JP3278574B2 - Color tone improver - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a color tone improving agent for improving the color tone of the epidermis, meat or produced eggs of seafood or poultry.

[0002]

2. Description of the Related Art Carotenoid compounds are pigments that are widely present in microorganisms such as bacterial cells, algae, and tissues and organs of plants and animals. Carotenoid compounds are food,
Food field as a colorant for beverages, salmon, trout, red sea bream,
Applications in the field of feed for the purpose of improving the color of meat, epidermis, egg yolk, and the like of meat or epidermis of fish and shellfish such as shrimp, and poultry such as chicken. In addition, carotenoid compounds are known to have antioxidant activity, and are expected to be used as antioxidants. In recent years, some carotenoid compounds have been found to have a carcinogenic inhibitory effect, and are expected to be used as pharmaceuticals in the future.

[0003]

One of the major uses of carotenoid compounds in the field of feed is as an agent for improving the color of animal epidermis, meat or their produced eggs. For example, astaxanthin, canthaxanthin, and the like are used as color enhancers for seafood such as salmon, trout, and red sea bream. It is also known to add canthaxanthin, astaxanthin, zeaxanthin, capsanthin and the like to feeds as a method for improving the color tone of epidermis, meat and egg yolk of poultry such as chickens.

[0004] Canthaxanthin is only a chemically synthesized product, and a natural product is not industrially produced. Chemically synthesized products are problematic in terms of safety and recent natural product oriented situations. Astaxanthin may be a chemically synthesized product or a natural product, but the chemically synthesized product has a problem in terms of safety and a natural product-oriented situation in recent years. Astaxanthin natural products are obtained by extraction from krill and crayfish, but have problems in that their content is small, stable supply is difficult, and costs are high. Also, the red yeast Phaffia rhodozyma producing astaxanthin (Paffia)
rhodozyma) has a strong cell wall, so even if it is supplied as it is, its absorption efficiency is extremely low, and it is necessary to pulverize the cell mechanically or decompose it with chemicals, enzymes, etc., which poses a problem in terms of cost. is there.

[0005] In addition, when the cells are decomposed, there is a problem that the stability of the carotenoid compound is reduced. In addition, green algae producing astaxanthin (Haematococc)
us) is a problem similar to Phaffia yeast in that the cell effect is low as it is because the cell wall is hard. Zeaxanthin is extracted from marigold and the like, but has a drawback in that its color tone is yellow and its use is limited. Capsanthin is a carotenoid contained in paprika, which is used for coloring egg yolk.However, it is extremely unstable to heat, light, etc., and the growing state of paprika is greatly affected by the weather, making it difficult to provide an industrially stable supply. It has disadvantages such as

[0006]

Means for Solving the Problems The present inventors have conducted various studies to solve the above problems, and as a result, completed the present invention.
That is, the microorganism E-396 strain (FERM BP-42)
83), its mutants, microorganism A-581-1 strain (FER
MBP-4671) and a mutant thereof, from a culture solution of the microorganism, the microorganism itself, a degradation product of the microorganism, a crushed product of the microorganism, and a carotenoid compound extracted or isolated therefrom. It relates to the selected color tone improver.

The microorganism used in the present invention is E-
396 strains can be mentioned. This strain was newly isolated by the present inventors, and was transferred to FERM BP-4 on April 27, 1993 by the National Institute of Bioscience and Biotechnology, Institute of Industrial Science and Technology.
283. This strain has the following mycological properties:

[0008]

[Table 1]

[0009]

[Table 2]

[0010]

[Table 3]

[0011]

[Table 4]

[0012]

[Table 5]

[0013]

[Table 6]

From the above results, the strain E-396 was thought to be a bacterium belonging to the genus Agrobacterium because it was an aerobic gram-negative bacillus and had hair peripheries. And DNA-D
Negative from the result of NA homology. Although the possibility of sphingomonas bacteria or photosynthetic bacteria was considered from the color tone of the colony, sphingolipids and bacteriochlorophyll were not detected, indicating that the strains were not strains of any genera. From the color tone, colony, GC content, and quinone of the colony, E-396 strain and the preservation strains of each bacterial species estimated to be related
Examination of NA-DNA homology revealed that no genus showing a high homology value was obtained. Furthermore, a molecular phylogenetic tree was prepared from the base sequence of the 16S ribosomal RNA of the E-396 strain by the neighbor joining method.

