WO2007023588A1 - Additive for livestock feeds - Google Patents

Abstract

It is intended to provide an additive for livestock feeds characterized by containing a microorganism capable of producing trans-vaccenic acid such as Butyrivibrio fibrisolvens MDT-10 strain and another microorganism capable of taking in trans-vaccenic acid such as Bifidobacterium adolescentis HF-11 strain. This additive for livestock feeds can inhibit further hydrogenation of trans-vaccenic acid in the lumen, elevate the amount of the accumulated trans-vaccenic acid and thus increase the amount of conjugated linoleic acid in livestock meat and milk.

Description

 Specification

 Animal feed additives

 Technical field

 The present invention relates to a livestock feed additive, a livestock feed, a method for producing an unsaturated compound, and a novel strain that can be used for them. The amount of conjugated linoleic acid in milk and beef can be increased by providing the animal feed additive of the present invention to cows and the like. Further, by using the method for producing an unsaturated compound of the present invention, the unsaturated compound can be produced efficiently even in an environment where microorganisms that reduce the unsaturated compound exist. Background art

 [0002] Conjugated linoleic acid (CLA), an isomer of linoleic acid (LA; cis9, cisl2-C18: 2), has attracted a great deal of attention as a bioactive substance, especially cis9, transl-CLA ( c9, tl l-C18: 2) has anticancer activity (Pariza and Hargraves, 1985; Chin et al, 1992), antitumor development activity (Ha et al, 1990; Ip et al, 1991), antiarteriosclerosis Various health promoting effects such as action (Lee et al, 1994), immune function activation (Cook et al., 1993; Miller et al "1994), improvement of hyperinsulinemia (Houseknecht et al" 1 998) (Pariza, 2004; Wahl e et al., 2004). Although CLA has also been reported to have a body fat reducing effect (Nicolosi et al., 1 997), a recent report confirms that this effect is observed in clO, tl2-CLA, but c9, ti l- CLA seems to be less prominent (Perfield et al., 2004; Wang and Jones, 2004) o Recently, clO, tl2-CLA has been pointed out to have a negative health impact (Rajakangas et al., 2003; Wahle et al., 2004).

[0003] As a food with high CLA content, milk and beef have the main CLA isomer, which is c9, tl l-CLA, which is LA by microorganisms in ruminant rumen. It is produced by the catabolism reaction (Harfoot and Hazlewood, 1997). In ruminants, a portion of this CLA flows down into the small intestine and is absorbed. In the lumen, most of the CLA undergoes a reduction (hydrogenation) reaction by microorganisms, resulting in transbataenoic acid (t-VA; tl 1-C 18: 1), and most of them are subjected to further hydrogenation to produce stearic acid (SA; C18: 0). However, if it flows down to the small intestine at the t-VA stage, it is absorbed into the body and then CLA is detected by Δ9 desaturase. (Griinari et al., 2000).

[0004] Recently, 78-93% of c9, tl l-CLA in milk was reported to be produced from t-VA by Δ9 desaturase in body tissues (Corl et al., 2001; Piperova et al., 2002). The CLA content in meat is also considered to increase with increasing t-VA absorption (Daniel et al., 2004). Therefore, if the t-VA production in the lumen is increased and the force is not subjected to further hydrogenation, the c9, tl l-CLA content in the milk can be increased. Let's go.

 [0005] As an attempt to increase CLA in milk and meat, when feed was supplemented with soybean oil, corn oil, peanut oil, sunflower oil, linseed oil, fish oil, etc., the amount of t-VA produced in the rumen Has been reported to result in an increase in c9, tl l-CLA content in milk 'meat (Dhiman et al., 2000; Chouinard et al "2001; Duckett et al" 2002). For these effects, polyunsaturated fatty acids inhibit the growth of microorganisms (Harfoot and Hazlewood, 1997), so the addition of fats and oils may inhibit the activity and growth of hydrogenated bacteria in the rumen. Forces that have been observed to have similar effects by ionophores such as monensin (Fellner et al., 1997; Sauer et al., 1998) and copper (Morales et al, 2000). It is thought that this was the result of inhibition of the added bacteria. Use of such means to suppress the growth of bacteria is not preferable because it may cause adverse effects such as a decrease in fiber digestibility (Hino and Asanuma, 2003).

[0006] Various bacteria have been reported to date to hydrogenate LA to t-VA! Among them, the ability is particularly high, but butyrivibrio fib risolvens (Butyrivibrio fib risolvens) (Kepler et al., 1966; Eyssen and Verhuist, 1984; Jiang et ai., 1998; Kim et al., 2002). This bacterium isomerizes LA to c9, tl l-CLA with LA isomerase (LA-I), and subsequently reduces CLA to t-VA with CLA reductase (CLA-R) (Kepler et al. , 1966). The present inventor has discovered a new strain (TH1 strain) of both B. vibrio fibrisolvens having both high LA-I activity and CLA-R activity, that is, high t-VA production ability (non-patent literature) 1). As mentioned above, clO, tl2-CLA can have a negative impact on health (Wahie et al. 2004) and can reduce milk fat in dairy cows (Peterson et al., 2004). Butybribrio fibrisolvens with high t-VA production capacity increased production of c9, tl l-CLA only It is a precious fungus for the purpose of calorie.

 [0007] Non-Patent Document 1: Fukuda, S., H. Furuya, Y. Suzuki, N. Asanuma, and T. Hino. 2005. A new strain of Butyriviono fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid J. Gen. Appl. Microbiol. 51: 105—113.

