyzed and the primers used were 27F (5 '- AGA GTT TGA TCC TGG CTC AG - 3') and 1492R (5 '- GGT TAC CTT GTT ACG ACT T - 3' ) (Weisburg et al., 1991, Journal of Bacteriology . 173: 697-703). Similarity analysis was performed using GenBank (NCBI) using a basic local alignment search tool (BLAST) (Altschul et al., 2002) 1990, Journal of Molecular Biology . 215: 403-410). The analyzed reference sequences were subjected to multiple sequence alignment using the CLUSTAL_W module of the MEGA4 program (Thompson et al., 1994, Nucleic Acids Research . 22: 4673-4680), and the whole strain was verified against the GenBank sequence. In addition, phylogenetic trees were constructed and phylogenetic trees were plotted according to their similarity (Tamura et al., 2004, Proceedings of the national academy of sciences of the United States of America . 101: 11030-11035; Tamura et al., 2007, Molecular Biology and Evolution . 24: 1596-1599).

The results of the above embodiment will be described below.

Of the 200 colonies randomly isolated from kimchi and salted fish, 26 candidate strains were screened for β-glucosidase positive colonies through MUG test, and 17 strains were isolated from salted fishes and 9 strains were isolated from kimchi. The final 6 strains were selected considering high β-glucosidase activity and growth in rich medium (MRS). The strains selected from salted fish were Weissella confusa NJ28 (Accession number KJ914897) and Weissella cibaria NJ33 (Accession number KJ914898). In kimchi, Lactobacillus curvatus NJ40 (Accession number KJ914899), Lactobacillus brevis NJ42 (Accession number KJ914900), Lactobacillus plantarum NJ45 (Accession number KJ914901) and Lactobacillus sakei NJ48 (accession number KJ914902) were selected. In the experiment, each strain was named NJ28, NJ33, NJ40, NJ42, NJ45 and NJ48 and used as a seed for the fermentation of medicinal plants.

Example  2: Selection of lactic acid bacterial strains suitable for fermentation of bivalves for physiological activity improvement

Fermentation conditions : Jeju-si, Jeollabuk-do provincial jungchu was secured. All of the lactic acid bacteria strains were agitated at 30 rpm at 150 rpm using MRS liquid medium (Difco, USA) and cultured for 24 hours. 3 g was weighed and dispensed into an Erlenmeyer flask containing 30 mL of MRS liquid medium and autoclaved (121 ° C, 15 min) using a household grinder. The previously prepared strains were inoculated into sterilized flasks and cultured at 30 ° C for 48 hours. 2 mL of the cultured medium was dispensed and used for counting viable cells. The remaining culture was taken out to an aluminum dish and then dried in a drying oven at 60 ° C for 24 hours. After drying, the culture was pulverized using mortar, weighed 1 g and divided into Erlenmeyer flask containing 99.9% ethanol and extracted for 20 hours. After the extraction, the culture solution was filtered through Whatman filter paper No. 1. 1, and then concentrated to 2 mL using a rotary evaporator (N-1110, EYELA, Japan) and stored in a -20 ° C cold freezer.

Measurement of viable cell count : The number of viable cells was measured in order to confirm the progress of the fermentation of the bacterium in which the bacterium and the strain were cultured together. 1 mL of the culture was diluted to 10 ^{-6 with} 10 mL of sterile 0.8% (w / v) NaCl solution (10-fold dilution) in 5 steps. The viable cell count was used to evaluate the fermentation progress of the culture solution.

Evaluation of antimicrobial activity : Antimicrobial activity was measured by using the prepared plant extract concentrate. Staphylococcus aureus (wild type), Listeria monocytogenes KACC0550, Salmonella gallinarum ATCC9184 and Mannheimia haemolytica (wild type) were selected as pathogens for antimicrobial activity. Each pathogen was inoculated with 1% in 10 mL of 0.8% water agar, mixed and applied to the surface of the LB plate medium and allowed to dry sufficiently. Paper disks (8 mm in diameter) were inoculated with 100 uL of plant extract, dried at room temperature, and then placed on a prepared medium. The clear zone was measured by incubation at 37 ° C for 24 hours in an incubator.

