Method for improving flavor and / or maintaining freshness of chicken meat by raising chickens

Abstract

This application provides: an agent for increasing free amino acids, derivatives thereof, and / or α-tocopherol in chicken meat, characterized in that the agent includes a Bacillus subtilis strain such as Bacillus subtilis (C-3102 strain) is included as an active ingredient and is administered to chickens; a flavor improving agent for chicken meat; a freshness improving agent for chicken meat; feed additives that include these agents; and a method for improving the flavor and / or maintaining the freshness of chicken meat.

Description

[Technical Field]

[0001] The present invention relates to a method for improving the flavor and / or maintaining the freshness of chicken meat by raising chickens, specifically, the method comprises feeding chickens a feed formulated with Bacillus subtilis bacteria. [Background technology]

[0002] Methods for improving the taste (especially the delicious taste) of meat include the commonly used use of seasonings, as well as post-slaughter treatments of raw meat, such as adding a concentrate of raw kiwifruit pulp (Patent Document 1) and adding advantame to meat or processed meat products (Patent Document 2). Feed-based methods include feeding chickens a feed containing 1.5 to 2.0% by weight of lysine to increase the amount of free glutamic acid in the meat (Patent Document 3), feeding poultry a feed containing 1.2% by weight or more of valine and 0.8% by weight or less of isoleucine to suppress an increase in free glutamic acid in the meat and the sourness of the meat (Patent Document 4), feeding a feed containing a protease to increase the free amino acids in the muscles of living animals (Patent Document 5), and a livestock breeding method characterized by feeding livestock a feed containing fermented milk to increase the unsaturated fatty acid and / or free amino acid content of the meat (Patent Document 6).

[0003] The technique of preserving meat quality by feeding vitamin E is also described in the Japanese Feeding Standards (Non-Patent Documents 1 and 2) and is widely used (Non-Patent Document 3). However, no method has been reported for increasing the α-tocopherol concentration in raw meat without feeding vitamin E or feed containing vitamin E.

[0004] Methods for maintaining the freshness of meat include storing it in a sealed container with an oxygen scavenger under negative pressure, low oxygen, and low temperature (Patent Document 7), immersing it in a fermented seasoning with an ethyl alcohol concentration of 30% or more and a sugar content of 1% or less for 2 to 60 seconds (Patent Document 8), and immersing it in an aqueous solution containing kojic acid or spraying it with said aqueous solution (Patent Document 9).However, all of these methods treat raw meat after slaughter, and require treatment agents, containers, and equipment.Maintaining the freshness of meat through feed, including the feeding of probiotics, is not known.

[0005] As an example of the use of a probiotic, a livestock meat quality improver containing the probiotic Bacillus subtilis C-3102 as an active ingredient is known, for example, from Patent Document 10. This meat quality improver is characterized by its ability to reduce fat in livestock meat.

[0006] The Bacillus subtilis C-3102 strain is also known to have effects on improving body weight gain and feed conversion ratio (Patent Document 11), and is also known to be used in feed for young pigs (Patent Document 12). However, it is not known that the Bacillus subtilis C-3102 strain improves the flavor, particularly the palatability, of meat. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 8-24544 [Patent Document 2] Patent No. 6593333 [Patent Document 3] Patent No. 4945762 [Patent Document 4] Patent No. 5260101 [Patent Document 5] Patent No. 3422955 [Patent Document 6] Patent Application No. 2017-163983 [Patent Document 7] Special Publication No. 62-27785 [Patent Document 8] Special Publication No. 1-30456 [Patent Document 9] Special Publication No. 5-30422 [Patent Document 10] Patent No. 3187911 [Patent Document 11] Japanese Patent Publication No. 63-209580 [Patent Document 12] Japanese Patent Publication No. 62-232343 [Non-patent literature]

[0008] [Non-Patent Document 1] Japan Feeding Standards for Beef Cattle (2000 edition), edited by the National Agriculture and Food Research Organization, Central Livestock Association (Tokyo, Japan) [Non-patent document 2] Japan Feeding Standards for Swine (2000 edition), National Agriculture and Food Research Organization, Central Livestock Industry Association (Tokyo, Japan) [Non-patent document 3] Mitsumoto, Mitsuru, 1996, Nihon Chikukaiho, 67(12), 1110-1126 Summary of the Invention [Problem to be solved by the invention]