 Disclosure of the invention

 Problems to be solved by the invention

[0008] As described above, t-VA production ability is high! Several strains are known! However, since the generated t-VA is reduced to stearic acid by hydrogenated bacteria in the rumen, simply giving the cow a strain with a high t-VA-producing ability will reduce the amount of CLA in beef and milk. It cannot be increased.

 [0009] The present invention has been made under the technical background as described above, and provides means for preventing further hydrogenation in the lumen of t-VA and increasing the amount of accumulated t-VA. The purpose is to do.

 Means for solving the problem

 [0010] As a result of intensive studies to solve the above-mentioned problems, the present inventor has administered t-VA-producing bacteria together with t-VA-incorporating bacteria into the lumen. It has been found that the reduction to stearic acid can be prevented and the amount of accumulated t-VA in the lumen can be increased. In addition, a microorganism having a higher production ability than the conventionally known t-VA producing bacteria was also found. The present invention has been completed based on these findings.

 That is, the present invention provides the following (1) to (10).

 [0012] (1) A feed additive for livestock, comprising a microorganism having an unsaturated compound or an unsaturated compound-producing ability, and a microorganism having an ability to take up an unsaturated compound.

 [0013] (2) The microorganism having an unsaturated compound-producing ability is a microorganism having a trans-paxenoic acid-producing ability, and the microorganism having an unsaturated compound-capturing ability is a microorganism having a trans-paxenoic acid-taking ability. The feed additive for livestock as described in (1) characterized by these.

[0014] (3) Microbial ability having the ability to produce transbatacenoic acid Butyrivibrio 'Fibriosolvens MDT-10 strain or a mutant thereof, characterized in that it is a feed additive for livestock according to (2) [0015] (4) Microbial power having the ability to take up transbataenoic acid Bifidobatterium 'adrecentise HF-11 strain or a mutant thereof The livestock according to (2) or (3) For feed additive.

 [0016] (5) The feed additive for livestock according to any one of (1) to (4), wherein the livestock is a ruminant.

 [0017] (6) A livestock feed comprising the livestock feed additive according to any one of (1) to (5).

[0018] (7) In an environment where microorganisms that reduce unsaturated compounds exist, cultivate microorganisms having an ability to produce unsaturated compounds and microorganisms having an ability to take in unsaturated compounds, and collect the unsaturated compounds. A method for producing an unsaturated compound.

[0019] (8) The method for producing an unsaturated compound as described in (7), wherein the unsaturated compound is transbatacic acid.

 [0020] (9) A butyribibrio fibrisolvens MDT-10 strain having the ability to produce transbatacenoic acid.

[0021] (10) Bifidobacterium 'adrecentis HF-11 strain having transbatacic acid uptake ability.

 The invention's effect

 [0022] By providing the livestock feed additive and livestock feed of the present invention to cattle and the like, the amount of CLA in milk and beef can be increased. In addition, the unsaturated compound production method of the present invention makes it possible to efficiently produce unsaturated compounds even in an environment where microorganisms that reduce unsaturated compounds exist.

 Brief Description of Drawings

[0023] [Fig. 1] Effects of butyrobibrio 'Fibriosolvens MDT-10 and bifu on the suppression of t-VA hydrogenation in batch culture of mixed rumen microorganisms (total viable count, 2 X 10 ¹¹ cfo / ml) The figure which showed the influence of the addition of Idbataterum vadrecentis HF-11 strain. A is an additive-free group (control group), B is a group with butyribibrio fibrisolvens MDT-10 (2 X 10 ⁹ cfo / ml), and a group with butyribibrio fibrisolvens MDT-10 (2 X 10 ⁹ cfo / ml) and Bifido butterium 'adrecentis HF-11 strain (2 X 10 ⁹ clu / ml) added. ○ indicates LA, ◊ indicates CLA, ▲ indicates t-VA, and □ indicates SA.

[Fig.2] Butyribibrio fibriso in t-VA accessory zone (lane 2) and non-attached zone (lane 1) A thin-layer chromatogram of lipid bodies of Rubens HF-11 strain. The lipids from t-VA-added cocoon were subjected to weak alkaline hydrolysis (0.1N NaOH, 100 ° C, 1 hour) and were scored in lane 3. As the developing solvent, black mouth form + acetone (96: 4) was used for A, and black mouth form + ethanol (95: 5) was used for B. Standard substances (Std) are triolein (TG), oleic acid (free FA), diolein (DG; mixture of 1,3-DG and 1,2-DG), 1-monoolein (1-MG), 2-molybdenum. Nolein (2-MG) and phosphatidylcholine (PL) were purchased from Sigma.

[Fig. 3] Diagram showing the effect of addition of Petribilli brio fibrisolvens MDT-10 and bifid baterilium adrecentis HF-11 on the t-VA content in the effluent in continuous culture of mixed rumen microorganisms ( (Total number of viable bacteria at the beginning, 1 X 10 ¹¹ ciu / ml) ₀ A is the group to which both bacteria are not added (control group), B is Butyrivibrio 'Fibriosolvens MDT-10 strain (1 X 10 ⁹ cfo / m 1 ), And C indicates the added force of butyricibrio 'Fibriosolvens (1 x 10 ⁹ cfo / ml) and bifid bacterium' addressestis (1 x 10 ⁹ cfo / ml), respectively. LA, carbohydrates, and protein are added every 6 hours, and LA (○), t-VA (A), which flows out during 0-1, 2-3, and 5-6 hours after LA addition. And the amount of SA (mouth) was plotted.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

 [0025] The feed additive for livestock of the present invention is characterized by containing a microorganism having an ability to produce an unsaturated compound and a microorganism having an ability to take up an unsaturated compound. In addition, instead of microorganisms having the ability to produce unsaturated compounds, the unsaturated compounds themselves should be included.