As a result, it was found that the E-396 strain was systematically independent of any closely related genera. Therefore E-
The 396 strain was confirmed to be a bacterium belonging to a completely new genus that is not a known genus. Other microorganisms used in the present invention include A-581-1 strain. This strain was newly isolated by the present inventors, and was deposited as FERM BP-4671 on May 20, 1994 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
This strain has the following mycological properties:

[0016]

[Table 7]

[0017]

[Table 8]

[0018]

[Table 9]

From the above results, the A-581-1 strain was found to be E-3
It was determined to be a bacterium belonging to the same new genus as the E-396 strain because it has the same properties as the 96 strains and has high DNA-DNA homology with the E-396 strain. The method of culturing these strains may be any method as long as it produces carotenoid compounds, and is, for example, as follows. That is, the producing bacterium contains a carbon source, a nitrogen source, an inorganic salt, and, if necessary, special required substances (for example, vitamins, amino acids, nucleic acid bases, etc.) necessary for growth.

Examples of the carbon source include sugars such as glucose, sucrose, fructose, trehalose, mannose, mannitol, and maltose; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, and malonic acid; ethanol, propanol; Butanol, pentanol,
Alcohols such as hexanol and isobutanol are exemplified. The addition ratio varies depending on the type of carbon source, but is usually 1 to 100 g, preferably 2 to 50 g per liter of the medium.
It is. Examples of the nitrogen source include one or two of potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, and urea.
More than one species is used. Although the addition ratio varies depending on the type of the nitrogen source, it is generally 0.1 g to 10 g, preferably 1 to 3 g per liter of the medium.

As the inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, zinc chloride, sulfuric acid One or more of copper, calcium chloride, calcium carbonate, sodium carbonate and the like are used. The addition ratio varies depending on the type of the inorganic salt, but is usually 0.001 per 1 L of the medium.
1 to 10 g. Specially required substances include one or two of vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, corn steep liquor, dried yeast, soybean meal, soybean oil, olive oil, corn oil, linseed oil, etc. More than one species is used. The addition ratio varies depending on the type of the substance, but usually, 1 g to 200 g per 1 L of the medium,
Preferably, it is 10 to 100 g.

The pH of the medium is 2 to 12, preferably 6 to 10.
Adjust to Culture conditions are 15 to 80 ° C., preferably 20 to 80 ° C.
The culture is carried out with shaking culture or aeration and agitation culture for 1 to 20 days, preferably 2 to 8 days.
In the cultured cells produced by the above method usually astaxanthin, adonixanthin, β-carotene, echinenone,
Carotenoid compounds such as canthaxanthin, zeaxanthin, β-cryptoxanthin, 3-hydroxyechinenone, asteroidenone, and adnirubin accumulate as a mixture. The production ratio of the carotenoid compound contained in these cells can be adjusted according to the use of the target animal or the like.

For example, by changing the aerobic conditions in the culture, the production ratio of the carotenoid compound can be changed. As an example, the production rate of adonixanthin can be increased by increasing the dissolved oxygen concentration in the culture solution. Alternatively, the producing strain can be mutated to preferentially produce a particular carotenoid pigment, e.g., artificially mutated so that one component is produced preferentially over the other. . Examples of such a mutation treatment include physical methods such as X-ray irradiation and ultraviolet irradiation, and chemical mutation agents such as NTG (N-methy).
1-N'-nitro-N-nitrosoguan
Idine and EMS (ethylmethane su
Chemical methods such as mutagenesis by lfonate) can be used.