[0009] The objective of the present invention is to improve the flavor (for example, palatability) of chicken meat by providing (or feeding) chickens with feed containing a live bacterial agent of Bacillus bacteria. [Means for solving the problem]

[0010] The present invention includes the following features. (1) An agent for increasing free amino acids, their derivatives and / or α-tocopherol in chicken meat, which comprises as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (international accession number: FERM BP-1096) or its derivatives, bred strains or mutant strains, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and which is administered to chickens. (2) The agent for enhancing the proliferation of Bacillus subtilis bacteria according to (1) above, wherein the Bacillus subtilis bacteria are in a spore state. (3) The Bacillus subtilis bacteria was 1 x 10 per 1 g of the enhancer. 5 cfu ~ 1 × 10 13 The agent for increasing the number of bacteria in the human body according to (1) or (2) above, wherein the number of bacteria is 1 or more. (4) A flavor improver for chicken meat, comprising as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or its derivatives, bred strains, or mutant strains, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and characterized in that the flavor improver is administered to chickens. (5) The chicken flavor improver according to (4) above, characterized in that the chicken flavor is delicious. (6) The chicken meat flavor improver according to (4) or (5) above, wherein the flavor improvement of chicken meat is achieved by enhancing the umami and rich umami flavor or bitter impurities. (7) The agent for improving chicken meat flavor according to any one of (4) to (6) above, wherein the improvement in chicken meat flavor is an increase in free amino acids in the chicken meat. (8) The agent for improving chicken meat flavor according to any one of (4) to (7) above, wherein the Bacillus subtilis bacterium is in the form of a spore. (9) The Bacillus subtilis bacteria was 1×10 per 1 g of the flavor improver. 5 cfu ~ 1 × 10 13 The chicken meat flavor improving agent according to any one of (4) to (8) above, wherein the chicken meat flavor improving agent is a chicken meat flavor improving agent having a virulence factor of 1 or more. (10) An agent for maintaining the freshness of chicken meat, which comprises, as an active ingredient, at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or a derivative, bred strain, or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and which is administered to chickens. (11) The agent for maintaining freshness of chicken meat according to (10) above, wherein the maintaining of freshness of chicken meat is achieved by increasing the α-tocopherol concentration in the chicken meat. (12) The agent for maintaining freshness of chicken meat according to (10) or (11) above, wherein the maintaining of freshness of chicken meat is achieved by lowering the K value (a constant for determining freshness) when the chicken meat is thawed after freezing. (13) The agent for maintaining freshness of chicken meat according to any one of (10) to (12) above, wherein the Bacillus subtilis bacterium is in the form of a spore. (14) The above Bacillus subtilis bacteria is 1 x 10 per 1 g of the freshness-keeping agent. 5 cfu ~ 1 × 10 13 The agent for maintaining freshness of chicken meat according to any one of (10) to (13) above, wherein the freshness of chicken meat is 0.01 to 0.01 cfu or more. (15) A feed additive for maintaining the flavor and / or freshness of chicken meat, comprising the increaser, flavor improver, or freshness maintainer according to any one of (1) to (14) above. (16) A feed for improving the flavor and / or maintaining the freshness of chicken meat, containing the feed additive according to (15) above in an amount of 1 ppm to 1000 ppm or more by weight. (17) The feed according to (16) above, which is an early-stage feed or a late-stage feed. (18) A method for improving the flavor and / or maintaining the freshness of chicken meat, comprising feeding chickens the feed described in (16) or (17) above. (19) The method according to (18) above, further comprising obtaining the chicken meat for consumption.

[0011] In the present invention, chickens fed a diet containing Bacillus subtilis bacteria (e.g., Bacillus subtilis C-3102 strain (hereinafter sometimes simply referred to as "C-3102 strain")) as an active ingredient have higher amounts of free amino acids and their derivatives than chickens (controls) that do not. Individual free amino acids or their derivatives contribute to various metabolic processes and, depending on their type, are expected to have anti-fatigue effects and various disease-ameliorating effects. Furthermore, free amino acids are known to affect taste, resulting in less odorous and more flavorful (especially delicious) meat, as well as maintaining freshness, which can benefit both chicken consumers and producers. In this specification, "Bacillus subtilis C-3102 strain" is used interchangeably with "Bacillus subtilis C-3102 strain" or "Bacillus subtilis C-3102 strain."