 [0026] Examples of unsaturated compounds include t-VA.

[0027] The microorganism having the ability to produce t-VA to be used is not particularly limited. For example, a microorganism belonging to the genus Butyribibrio can be used, and the genus Eubacterium (Evssen et al., 1984), the genus Fusobacterium (Fusobacterium) (Harfoot et al, 1997), Megasufera (MeeasphaeralJ¾ (Kim et al, 2002)), etc. Microorganisms belonging to the genus Butyribibrio can be used as microorganisms belonging to Butyrivibrio 'Fibrisolvens. As a strain belonging to Fibriosorbens, Butyrivibrio 'Fibrisolvens TH1 Strain (Fukuda et al., 2005), butyribibrio 'Fibrio sorbens MDT-10, butyribibrio' Fibrino sorbens ATCC19171 (Fukuda et al., 2005), butyribibrio 'Fiber solvens ATCC 51255 (Fukuda et al. , 2005), butyribibrio 'Fibriosolvens A 38 strain (Fukuda et al., 2005), butyribibrio' Fibrisolvens OB156 strain (Fukuda et a 1., 2005), etc. It is preferable to use the MDT-10 strain. Butyri vibrio 'Fiber sorbens MDT-10 strain was isolated by the inventor and is a patent biological deposit center of the National Institute of Advanced Industrial Science and Technology (Tsukuba Sakaihigashi 1-chome 1-chome 1 6) is deposited under the deposit number FERM BP-10540 (date of deposit: July 8, 2005). Further, a mutant strain of this strain may be used in place of the butyribibrio 'fibrinosorbens MDT-10 strain. Here, the “variant strain” refers to a strain originating from butyribibrio fibrisolvens MDT-10 strain, which has one or more mycological properties and is different from the above strain.

[0028] The microorganism having t-VA uptake ability to be used is not particularly limited. For example, enteric bacteria such as Bifidobacterium, Escherichia, and Lactob adll are used. Can do. Among intestinal bacteria, it is particularly preferable to use a microorganism belonging to Bifidobacterium adolescentis, which preferably uses a microorganism belonging to the genus Bifidobacterium. Among the microorganisms of Bifidobacteria adrecentis, it is preferable to use Bifidobacterium adrecente strain HF-11. Bifido Batterium vadrecentis strain HF-11 is a strain isolated by the present inventor and deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-10539. Day: 2 July 8, 005). In addition, a mutant strain of this strain may be used in place of the Bifido Batterium 'adrecentis HF-11 strain. The meaning of the “mutant strain” is the same as in the case of butyribibrio fibrisolvens MDT-10.

[0029] As unsaturated compounds other than t-VA, unsaturated fatty acids such as conjugated linoleic acid, linoleic acid, linolenic acid, arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, vitamin A, β-power rotin, Examples thereof include carotenoids such as xanthophyll. Several bacteria that produce these compounds are known. For example, conjugated linoleic acid As a microorganism having a production ability, a strain (MDT-5 strain) belonging to Butyrivibrio fibrisolvens has been isolated by the present inventor. In addition, microorganisms capable of producing docosahexaenoic acid or eicosapentaenoic acid include Vibrio genus T3615 (Yano et al., 1994), Moritella marina (M2l ki li l (Morita et al., 2005). ), And deep-sea microorganisms such as Shewanella marinintestina (Morita et al., 2005) and algae-like marine microorganisms collectively called Labyrinthura (Hiroshi Akikuma 'Kazuhisa Komine. 2005. Production of useful lipids by marine micro-organisms Application, Foods Food Ingredients J. Jpn., 210: 758-764.) In addition, as a microorganism capable of taking up docosahexaenoic acid or eicosapentaenoic acid, Shuwanella benthica (Shewanella benthica) is known. Shewanella violacea and Moritella vavanosii are considered to have such abilities (Fang et al, 2004) ₀

 [0030] The microorganism used in the feed additive for livestock of the present invention may be a living bacterium, but is preferably a frozen and dried living bacterium. Moreover, the feed additive for livestock of the present invention may contain components other than the microorganisms described above.

 [0031] The mixing ratio of the microorganism having the ability to produce unsaturated compounds and the microorganism having the ability to take up unsaturated compounds is not particularly limited. For example, when the unsaturated compound is t-VA, the weight ratio of the former to the latter is 1 : 3 to: L: preferably in the range of 5.

 [0032] The feed additive for livestock of the present invention is preferably used for an animal having rumen, that is, a ruminant such as a sushi, a hedge, a goat, etc., which has a strength that can be used for general livestock.

 [0033] The amount of the livestock feed additive of the present invention added to the feed is not particularly limited. For example, when the unsaturated compound is t-VA, the weight of both microorganisms relative to 100 g of a general mixed feed It is preferable to add so that the force is about 5 to 1 lg.

[0034] The feed additive of the present invention increases the amount of unsaturated compounds in the digestive tract of livestock by incorporating unsaturated compounds into microorganisms and preventing further reduction reaction. The technique of incorporating such unsaturated compounds into microorganisms to prevent reduction produces unsaturated compounds in an environment where microorganisms that reduce unsaturated compounds exist (for example, juice of rumen contents). It can also be applied to methods. That is, in an environment where microorganisms that reduce unsaturated compounds exist, unsaturated microorganisms and unsaturated microorganisms By culturing both microorganisms having the ability to take up compounds, unsaturated compounds can be produced efficiently.