In the present invention, the microorganism E-396 is added to the target animal.
Strain (FERM BP-4283), a mutant thereof, a culture solution of a microorganism selected from the microorganism A-581-1 strain (FERM BP-4671) and its mutant, the microorganism strain itself, a decomposition product of the microorganism cell, One or more color tone improvers selected from the crushed microbial cells and the carotenoid compounds extracted or isolated therefrom are provided, but may be provided alone or as a mixture with feed. .

When the water content of the culture solution is an obstacle, it can be used after drying with a spray drier or the like. When only the cells are separated from the culture solution, the separation can be performed by a conventional filtration method, centrifugation method or the like. The cells containing water obtained by the separation may be supplied as they are, or may be used after drying with a spray drier or the like.
The cells of the bacterial cells may be decomposed by chemical treatment such as enzymatic treatment or acid hydrolysis or crushed by physical treatment such as ultrasonic treatment or pressure crushing before use.

Since the carotenoid compound is formed inside the cells, the carotenoid compound can be extracted from the cells with a solvent. As the solvent used here, any solvent can be used as long as the compound can dissolve the carotenoid compound. For example, organic solvents such as acetone, chloroform, dichloromethane, hexane, tetrahydrofuran, cyclohexane, ethanol, methanol, isopropyl alcohol, benzene, carbon disulfide, and diethyl ether are used.Preferably, chloroform, dichloromethane, tetrahydrofuran, acetone, ethanol, methanol, Isopropyl alcohol is used. Also, each carotenoid compound can be isolated and purified. Conventional methods such as adsorption, elution, and dissolution can be used for the generation.

Although the color tone improving agent of the present invention may be used as feed as it is, an antioxidant such as BHT (butylhydroxytoluene), ethoxyquin, and vitamin E is added for the purpose of preventing the decomposition of the carotenoid contained therein. It is also preferably adopted. Further, these surfaces may be coated with gelatin or the like. The animal to be colored may be any one as long as the color tone enhances its value.For example, if it is a seafood, red sea bream, coho salmon, rainbow trout, swordfish, trout, hamachi, goldfish, western prawn, prawn, lobster, etc. Improves skin, meat and egg color. In poultry, chicken, quail and the like are mentioned, and the color tone of epidermis, meat and egg yolk is improved.

When the color tone improving agent of the present invention is mixed with feed, it can be mixed with the feed in powder form, pelletized or flaked. An advantage of providing the cells of the present strain without decomposition or pulverization is that absorption efficiency is extremely good. That is, the bacterial cells of the present invention belong to bacteria, have a thin cell wall, and are not strong, so they are easily decomposed in the animal body and have extremely high absorption efficiency. In contrast,
Since the red yeast Phaffia rhodozyma has a strong cell wall, the cell wall needs to be decomposed in order for the carotenoid compound to be absorbed in the animal body, so that the absorption efficiency is poor.

The astaxanthin produced by the strain used in the present invention is (3S, 3'S) -astaxanthin, and its purity is almost 100%. It is known that astaxanthin, which is a natural product, exists in crayfish, haematococcus, salmon, trout, and red sea bream, and has a high (3S, 3'S) content. On the other hand, Fafia Rodchima (3
It is known that the content of the (R, 3′R) -form is large and has an absolute configuration opposite to that of naturally occurring astaxanthin. Astaxanthin produced by the strain of the present invention is 100
% (3S, 3'S) -astaxanthin and having the same absolute configuration as astaxanthin, which is predominant in nature, is of great industrial value.

[0030]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Embodiment 1 FIG. 10 mL of a medium having the composition shown in Table 10 was added to a medium having a diameter of
The mixture was placed in a test tube having a size of 12 mm and steam sterilized at 121 ° C. for 15 minutes. One loopful of the E-396 strain (FERM BP-4283) was inoculated into this, and cultured at 30 ° C. for 2 days with reciprocal shaking at 300 rpm. 10 mL of this culture was added to a medium having the same composition as above.
Inoculate 6 500 mL Sakaguchi flasks containing 0 mL
Reciprocal shaking culture was performed at 0 ° C. for 2 days at 100 rpm.