Antioxidant activity : Juan and Chou (2010, Food Microbiology . 27: 586-591), the free radical scavenging activity assay was used to measure antioxidant activity. (DPPH) (2-di (4-tert-octylphenyl) -1-picrylhydrazyl, Sigma, USA), and 500 μL of the diluted extract was mixed with 2 mL of DPPH solution. For 15 minutes. After the reaction was completed, 2.5 mL of distilled water was added and the optical density was measured at 525 nm using a spectrophotometer (Optizen UV2120, Mecasis, Korea). For the evaluation of antioxidant activity, the difference between the absorbance of distilled water and the medicinal plant extract was used instead of the extract.

Scavenging effect,% = (1 - absorbance of sample / absorbance of control) X100

The results of the above embodiment are described below.

The growth of microorganisms in the mung bean (10% added to the MRS liquid medium) is shown in Fig. The strains except W. cibaria NJ33 and L. sakei NJ48 were found to grow at a level of 10 ⁸ CFU / mL. The growth rates of W. confusa NJ28, L. curvatus NJ40 and L. plantarum NJ45 were significantly higher. Based on the results, it was concluded that some strains were suitable for fermentation.

The antimicrobial activity of the fermented mugwort extract was as shown in Table 1. All four pathogens have antibacterial activity against L. curvatus It was shown that it was a mulberry extract fermented with NJ40 strain. Also, L. plantarum The extracts of L. monocytogen and M. haemolytica fermented with NJ45 were found to have antibacterial activity against S. aureus and S. gallinarum . Especially, the antimicrobial activity was not observed in CON (control) obtained by culturing without inoculating the strain. L. curvatus NJ40 and L. plantarum It is considered that NJ45 has a direct effect on the antibacterial activity.

Pathogen Test section CON NJ28 NJ33 NJ40 NJ42 NJ45 NJ48 Growth inhibition, mm S. aureus ND ND ND 9.39 ND 9.17 ND L. monocytogenes ND ND ND 10.63 ND ND ND M. haemolytica ND ND ND 10.93 ND ND ND S. gallinarum ND ND ND 9.21 ND 9.06 ND

Table 1 shows the antimicrobial activity evaluation results of the mulberry extract and fermented mulberry extract

In the table, the test-Con: non-fermented mulberry extract, NJ28: W. confusa, NJ33: W. cibaria, NJ40: L. curvatus, NJ42: L. brevis; NJ45, L. plantarum, NJ48: L. sakei.

To evaluate antioxidant activity, free radical scavenging activity measurement was used and DPPH was used as the evaluation standard. The degree of antioxidant activity was evaluated using the difference in absorbance of the DPPH solution from purple to yellow. The results of measuring the antioxidative activity of DPPH are shown in FIG. Con ( L. curvatus NJ40 and L. plantarum NJ45) compared to CON (non fermented extract) showed low in extracts of L. curvatus and L. plantarum NJ45, whereas L. brevis NJ42 .

Example  3: Effect of fermented mushroom extracts on rumen fermentation

This experiment was carried out to investigate the effect of the extracts of two or three fermented lactic acid bacteria on ruminal fermentation in ruminant feed, and the experiment was carried out in vitro.

Disclosure axis and specification management: in North Jeolla North Jeolla Province Livestock Hygiene Institute of the Gimje site bovine rumen cannula were attached material disclosing beef steers (weighing 400 kg ± 30 kg) 2 two, in Gastric juice for the rumen fermentation test in vitro was collected. The announcement axis was fed with 4 kg of rice straw and 4 kg of poultry mix feed twice a day (08:00) and afternoon (17:30). Mineral blocks and water were allowed to eat freely.

Test design and sample preparation: The orchardgrass used as the test substrate was milled using a laboratory grinder equipped with a 2 mm sieve (Cutter mill, ICA MF10.1, Staufen, Germany). NJ40 ( L ) was used for the control of the test. The experimental design was as follows: No added control, garlic oil additive (control reference for results), ethanol additive (for analysis of alcohol use effect on extraction), non fermentation (no fermentation) curvatus NJ40), NJ45 ( L. and plantarum NJ45). Each plant extract was inoculated with 1% (w / v) of feed to the total treatments used in the test.