[0012] This specification includes the disclosure of Japanese Patent Application No. 2020-039264, from which this application claims priority. [Brief explanation of the drawings]

[0013] [Figure 1A-B] This figure shows the effect of calsporin (CS)-supplemented diet on the amounts of various amino acids in the superficial pectoral muscle of chickens (broilers). The amino acids measured were (A) threonine, tyrosine, serine, glutamic acid, glutamine, taurine, alanine, and arginine, and (B) methionine, lysine, leucine, isoleucine, valine, phenylalanine, and histidine. [Figure 1C] This figure, a continuation of Figure 1A-B, shows the effect of calsporin (CS)-supplemented diet on the amount (µmol) of amino acid derivatives per gram of the superficial pectoral muscle (wet tissue) of broiler chickens. The amino acid derivatives measured were o-phosphoethanolamine, hydroxyproline, and 1-methylhistidine. [Figure 2]This figure shows the results of an odor analysis showing the effect of a diet containing 100 ppm calsporin (CS) (CS group) and a diet without CS (control group) on the odor (smell) of the superficial pectoral muscle of chickens (broilers). In the odor analysis, graph B shows the odor index equivalent value (vertical axis) and the results of an analysis of the contribution of each item (i.e., hydrogen sulfide, sulfur-based compounds, amine-based compounds, organic acids, aldehydes, esters, aromatic compounds, and hydrocarbons) (horizontal axis) to the odor. Meanwhile, graph A shows the results of a comprehensive evaluation of the odor of all of the above items for the CS group and the control group. In the figure, the odor index equivalent value indicates the evaluation of odor intensity. [Figure 3] This figure shows the results of evaluation using a taste sensor of the "bitterness and off-flavor," "umami," and "umami richness" of the superficial pectoral muscle of chickens (broilers) after feeding diet containing calsporin (CS). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will now be described in further detail. 1. Agents for increasing amino acids, their derivatives or α-tocopherol in chicken meat, agents for improving chicken flavor, agents for maintaining chicken freshness, and feed additives In a first aspect, the present invention provides an agent for increasing amino acids, derivatives thereof and / or α-tocopherol in chicken meat, the agent comprising, as an active ingredient, at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or a derivative, bred strain or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and the agent is administered to chickens.

[0015] In a second aspect, the present invention provides an agent for improving the flavor of chicken meat, which comprises as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or a derivative, bred strain, or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and which is to be administered to chickens.

[0016] The term "flavor" as used herein refers to the delicious taste perceived by the senses of taste and smell when eating chicken or chicken soup. Deliciousness is a taste created by the harmony of umami, umami-rich flavor (also called "rich flavor"), and, in some cases, bitterness, off-flavors, sourness, saltiness, astringency, etc.

[0017] In a third aspect, the present invention provides an agent for maintaining the freshness of chicken meat, which comprises as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or a derivative, bred strain, or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and which is to be administered to chickens.

[0018] As used herein, "maintaining freshness" refers to, for example, lowering the K value (a constant for determining freshness) when thawed after freezing. Specifically, maintaining freshness prevents the freshness of chicken meat from decreasing.

[0019] In a fourth aspect, the present invention provides a feed additive for improving the flavor and / or maintaining the freshness of chicken meat, comprising the above-mentioned increasing agent, the above-mentioned flavor improving agent, or the above-mentioned freshness maintaining agent.

[0020] In this specification, the "Bacillus subtilis C-3102 strain" has the international deposit number FERM BP-1096 and is currently preserved at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE). This strain is the same as the active ingredient of the livestock meat quality improver characterized by reducing meat fat, as described in Patent Document 10. However, this document does not state or suggest the use of this strain to increase amino acids, their derivatives, and / or α-tocopherol in chicken meat, improve the flavor of chicken meat, or maintain its freshness, for example, to enhance the umami and richness of chicken meat.