Next, 600 mL of this culture solution was inoculated into a 30-L fermenter containing 20 L of a medium having the same composition as above, and inoculated at 30 ° C.
Aerobic culture was performed at 300 rpm and 1.0 vvm for 76 hours.
Cells (wet weight: 550 g) were obtained from 18 L of the culture solution using a Sharpless centrifuge. Add 10 L of tap water to these cells
After the suspension was added and sufficiently suspended, the cells (wet weight: 540 g) were obtained again by a Sharpless centrifuge. Next, the cells 54
After adding 1000 L of tap water to 0 g and sufficiently suspending, the cells were dried using a spray drier to obtain 185 g of dried cell bodies. The operating conditions were an inlet air temperature of 200 ° C., an outlet air temperature of 100 ° C., and a suspension supply rate of 46 mL / min. The carotenoid content in the dried cells was analyzed by high performance liquid chromatography, and the composition was as shown in Table 11.

[0032]

[Table 10]

[0033]

[Table 11]

Next, 40-week-old white leghorns (10 birds)
Was put in one cage and fed with a diet obtained by adding 0.5 mass% of the dried bacterial cell obtained by the above method to a standard diet for layer test manufactured by Japan Combined Feed Co., Ltd. for 3 weeks. The color tone of the egg yolk was visually observed, and the red color was clearly observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

Embodiment 2 FIG . 10-week-old white leghorn (10
The feathers) were put in one cage, and fed with 1.5 mass% of the dried bacterial cell obtained in Example 1 to a standard feed for layer test manufactured by Japan Combined Feed Co., Ltd. for 3 weeks. When the color tone of the epidermis and meat was visually observed, red coloration was clearly observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

Embodiment 3 FIG . Red sea bream (60-70g)
The dried bacterial cell obtained in Example 1 was applied to 20 mice at 4.0 ma.
The feed to which ss% was added was adjusted and added continuously for 90 days. When the color tone of the body surface was visually observed, red coloration was observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

Embodiment 4 FIG . E-396 strain (FERM BP
-4283) to NTG (N-methyl-N'-ni).
(Tro-N-nitrosoguanidine), and colonies having different color tones were selected. The carotenoid compounds in the cultures of these strains were analyzed, and strains that did not produce astaxanthin but produced canthaxanthin (canthaxanthin-producing mutants) were selected. Put 10 mL of medium having the composition shown in Table 1 into a test tube having a diameter of 18 mm.
Steam sterilization was performed at 21 ° C. for 15 minutes. One loopful of a canthaxanthin-producing mutant was inoculated into the loop, and 300 rp for 2 days at 30 ° C.
m 2 reciprocal shaking culture was performed. 10 mL of this culture solution was inoculated into six 500 mL Sakaguchi flasks each containing 100 mL of a medium having the same composition as above, and subjected to reciprocal shaking culture at 100 rpm at 30 ° C. for 2 days.

Next, 600 mL of this culture was inoculated into a 30-L fermenter containing 20 L of a medium having the same composition as above, and was inoculated at 30 ° C.
Aerobic culture was performed at 300 rpm and 1.0 vvm for 76 hours.
Cells (wet weight: 500 g) were obtained from 18 L of the culture solution by a Sharpless centrifuge. Add 10 L of tap water to these cells
After the suspension was sufficiently added and suspended, the cells (wet weight: 480 g) were obtained again by a Sharpless centrifuge. Next, the cells 48
After adding 1000 L of tap water to 0 g and sufficiently suspending, the cells were dried using a spray drier to obtain 170 g of a dried cell. The operating conditions were an inlet air temperature of 200 ° C., an outlet air temperature of 100 ° C., and a suspension supply rate of 46 mL / min. The carotenoid content in the dried cells was analyzed by high performance liquid chromatography, and the composition was as shown in Table 12.