 Example

 Hereinafter, the present invention will be described in more detail with reference to examples.

 [0036] The materials and methods used in the experiment will be described first.

 [0037] (1) Test bacteria and culture conditions

Fukuda et al. (Fukuda et al., 2005) have reported the origin of the Butyri vibrio 'Fiber sorbens TH1 strain. Butyribibrio 'Fiber sorbens MDT-10 strain was newly isolated from the goat (Japanese native species) lumen by the roll tube method (Ogimoto and Imai, 1981) and identified according to Bryant's criteria (Bryant, 1986). Similarly, Biff id butter Teri © arm 'add Resentisu HF-11 strain (Scardovi, 1986), Eshierikia' co ¹ Escherichia coli) HF- 23 strain (Orskov, 1986), Biff id Agrobacterium 'base blade (Bifidobacterium breve) HF -31 strains (Scardovi, 1986) and Lactobacillus fermentum HF-3 strains (Kandler and Weiss, 1986) were newly isolated and identified from adult feces. Fukuda et al. (Fukuda et al, 2004) have reported the origin of bifidobacterium 'breve TH1 strain. Bifidobacterium 'Adresentis JCM1275, Bifidobacterium bifidum TCM1255, Bifidoba cterium infantis JCM1222, Bifidobacterium longum J CM12 'Coli JCM 1649 and Lactobacillus' Farmentum JCM 1137 were purchased from the RIKEN Microbial System Storage Facility (JCM).

 [0038] In principle, each strain was cultured in a 30 or 50 ml vial with 15 or 30 ml of growth medium. The growth medium is a basal medium mixed with rumen centrifuge supernatant 3: 1 (Miyazaki et al., 1992). The composition of the basal medium (g / L) was as follows. K HPO, 0.45; KH PO, 0.45; (NH) SO, 0.9; NaCl, 0.9; CaCl · 2Η

 2 4 2 4 4 2 4 2 2

Ο, 0.12; MgSO · 7Η Ο, 0.19; glucose, 2.0; cellobiose, 2.0; trypticase

 4 2

Puton (BBL; Becton Dickinson, Cockeysville, MD, USA), 1.0; Yeast extract (Difco Laboratories Inc., Detroit, MI, USA), 1.0; and Sistine 'HC1, 0.6. The pH of the medium was adjusted to 7.0 (Miyazaki et al., 1992). LA and t-VA are bovine blood unless otherwise stated After mixing with clear albumin (BSA) at a ratio of 1: 3 (abbreviated as LA / BSA and t-VA / BSA, respectively), it was added to the culture (Fukuda et al, 2005). The culture was anaerobically performed at 39 ° C, and in principle, performed in triplicate.

 [0039] The growth of the bacteria was estimated by the OD value. When fatty acids (FA) are added to the culture,

 600

 Growth was estimated by measuring the amount of nitrogen (N). The amount of bacterial cells N was quantified by the Kjeldahl method. The viable count of butyribibrio fibrisolvens and intestinal bacteria in mice was measured by a roll tube method using a growth medium supplemented with 2% (wt / vol) agar. Five test tubes were used for each sample, and the number of each colony was counted (Fukuda et al., 2005).

[0040] (2) Culture of mixed rumen microorganisms

 Luminous strength of goats (Japanese native species) The middle layer of the lower layer solution was obtained by incubating the juice of the collected contents with quadruple gauze for 30 minutes in C 0 gas phase, and then removing the lifted feed pieces by suction.

2

 Was used as a mixed rumen microorganism (Hino et al, 1993). Notch culture was performed in principle using the medium and method described above.

[0041] An apparatus having three overflow type fermenters was used for continuous culture, and the structure and operation method were in accordance with the report of Hino and Hamano (Hino and Hamano, 1993). CO ki

 2 Under the flow, the juice of the rumen contents and the modified basal medium (liquid excluding the basal medium sugar and trypticase 'peptone) were mixed at a ratio of 1: 2 and injected into the fermenter (300 ml capacity). CO always

 2 The modified basal medium was fed continuously with aeration. The dilution rate was set to 0.1 / h. Carbonate and protein were added to the fermentor every 6 hours. That is, 3 g of a mixture of 50 g starch, 10 g frutatooligosaccharide (Meiji Seika, Tokyo), 10 g cellulose powder (Advantech, Tokyo), 20 g casein, and 5 g trypticase 'peptone (BBL) Added. These substrates can be clarified by placing them in a nozzle previously substituted with CO for 24 hours.

 2

 Most of the zeros were removed. LA is adsorbed on these substrates and every 6 hours 2

2

0 mg (70 μmol) was added. The culture was performed at 39 ° C for 24 hours, and the pH was maintained at around 6.8. During cultivation, the effluent accumulated every hour was collected, and LA, t-VA, and SA in the effluent were quantified. Butyl vibrio fibrisolvens MDT-10 and bifidobacterium adrecentis HF-11 are added to the total viable count of mixed rumen bacteria (1 X loH cfo / ml) at the start of culture. It was poured into the fermenter so as to be% (1 × 10 ⁹ cfo / ml). Continuous culture The same test was repeated twice, with 3 test sections performed simultaneously in 3 fermenters.