[0021] As used herein, the "derivative, bred, or mutant" of Bacillus subtilis C-3102 can be obtained by culturing the parent strain C-3102 in the presence of chemical mutagens such as nitrosoguanidine, nitrosourea, methyl ethanesulfonate, or their derivatives, or by culturing the parent strain after irradiating it with high-energy radiation such as ultraviolet light or X-rays, or by allowing the strain to mutate naturally during storage. Among the resulting strains, those that increase the free amino acids (e.g., proteinogenic amino acids and other amino acids (e.g., taurine)), amino acid derivatives, and α-tocopherol in chicken meat when fed a diet containing the strain, compared to a control (raised without the strain), thereby improving the flavor of chicken meat (e.g., enhancing umami and richness of umami) or maintaining its freshness, are selected.

[0022] As used herein, "another Bacillus subtilis strain" refers to a Bacillus subtilis strain that has properties equivalent to those of Bacillus subtilis strain C-3102 but is different from C-3102 or its derivatives, bred strains, or mutant strains. Here, "equivalent properties" refers to the properties possessed by the C-3102 strain, i.e., the property of increasing the free amino acids, amino acid derivatives, and α-tocopherol in the meat of chickens fed the Bacillus subtilis strain, thereby improving the flavor of the chicken meat or maintaining its freshness, specifically, for example, the property of enhancing at least the umami and rich flavor of the chicken meat. In some cases, other properties, as described below, may also be included. The "other Bacillus subtilis strain" may be isolated from nature using known Bacillus subtilis isolation methods, or may be selected from known strains.

[0023] A preferred example of the Bacillus subtilis bacterium is Bacillus subtilis strain C-3102 or a derivative, bred strain or mutant strain thereof.

[0024] In one embodiment of the present invention, the Bacillus subtilis bacteria in the agent for increasing free amino acids, derivatives thereof, and / or α-tocopherol in chicken meat, the agent for improving chicken flavor, the agent for maintaining chicken freshness, or the feed additive are preferably in the form of spores. When chickens ingest the agent, flavor improver, freshness-maintaining agent, or feed additive in the form of spores, the Bacillus subtilis bacteria germinate and become vegetative cells, consuming oxygen during this process, which is thought to enhance anaerobic conditions in the chicken's digestive tract, creating a favorable environment for the proliferation of beneficial bacteria such as bifidobacteria and lactic acid bacteria and for the production of acid.

[0025] With regard to the property of increasing free amino acids, their derivatives, and / or α-tocopherol in meat in chickens, the increase means an increase compared to a control (raised without feeding the strain). Examples of amino acids that are increased include threonine, tyrosine, serine, glutamic acid, glutamine, taurine, alanine, arginine, methionine, lysine, leucine, isoleucine, valine, phenylalanine, and histidine.

[0026] Taurine is a type of sulfur-containing amino acid, and 50-80% of the taurine in the body is found in muscles, where it is said to play an important role in maintaining the body's homeostasis (for example, maintaining liver function, lowering blood cholesterol (preventing arteriosclerosis), maintaining blood pressure, maintaining normal body temperature, and maintaining hydration). Adding feed additives to the feed increases taurine in the superficial pectoral muscle by more than twice the control level.

[0027] Other amino acid derivatives, such as hydroxyproline and 1-methylhistidine, also increase.

[0028] Alpha-tocopherol also increases in chicken meat when chickens are fed diets containing the feed additive, compared to controls, regardless of whether the diet is supplemented with vitamin E. Alpha-tocopherol has the strongest antioxidant or peroxidation inhibitory effect of any component of vitamin E. Therefore, alpha-tocopherol contributes to the inhibition of peroxidation in chicken meat.

[0029] In one embodiment of the present invention, the feed additive further has the property of reducing the odor (bad smell) of chicken meat when fed to chickens. Specifically, analysis of the odor components of chicken meat shows a tendency to reduce odor components (e.g., hydrogen sulfide, sulfur-based, amine-based, organic acid-based, aldehyde-based, ester-based, aromatic, hydrocarbon-based, etc.). Because the odor of chicken meat impairs the flavor of the meat, it is preferable to minimize the amount of components that cause this odor.

[0030] In another embodiment of the present invention, the feed additive further has the property of maintaining the freshness of chicken meat (e.g., suppressing loss of freshness) when fed to chickens. The freshness of chicken meat can be determined by measuring the K value (a constant for determining freshness), with a lower K value indicating higher freshness. The feed additive of the present invention reduces the K value of chicken meat. This maintenance of freshness of chicken meat is partly related to the increased concentration of α-tocopherol in the chicken meat.