[0039]

[Table 12]

Next, 40-week-old white leghorns (10 birds)
Was put in one cage, and fed to the standard feed for layer test manufactured by Japan Combined Feed Co., Ltd., to which 0.3% by mass of the dried bacterial cells obtained by the above method was added, was fed for 3 weeks. When the color tone of the yolk was visually observed, orange coloring was clearly observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

Embodiment 5 FIG . 10-week-old white leghorn (10
The feathers) were placed in one cage, and fed to the standard feed for layer test manufactured by Japan Combined Feed Co., Ltd., to which 0.9% by mass of the dried bacterial cell obtained in Example 1 was added, for 3 weeks. The color tone of the epidermis and the meat was visually observed. As a result, the orange color was clearly observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

Embodiment 6 FIG . Medium 10 having the composition of Table 13
The mL was placed in a test tube having a diameter of 18 mm and steam sterilized at 121 ° C. for 15 minutes. A-581-1 strain (FERM BP-
4671) and inoculated with one platinum loop at 300 rpm for 2 days at 30 ° C.
Was performed. 10 mL of this culture was inoculated into six 500 mL Sakaguchi flasks containing 100 mL of a medium having the same composition as in Table 1 and subjected to reciprocal shaking culture at 100 rpm at 30 ° C. for 2 days. Next, 600 mL of this culture solution was inoculated into a 30 L fermenter containing 20 L of a medium having the same composition as in Table 1,
Aerobic culture was performed at 0 ° C., 300 rpm, and 1.0 vvm for 80 hours. Cells (wet weight: 400 g) were obtained from 18 L of the culture solution by a Sharpless centrifuge.

After 10 L of tap water was added to the cells and suspended sufficiently, the cells (wet weight: 380 g) were obtained again by a Sharpless centrifuge. Next, 1000 L of tap water was added to 380 g of the cells, and after sufficient suspension, the cells were dried using a spray drier to obtain 120 g of a dried cell. The operating conditions were an inlet air temperature of 200 ° C, an outlet air temperature of 100 ° C, and a suspension supply rate of 46 mL / min. The carotenoid content in the dried bacterial cells was analyzed by high performance liquid chromatography to find the composition shown in Table 14. Next, 40-week-old white leghorns (10 birds) were placed in one cage, and 1.2 mass% of the dried bacterial cells obtained by the above method was added to a standard feed for layer test manufactured by Japan Combined Feed Co., Ltd. The feed was given for 3 weeks. The color tone of the egg yolk was visually observed, and the red color was clearly observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

[0044]

[Table 13]

[0045]

[Table 14]

Embodiment 7 FIG . 10-week-old white leghorn (10
The feathers) were placed in one cage and fed with 3.5 mass% of the dried bacterial cell obtained in Example 6 to a standard feed for layer test manufactured by Japan Combined Feed Co., Ltd. for 3 weeks. When the color tone of the epidermis and meat was visually observed, red coloration was clearly observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

Embodiment 8 FIG . Red sea bream (60-70g)
The dried bacterial cell obtained in Example 6 was applied to 20 mice at 9.0 ma.
The feed to which ss% was added was adjusted and added continuously for 90 days. When the color tone of the body surface was visually observed, red coloration was observed and the color tone was improved as compared with the control group in which the dried bacterial cells were not added.