[0042] (3) Search for bacteria with high t-VA uptake capacity

 Bacteria were isolated from more than 500 colonies each from goat lumen and adult feces by the roll tube method described above. Each isolate was cultured overnight in a growth medium (15 ml) supplemented with 5 μmol of t-VA / BSA and then spun down (20,000 × g, 10 minutes, 4 ° C.). Pellet twice with 15 ml of 50 mM potassium phosphate (KPi) buffer supplemented with 0.1% (vol / vol) Triton X-100 to remove t-VA attached to the surface of the precipitated cells. Wash 'centrifuged. Similarly, the pellet was washed with 15 ml of KPi buffer and spun down to remove Triton X-100. Next, 10 ml of black mouth form + methanol (2: 1) was added to the pellet, and the total cell lipid was extracted by vigorous stirring for 30 minutes. 5 ml of water was added, stirred for 5 minutes, and spun down (20,000 x g, 10 minutes, 4 ° C). After transferring the lower layer to the sample vial and volatilizing the black mouth form by aeration of N gas,

 2

 T-VA was quantified by gas chromatography (GLC) as described below.

[0043] Next, bacteria identified as having high t-VA uptake ability and the top 4 species were identified, and then the t-VA uptake rate and the presence or absence of hydrogenation ability were examined. The t-VA uptake rate was determined by culturing each bacterium to an OD value of 1.0, and then adding 5 μmol of t-VA / BSA to 15 ml of medium.

 600

 Re-cultured to a D value of 1.5 (late log phase) (2-3 hours). After incubation, use the same method as above

600

 The amount of t-VA was quantified. For the uptake rate, the amount of t-VA taken up during this time was divided by the average value of the amount of N cells before and after re-culture, and the value was expressed per hour. On the other hand, regarding the hydrogenation ability, LA-I and CLA-R activities were measured using cells cultured separately by a usual method (described later).

 [0044] (4) Analysis of molecular species of t-VA taken up into Bifidobacterium 'adrecentis HF-11 strain

Bifidobacterium 'adrecentiise HF-11 strain was cultured for 5 hours (until late logarithmic growth) in a growth medium (30 ml) supplemented with 5 μmol of t-VA / BSA. Cell strength after washing with% Triton X-100 solution Total lipid was extracted with black mouth form + methanol (2: 1). This total lipid was separated by thin layer chromatography (TLC), and the molecular species into which t-VA was incorporated was examined. Chromium form + acetone (96: 4) was used as the TLC developing solvent, and 50% (vol / vol) H 2 SO 4 was sprayed and heated for spot color development (Fukuda et al., 2002). Re For detection of lipid (PL), coloration with Dittmer-Lester reagent was also performed (Dittmer and Lester, 1964). That is, 4 g of MoO ³ was added to 100 ml of 25 N-sulfuric acid and dissolved while gently boiling to make A solution. A solution of 0.18 g of powdered Mo in 50 ml of solution A, slowly boiled for 15 minutes and dissolved is used as solution B, and a mixture of solution A + solution B + H ² 0 (1: 1: 2) 0 辻 1 ^ 61-1 ^ 3 6 r reagent was used. When the amount of t-VA in each spot was examined, spot color development was omitted, the spots were scraped based on the Rf value, lipids were extracted with black mouth form + methanol (2: 1), and analyzed by GLC.

 [0045] From the results of TLC and GLC, t-VA was incorporated in the spots presumed to be monoglyceride (MG) and diglyceride (DG). The ratio of glycerol was measured. That is, after spotting MG and DG spots, extraction was performed several times with black mouth form + methanol (2: 1). The extract was divided into two equal parts, one for fatty acid (FA) quantification (described below) and the other for glycerol quantification. For the determination of glycerol, after distilling off the solvent, a blood triglyceride determination kit (Triglyceride E-Test Soichi, Wako Pure Chemicals, Tokyo) was used. That is, MG and DG were hydrolyzed with lipoprotein lipase, and the produced glycerol was phosphorylated with glycerol kinase. The resulting glycerol-3-phosphate is acidified with glycerol-3-phosphate oxidase, and H 0 produced in the reaction is acidified with 4-aminoantipyrine with peroxidase to produce a blue color.

twenty two

 Let The blue light absorbance was measured at a wavelength of 600 to determine the amount of glycerol.

[0046] (6) Determination of FA and organic acids

The total lipid or various lipids described above were transmethylated as follows. On the other hand, in the case of batch culture of mixed rumen microorganisms, the reaction is stopped by cooling the culture vial with ice, and in the case of continuous culture, the effluent is recovered in ice water and then chloroform + methanol (2 1) An equal amount of the mixed solution was added, lauric acid was added as an internal standard, and the mixture was vigorously stirred for 5 minutes. After centrifuge (5,000 X g, 5 minutes), the black mouth form layer was recovered. After adding the same amount of Kuroguchi form to the recovered Kuroguchi form and stirring again, the Kuroguchi form layer was recovered in the same manner, and both recovered solutions were combined to obtain total lipid. Next, 5% hydrochloric acid-methanol was added to the total lipid distilled off from the solvent, transmethylated at 60 ° C for 20 minutes, and each FA was identified and quantified by GLC and GLC-mass spectrum analysis ( Fukuda et al., 2002) ₀ of isolates The generated organic acids were analyzed by HPLC for identification and to know the aspects of fermentation in mixed rumen microbial culture systems (Fukuda et al, 2002) ₀