Preparation of ruminal juice: 30 minutes before the feeding of the rats on the morning of the day, the rats were harvested through a cannula attached to the rumen. The collected gastric juice was filtered through a 4-ply cheese cloth, washed with 2 L flask (39 ° C) filled with O ₂ -free CO ₂ , And the anaerobic condition was maintained by blocking the entry of oxygen. 30 minutes before the start of the experiment, the ruminal fluid was bubbled with O ₂ -free CO ₂ to adjust the pH to 6.5, and NaHCO _{3 9.8 g, Na 2 HPO 4 ·} 2H 2 O 4.62 g, KCl 0.57 g, NaCl 0.47 g, MgSO 4 · 7H 2 O 0.12 g, CaCl 2 (Trolesen and Hanel, 1966) with 4: 1 ratios of rumen inoculum with a mixture of McDougall's buffer solution (Trolesen and Hanel, 1966) consisting of (CaCl ₂ · 2H ₂ O) / 100 mL 4 During the dilution and filtration of gastric juice, O ₂ -free CO ₂ was injected to maintain the anaerobic state so that gastric juice was not exposed to oxygen. Tilley and Terry (1963, Grass and Forage Science . 18: 104-111).

Analytical Items and Analytical Methods: Total gas production by incubation time was measured by using a glass syringe for experimental purposes. The measured gas was collected in an aluminum pack equipped with a rubber stopper to measure hydrogen and methane emissions. After fermentation, the pH of the rumen juice was measured using a pH meter (S20 Seven Easy ^™ , Mettler-Toledo). The ammonia nitrogen content of the rumen juice was determined according to the method of Chaney and Marbach (1962, Clinical Chemistry , 8: 130-132), and centrifuged at 4,000 rpm for 15 minutes to remove 20 μL of the supernatant 1 mL of phenol color reagent and 1 mL of alkali-hydrorite reagent were mixed and reacted at 37 ° C for 15 minutes and then measured at an optical density of 630 nm using a spectrophotometer (Optizen UV2120, Mecasis, Korea). Volatile fatty acids were prepared according to the method of Erwin et al. (1961). After addition of 200 μL of metaphosphoric acid to 1 mL of the supernatant of the ruminal juice from which the feed particles had been removed, the sample was subjected to centrifugation at 13,000 rpm for 30 minutes, and the sample was subjected to Nukol ^™ and fused silica capillary columns (oven = 180 ° C, injector = 220 ° C and detector = 200 ° C) with gas chromatography (HP7890, Agilant, CA. Hydrogen and methane emissions were analyzed by gas chromatography (HP7890, Agilant, CA. USA) equipped with Carboxen ^™ , fused silica capillary column (0.53 mm id × 30 m length, SUPELCO, USA) 150 [deg.] C and TCD = 150 [deg.] C). The results of this experiment were analyzed and evaluated according to the General Linear Model of SPSS program (Version 18, IBM, New York, USA). Analysis of variance (ANOVA) was performed to verify significance of each test section, followed by Duncan's multiple range test. The significance level was 5%.

Fermented ruminal fermentation characteristics of dietary fermented diarrhea extracts were investigated using different lactic acid bacteria strains.

The rumen pH of the rumen was not statistically significant during the incubation time (Table 2). However, it was numerically investigated in the range of 6.60 - 6.64, which is the optimum range of rumen fermentation (5.80 - 7.20) (Hiltner and Dehority, 1983, Applied and Environmental Microbiology . 46: 642-648). Therefore, it is considered that the addition of mulberry extract does not have negative effect on rumen fermentation. Ammonia nitrogen, one of the most important factors in the growth of rumen microbes, represents the growth of microorganisms in the rumen. Production of ammonia nitrogen was shown to increase significantly in the ethanol impregnated furniture, followed by high appeared in extracts (NJ45) of Eucommia fermented using L. plantarum NJ45 (p <0.05) . On the other hand, the extracts of non - fermented mugwort (non - fermented) and fermented L. curvatus NJ40 (NJ40) were significantly lower. The decrease in the amount of ammonia nitrogen produced by the rumen can be attributed to the smooth utilization of ammonia nitrogen from the feed for the growth of microorganisms. This is because the growth of the microorganisms is so rapid that a large amount of ammonia nitrogen is introduced into the microorganisms . Therefore, it was shown that the extract of L. persimilis and L. persimilis (L.curvatus NJ40 fermentation) can promote the growth of rumen microorganisms.