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1452 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear sequence AGTTTGATCC TGGCTCAGAA CGAACGCTGG CGGCAGGCTT AACACATGCA AGTCGAGCGA 60 GACCTTCGGG TCTAGCGGCG GACGGGGG TCACGGAG TAACGCGTGGTCACGGTCTAGGAG GCCCGCGTTG GATTAGGTAG TTGGTGGGGT AATGGCCCAC CAAGCCGACG 240 ATCCATAGCT GGTTTGAGAG GATGATCAGC CACACTGGGA CTGAGACACG GCCCAGACTC 300 CTACGGGAGG CAGCAGTGGG GAATCTTAGA CAATGGGGGC AACCCTGATC TAGCCATGCC 360 GCGTGAGTGA TGAAGGCCTT AGGGTTGTAA AGCTCTTTCA GCTGGGAAGA TAATGACGGT 420 ACCAGCAGAA GAAGCCCCGG CTAACTCCGT GCCAGCAGCC GCGGTAATAC GGAGGGGGCT 480 AGCGTTGTTC GGAATTACTG GGCGTAAAGC GCACGTAGGC GGACTGGAAA GTCAGAGGTG 540 AAATCCCAGG GCTCAACCTT GGAACTGCCT TTGAAACTAT CAGTCTGGAG TTCGAGAGAG 600 GTGAGTGGAA TTCCGAGTGT AGAGGTGAAA TTCGTAGATA TTCGGAGGAA CACCAGTGGC 660 GAAGGCGGCT CACTGGCTCG ATACTGACGC TGAGGTGCGA AAGCGTGGGG AGCAAACAGG 720 ATTAGATACC CTGGTAGTCC ACGCCGT AAA CGATGAATGC CAGACGTCGG CAAGCATGCT 780 TGTCGGTGTC ACACCTAACG GATTAAGCAT TCCGCCTGGG GAGTACGGTC GCAAGATTAA 840 AACTCAAAGG AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGAAGC 900 AACGCGCAGA ACCTTACCAA CCCTTGACAT GGCAGGACCG CTGGAGAGAT TCAGCTTTCT 960 CGTAAGAGAC CTGCACACAG GTGCTGCATG GCTGTCGTCA GCTCGTGTCG TGAGATGTTC 1020 GGTTAAGTCC GGCAACGAGC GCAACCCACG TCCCTAGTTG CCAGCAATTC AGTTGGGAAC 1080 TCTATGGAAA CTGCCGATGA TAAGTCGGAG GAAGGTGTGG ATGACGTCAA GTCCTCATGG 1140 GCCTTACGGG TTGGGCTACA CACGTGCTAC AATGGTGGTG ACAGTGGGTT AATCCCCAAA 1200 AGCCATCTCA GTTCGGATTG TCCTCTGCAA CTCGAGGGCA TGAAGTTGGA ATCGCTAGTA 1260

ATCGCGGAAC AGCATGCCGC GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGTCAC 1320 ACCATGGGAG TTGGTTCTAC CCGACGACGN TGCGCTAACC TTCGGGGGGC AGGCGGCCAC 1380 GGTAGGATCA GCGACTGGGG TGAAGTCGTA ACAAGGTAGCGTCCGTCGACGGATCCGCGTCCGGATCCGCGTCCGCGTCGATCGATCGATCGATCGACGTGCCTCCGCGTCGA

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12N 1/20 ZNA C12N 1/20 ZNAA C12P 23/00 C12P 23/00 // C12N 15/09 C12N 15/00 A (72) Inventor Minoru Mizuta 8 Chidori-cho, Naka-ku, Yokohama-shi, Kanagawa Pref. Japan Petroleum Corporation Central Research Laboratory (56) References JP-A-5-76347 (JP, A) JP-A 5-219983 (JP, A) 58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1/00-1/38 C12P 23/00 A23K 1/16 A23K 1/18 A23L 1/272 C09B 61/00

Claims (2)

(57) [Claims] 1. The microorganism E-396 strain (FERM BP-
4283) or a culture solution of a mutant thereof having the same color tone improving effect, the microbial cell itself, a decomposition product of the microbial cell, a crushed product of the microbial cell, and a carotenoid compound extracted or isolated therefrom A color improver selected from: 2. The microorganism A-581-1 (FERM B)
P-4671) or a culture solution of the mutant having the same color tone improving effect, the microbial cell itself, a decomposed product of the microbial cell, a crushed product of the microbial cell, and extracted or isolated therefrom A color improver selected from carotenoid compounds.