 [0047] (7) Measurement of LA-I and CLA-R activities

 LA-I and CLA-R activities were measured using live bacteria. That is, the bacteria cultured until late in logarithmic growth were collected by centrifugation (20,000 × g, 10 minutes, 4 ° C.), suspended in 50 mM KPi buffer (pH 7.0), and used immediately. For LA-I activity measurement, add 0.1 ml of LA dimethyl sulfoxide (DMSO) solution (1.0 / z mol / ml reaction mixture) to KPi buffer (2.7 ml), and then the cell suspension (0.2 ml) was added to initiate the reaction and incubated at 30 ° C for 3 minutes (Hunter et al, 1976). Thereafter, the reaction mixture was ice-cooled, and 3 ml of the above lipid extraction solvent was added to stop the reaction. Next, after adding 0.5 mg of lauric acid as an internal standard, lipid extraction and FA quantification were performed as described above to determine the amount of LA decrease and the amount of CLA produced. Since the LA isomerization reaction was linear for 3 minutes, this numerical force was also determined for enzyme activity.

 [0048] For measurement of CLA-R activity, a reaction mixture containing the above KPi buffer, CLA in DMSO (1.0 μmol / ml), 30 mM methylviologen, and 90 mM sodium dithionite solution The cell suspension (0.2 ml) was added to (2.8 ml) and the reaction was started. The reaction was performed anaerobically by incubating at 40 ° C for 5 minutes. The reaction was stopped as described above, and the amount of CLA decreased and the amount of t-VA produced were measured.

 [0049] (8) Data evaluation

 Using the Sigma Stat statistical analysis system Qandei Scientinc, San Rafael, CA), the analysis was performed by Tukey's test method or Student's t test method.

 [0050] [Example 1] Isolation of a new strain (MDT-10) of butyribibrio fibrisolvens with high t-VA production ability, and effect of addition of MDT-10 strain to batch culture system of mixed rumen microorganisms

Fukuda et al. (Fukuda et al, 2005) reported the discovery of a new strain (TH1) of butyribibrio 'Fibriosolvens that has a high t-VA-producing ability, but the newly isolated strain (MDT-10) The t-VA production rate was about 30% higher than that of the TH1 strain (1.6 μmol / h / mg fungus-N versus 1.2). LA-I activity of MDT-10 strain was not much different from TH1 strain (0.53 μmol / min / mg cell-N compared to 0.45), but CLA-R activity was nearly 40% higher (to 0.19) 0.26 μmol / min / mg bacterial cell-N). Therefore, this strain rapidly accumulates LA without tapping CLA. Converted.

[0051] MDT-10 strain is added to the mixed rumen microorganism batch culture system so that the total viable count of mixed bacteria is 1% (2 X 10 ⁹ cfo / ml), and cultured in LA-added medium. As a result, the conversion rate from LA to t-VA was remarkably increased, and the t-VA peak strength increased more than twice that of the case where no SMDT-10 strain was added (Figures 1-A and B). However, 10 hours later, most of the t-VA produced was reduced to SA.

 [Example 2] Isolation of bacteria having high t-VA uptake ability

 As mentioned above, in the rumen microorganism mixed system, even if t-VA production increases, it will eventually be subjected to hydrogenation, so t-VA can be reduced by incorporating the produced t-VA into bacteria. We tried to protect the action of hydrogenated bacteria. Rumen bacteria generally have a low FA uptake capacity (Hino et al, 1993), but we could not find a high t-VA uptake capacity from rumen bacteria. The butyribibrio 'Fibriosolvens MDT-10 strain was also highly capable of t-VA uptake in spite of its very high ability to produce t-VA (Table 1). The same was true for other strains of Butyri vibrio fibrisolvens (data not shown).

 [0053] However, some bacteria with high t-VA uptake ability were isolated from adult feces. Of these, the top four fungi with the highest potential were identified. As a result, we found Bifidobacterium 'adrecentis HF-11 strain as a bacterium with extremely high t-VA uptake capacity (Table Do. Also, this strain has not only t-VA but also other CLA uptake capacity. Much higher than that of Escherichia coli HF-23 (data not shown) .The HF-11 strain is more than Bifido Batterium 'adrecentis JCM1275 (standard strain) About 10% more t-VA was captured (data omitted).

 [0054] However, Bifido Batterium 'Bifidum JCM1255 strain (standard strain), Bifido Batterium

'Infantes JCM1222 strain (standard strain) and Bifidobatterum longum JCM12 17 strain (standard strain) had a t-VA uptake capacity of less than half that of Bifidobacterium adrecentis HF-11. (Data omitted) The bifidobatterium genus does not seem to have high t-VA uptake in general. The t-VA uptake capacities of Escherichia coli JCM 1649 (standard strain), Bifidobataterum breve TH1 and Lactobacillus fermentum JCM 113 7 (standard strain) One strain and large difference There was no. Based on the above, it can be said that the bifidobatterium 'adrecentis HF-11 strain is particularly an enteric bacterium with a high t-VA uptake capacity.