Contents Control Garlic oil spirits Mulberry extract SEM ² Non fermentation Fermented mulberry extract NJ40 NJ45 pH 6.60 6.63 6.63 6.63 6.64 6.62 0.004 NH ₃ -N, mg / 100 mL 6.24 ^ab 6.23 ^ab 7.07 ^c 5.86 ^a 5.86 ^a 6.61 ^b 0.114

Table 2 shows the effect of dietary supplementation of fermented mugwort extract on ruminal fermentation pH and ammonia nitrogen

The results of analysis of volatile fatty acids (VFA) of the plant extracts using Dioscorea are shown in Table 3. Total volatile fatty acids were significantly higher (p <0.05) in the NJ45 treatments than the fermented L. plantarum NJ45 fermented and fermented only. Volatile fatty acids produced during the fermentation process in the rumen act as a direct energy source for ruminants. Therefore, it can be predicted that the productivity of the ruminant animal is improved when the amount of volatile fatty acid produced is large. The amount of volatile fatty acids produced in each volatile fatty acid was higher in the treatments with the mugwort extract and fermented mugwort extract than the control. The A / P ratio, which is the ratio of Acetate to propionate, is generally known to be the most stable fermentation when the ratio is 2: 1 or more. As a result of this experiment, rumen fermentation seems to be stable because the A / P ratio of the whole treatment was 2: 1 or more.

Contents Control Garlic oil spirits Mulberry extract SEM ² Non fermentation Fermented mulberry extract NJ40 NJ45 Total VFA, mM 60.35 ^ab 61.19 ^ab 57.86 ^a 66.77 ^c 63.85 ^bc 65.78 ^c 0.853 Acetate, mM 36.21 ^a 37.16 ^ab 35.51 ^a 40.07 ^c 38.84 ^bc 39.75 ^c 0.485 Propionate, mM 12.76 ^b 12.34 ^ab 11.72 ^a 13.76 ^c 13.05 ^bc 13.58 ^c 0.189 iso-Butyrate, mM 0.69 ^b 0.69 ^b 0.62 ^a 0.76 ^c 0.69 ^b 0.71 ^bc 0.012 n-Butyrate, mM 7.83 ^bc 7.75 ^b 7.24 ^a 8.59 ^d 7.98 ^bc 8.29 ^bc 0.117 iso-Valerate, mM 1.69 ^a 2.07 ^b 1.77 ^a 2.30 ^c 2.10 ^b 2.17 ^bc 0.055 n-Valerate, mM 1.18 ^b 1.18 ^b 1.00 ^a 1.28 ^c 1.20 ^b 1.27 ^c 0.023 A / P ratio 2.84 ^a 3.01 ^cd 3.03 ^d 2.91 ^b 2.98 ^c 2.93 ^b 0.016

Table 3 shows the effects of dietary supplementation of fermented squid extract on the production of volatile fatty acids in rumen

Fermentation gas in the rumen is an indicator of microbial growth and metabolic efficiency. In other words, the production of a large amount of ruminal fermentation gas can be regarded as the growth and metabolism of microorganisms. The total fermentation gas production was significantly higher in NJ40 and NJ45 treated with lactic acid bacteria.

Contents Control Garlic oil spirits Mulberry extract SEM ² Non fermentation Fermented mulberry extract NJ40 NJ45 Total gas, mL 76.67 ^cd 75.00 ^b 61.67 ^a 76.00 ^c 77.67 ^e 77.33 ^d 1.364 Hydrogen, mL 0.04 0.04 0.04 0.04 0.04 0.04 0.001 Methane, mL 6.84 ^b 6.13 ^a 6.91 ^b 7.25 ^bc 7.22 ^bc 7.87 ^c 0.139

Table 4 shows the effect of dietary supplementation on fermentation gas production and composition in rumen.

Claims (5)

Wherein the effective component is a fermented product obtained by inoculating and fermenting at least one strain selected from the group consisting of Lactobacillus brevis, Lactobacillus caballus, and Lactobacillus plantarum. The composition according to claim 1, wherein the antimicrobial composition has antibacterial activity against at least one bacterium selected from the group consisting of Staphylococcus aureus, Listeria monocytogenes, Salmonella gallinarum, and Mannheimia haemolytica. A composition for antioxidation, comprising as an active ingredient, a fermented product obtained by inoculating a strain of L. brevis (L. brevis) and fermenting it. A composition for adding a livestock feed, comprising as an active ingredient, a fermented product obtained by inoculating and fusing at least one strain selected from the group consisting of Lactobacillus brevis, Lactobacillus caballus, and Lactobacillus plantarum. A method for adding a livestock feed composition comprising the step of adding to a feed a bacterium fermented by inoculating a bacterium selected from the group consisting of Lactobacillus brevis, Lactobacillus caballus, and Lactobacillus plantarum, Gt;

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