[0055] After culturing the HF-11 strain, the amount of t-VA recovered from the cells and the culture supernatant was almost equal to the amount added to the medium (data not shown). As described below, this strain does not synthesize t-v A by itself (Table 2). Therefore, it is considered that the HF-11 strain does not hydrogenate t-VA. In other words, it can be said that this strain has a precious property that it has a high ability to take up t-VA into the cells without hydrogenation. The HF-11 strain did not have both LA-I and CLA-R (Table D o

 [0056] When Bifido Butterium adrecentis HF-11 was cultured in growth medium (30 ml) supplemented with 5 μmol t-VA for 8 hours (until growth stopped), 4.5 μmol or more T-VA was taken up into the fungus body. T-VA incorporated into the HF-11 strain is 62, 3, respectively, in free FA, DG (1,2-DG + 1,3-DG), MG (1-MG + 2-MG), and PL. 19 and 15% were incorporated (Figure 2, Table 2). In addition, the proportion of t-VA in total FA for each of the above lipid molecular species was 89, 78, 84, and 89%, respectively. When cultured in a culture medium without t-VA, t-VA was not detected not only in the cells (Table 2) but also in the culture supernatant (data not shown). Therefore, it is considered that the HF-11 strain does not synthesize t-VA.

 [0057] The fact that the TLC spots were DG and MG also confirmed the specific force of FA and glycerol after hydrolysis with lipoprotein lipase. In addition, the results of weak alkaline hydrolysis (0.1 N NaOH, 95 ° C, 1 hour) suggested that most of PL would be glyceose PL (Fig. 2). Since these glyce mouth lipids can be digested in the small intestine, most of the t-VA in HF-11 cells is thought to be absorbed by the host.

[0058] The HF-11 strain has a force of uptake of t-VA and oleic acid (c9-C18: l) into the cells at similar rates. The uptake rate of SA (18: 0) is 1/3 that of C18: l. It was about. The uptake rate of C18: 2 (LA and CLA) was about 1/10 of C18: 1, and the uptake of C18: 3 (linolenic acid and conjugated linolenic acid) was even slower. In this way, it is interesting that C18: l has a very high uptake capacity and is not necessarily in parallel with the number of double bonds. In addition, the HF-11 strain seemed to suppress de novo synthesis of FA when there was a large amount of externally supplied FA (Table 2). This is very interesting for the regulation of FA synthesis and the viewpoint power. [0059] [Table 1] ί-VA uptake capacity of isolated bacteria and specific activities of LA-I and CLA-R. ^Υ uptake of bacteria ί-VA Activity ¾

 (mol / mg cell N / 5 h) LA-I CLA- R

A. Adolescentis Hi ¹ -11 0.76 ND ND

 E. coli HF-23 0.18 ND ND

 B. breve HF-31 0.09 0.07 ND

 L. fermentum HF-3 0.07 ND ND

B. fibrisolvens MDT- 10 ND 0.52 0.29

¹⁾ Four bacteria with high ί-VA uptake ability isolated from human feces. ND means below detection limit (<0,01).

²⁾ Activity is expressed as specific activity (/ • i mol / min / mg bacterial cell N).

[0060] [Table 2]

 FA composition in the lipid fraction of B. adolescentis HF-11 cultured in medium with or without ί-VA, and the amount of f-VA incorporated into each fraction Lipid fraction i-VA FA Morpa -Addition of FA total amount f-VA uptake amount

 14: 0 16: 0 18: 0 C9-18: l To VA Other (/ i mol / container) (mol / container) (%) Free FA 4 34 38 22 0 1 2.11

 + 0 8 3 0 89 0 3.03 2.79 62

DG 17 48 35 0 0 0 0.73

 + 0 8 8 0 84 0 0.23 0.15 3

MG ― 15 42 43 0 0 0 0.20

 + 0 5 6 0 89 0 0.97 0.87 19

PL ― 13 58 6 7 0 16 1.90

 + 0 7 2 0 78 13 0.88 0.69 15

(+) And (-) indicate addition and no addition, respectively.

[Example 3] Coexistence of Butyri vibrio fibrisolvens and bifid butterium adrecentis

When single cultures of Butyri vibrio 'Fibriosolvens MDT-10 and Bifidobatatrium adorescens HF-11 were grown individually, the growth rate as measured by OD was almost equal. . However, the cell length of Butyrivibrio fibrisolvens is much shorter than that of bifidbacterium adrecentis.

 600

 The number of bacteria was about 2.5 times more vigorous in Butyri vibrio fibrisolvens than in Bifidobatterum adrecentis. When both bacteria were mixed and co-cultured at a ratio of 1: 1 in terms of OD value,

 600

 The ratio of both bacteria by microscopic examination was always about 2.5, which did not change until the growth was stopped (data not shown). Therefore, the growth rates of both bacteria are almost equal, and there is no antagonism between the two bacteria.

 [0061] [Example 4] Uptake of t-VA produced by Butyri vibrio fibrisolvens by bifidobacterium 'adrecentis'

 Buty vibrio 'Fibrinosolvens MDT-10 strain and bified butteryrum adorestis HF-11 strain were separately cultured in the presence of LA, t-VA was not detected in the cells after washing (Table 3). This is considered to be due to the lack of the ability to take the former bacterial case-VA into the cells and the ability to produce the latter bacterial case-VA. However, when both bacteria were co-cultured, 51% of the total FA in the cells combined with both bacteria was t-VA. At the end of the culture (16 hours), it was converted to about 80% strength-VA of the added LA (18 μmol / 30 ml medium) and was present in the cells. In another experiment, when both bacteria were cultured in the presence of 5 μmol / 30 ml LA for 5 hours (until the late logarithmic growth phase), more than 95% of them were recovered from the cells in the form of t-VA. (Data omitted). These results indicate that the HF-11 strain efficiently incorporated t-VA produced by the MDT-10 strain.

[0062] [Table 3]

Fatty acid composition of total lipid in cells of β. / Ifen 'o / ve / w MDT-10 and β. Adolescentis HF-11 cultured in the presence of LA. 囷 Fatty acid composition

 14: 0 16: 0 18: 0 c9-18: l ill-18: l 18: 2 Other

MDT-10 24 38 8 0 0 0 30

HF-11 0 39 40 2 0 8 11

MDT-10 and HF-11 10 21 4 1 51 0 13 l After 16 hours of single culture or co-culture in the presence of LA (18 mol / 30 ml medium), 0.1% (v / v) Triton X Washed twice with buffer containing -100. The table shows the percentage of fatty acids in total lipids.

[Example 5] Effect of simultaneous loading of butyricum vibrio fibrisolvens and bifid baterium adorecentites on t-VA production and accumulation in a mixed rumen microbial culture system

 In a batch culture system of mixed rumen microorganisms, butyral vibrio fibrisolvens MDT-10 and bifid butterium vadrentense HF-11 are combined with 1% of the total viable count of mixed rumen bacteria (2 X loH cfo / ml). When added to the culture medium and cultured in LA-added medium, the rate of decrease of accumulated t-VA was smaller than that of the MDT-10 strain alone (Figure 1-B vs. C). The amount of accumulated t-VA after 10 hours increased more than 10 times (0.02 mM vs. 0.23 mM). In actual lumens, the contents always flow down, so the larger the amount of t-VA in the lumen at each time point, the greater the amount of t-VA flowing. Therefore, it is clear that the amount of t-VA flowing increases with the addition of HF-11 strain, and the addition effect of both strains seems to be extremely large compared to the control group to which neither strain is added. (Figure 1-A vs. C).

When 1% of the total viable count of mixed rumen bacteria was added to the continuous culture system of mixed rumen microorganisms, the total amount of t-VA in the effluent in 24 hours increased by about 30% ( Figure 3-A, B). In addition, the later LA was added, the faster the LA decreased in the effluent (the slope of the LA graph in Fig. 3-B became larger). It shows that the conversion rate to VA increased later. The reason for this is thought to be an increase in the number of MDT-10 strains. In other words, the MDT-10 strain grows at a rate greater than the dilution rate. Suggest that This corresponds to the increase in t-VA outflow at a later time. As the amount of t-VA increased much more than the amount of SA (Fig. 3-A vs. B), compared to the hydrogenation ability of t-VA by mixed rumen microorganisms, MDT-10 strain It is thought that the increase in t-VA production due to the was greater.

 [0064] In addition to the MDT-10 strain, when 1% of the total viable count of the mixed rumen bacteria was added, the amount of t-VA that flowed out in 24 hours was reduced to the MDT-10 strain. It increased approximately twice as much as the case of only (Figure 3-C). In addition, the decrease in SA even though t-VA increased, which significantly reduced SA outflow. The fact that HF-11 stock power ¾-VA was taken in, so t-VA was hydrogenated. This is probably due to the action of the fungus. When both strains were added, the amount of t-VA efflux was 2.5 times that of the control group to which neither was added. When both strains were added, the t-VA efflux increased significantly over time compared to the MDT-10 strain alone. Presumed to have proliferated. In this experiment, it is possible that the effect of adding both bacteria to 1% of the total number of viable bacteria will increase the number of bacteria over time, even if the number of added bacteria is small.

 [0065] During continuous culture, the appearance of fermentation product (organic acid) production did not change much, and no significant change was observed due to the addition of both bacteria (data not shown). In addition, the number of protozoa by microscopic examination did not decrease so much (data not shown). Therefore, when cultured for about 24 hours under these culture conditions, it is considered that the microbial flora did not change so much except for the added bacteria.

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 This specification includes the contents described in the specification and Z or drawing of the Japanese patent application (Japanese Patent Application No. 2005-239274) which is the basis of the priority of the present application. In addition, all publications, patents, and patent applications cited in the present invention are incorporated herein by reference as they are.

Claims

The scope of the claims

 [1] A feed additive for livestock, comprising a microorganism having an unsaturated compound or an unsaturated compound-producing ability, and a microorganism having an ability to take up an unsaturated compound.

 [2] The microorganism having an ability to produce unsaturated compounds is a microorganism having an ability to produce transpaxenoic acid, and the microorganism having an ability to take unsaturated compounds is a microorganism having an ability to take up transpaxenoic acid. Item 2. The animal feed additive according to Item 1.

 [3] The microbial power having the ability to produce transbatacenoic acid is the feed additive for livestock according to claim 2, which is a butyrivibrio 'Fibriosolvens MD T-10 strain or a mutant thereof

[4] The feed additive for livestock according to claim 2 or 3, wherein the microorganism has a transbatacenoic acid uptake ability, and is Bifidobataterum 'Adrescentis HF-11 strain or a mutant thereof.

 [5] The livestock feed additive according to any one of claims 1 to 4, wherein the livestock is a ruminant.

 [6] A livestock feed comprising the livestock feed additive according to any one of claims 1 to 5.

[7] Cultivate microorganisms capable of producing unsaturated compounds and microorganisms capable of incorporating unsaturated compounds in an environment where microorganisms that reduce unsaturated compounds exist, and collect culture force unsaturated compounds. A method for producing an unsaturated compound.

[8] The method for producing an unsaturated compound according to [7], wherein the unsaturated compound is transpacsenoic acid.

 [9] Butyrivibrio 'Fibriosolvens MDT-10 strain capable of producing transbatacenoic acid.

[10] Bifidobataterum adrecentis HF-11 strain capable of incorporating transbatacenoic acid.