[0031] In another embodiment of the present invention, the feed additive has the property of enhancing bitter off-flavors in chicken meat when fed to chickens. The bitter off-flavors are derived from bitter substances, and at low concentrations correspond to richness or a secret flavor.

[0032] In another embodiment of the present invention, the Bacillus subtilis bacterium in the growth agent, flavor improving agent, freshness-maintaining agent or feed additive is 1×10 per 1 g of the growth agent, flavor improving agent, freshness-maintaining agent or feed additive. 5 cfu ~ 1 × 10 13 cfu or more, preferably 1 x 10 8 cfu ~ 1 × 10 12 cfu.

[0033] The feed additive may be, for example, Calsporin®. Approximately 1 x 10 per gram of Calsporin 10 It contains cfu of Bacillus subtilis C-3102 strain. Calsporin (registered trademark) (CALSPORIN; manufactured by Asahi Biocycle Co., Ltd.) is a feed additive for livestock (e.g., cattle, pigs, and chickens) containing the Bacillus subtilis C-3102 strain, a Bacillus subtilis strain, as its active ingredient. It is commercially available and is useful for improving meat and egg productivity by improving weight gain and feed conversion ratio. It is known that feeding an appropriate amount of Calsporin to livestock daily increases the number of beneficial intestinal bacteria (e.g., bifidobacteria and / or lactic acid bacteria) and optimizes the intestinal flora.

[0034] Bacillus subtilis bacteria can be cultured in liquid or solid media containing carbon sources, nitrogen sources, inorganic substances, and the like commonly used in the cultivation of Bacillus bacteria. Carbon sources include assimilable carbon sources such as glucose, fructose, sucrose, starch, and molasses. Nitrogen sources include peptone, meat extract, casein hydrolysate, and ammonium sulfate. If necessary, inorganic components such as phosphate, magnesium, potassium, sodium, calcium, iron, and manganese salts, vitamins, amino acids, antifoaming agents, and surfactants can also be added. Culture is preferably carried out under aerobic conditions, with the initial pH of the medium at 5 to 9, preferably 6 to 8, at a culture temperature of 20 to 50°C, preferably 35 to 40°C, and for a culture time of, for example, 12 hours to 7 days. After cultivation, the bacterial cells can be recovered by a separation method such as centrifugation. The recovered bacterial cells may be in any form, such as a culture, concentrate, dry product, liquid, granules, powder, pellets, rods, or spheres.

[0035] 2. Feed In a fifth aspect, the present invention provides a feed for improving the flavor (e.g., palatability) of chicken meat (preferably enhancing the umami, rich umami flavor, and / or bitter and off-flavors of chicken meat) and / or maintaining the freshness of chicken meat, the feed comprising a feed additive containing the increaser, flavor improver, or freshness-maintaining agent described in Section 1 above.

[0036] The feed may be any feed for chicken production, and is generally divided into early-season feed and late-season feed, each with a different composition of feed ingredients. Examples of feed ingredients include corn, grain sorghum, soybean meal, corn gluten meal, fish meal, animal fats and oils, L-lysine hydrochloride, DL-methionine, L-arginine, dicalcium phosphate, calcium carbonate, salt, vitamin K3, vitamin A, vitamin D, vitamin B, minerals, copper sulfate, and, as needed, vitamin E. The crude protein and metabolic energy content of the early-season feed are, for example, 22.0% and 3.20 Mcal / kg, respectively, and those of the late-season feed are, for example, 19.0% and 3.25 Mcal / kg. The early-season feed is fed to chickens, for example, from 0 to 20 days of age, and the late-season feed is fed to chickens, for example, from 21 days of age until shipping, for example, from 21 to 40 days of age or 21 to 50 days of age.

[0037] The feed additive of the present invention can be added to (or blended with) chicken feed, for example, early feed and late feed, or late feed. The amount of the feed additive added to the feed is not limited as long as it can achieve the properties described in Section 1 above, i.e., at least the property of increasing free amino acids, their derivatives, and α-tocopherol in chicken meat. In addition, the amount of Bacillus subtilis bacteria in the feed additive (e.g., 1 x 10 per 1 g of the feed additive) can be increased. 5 cfu ~ 1 × 10 13 cfu or more, preferably 1 x 10 8 cfu ~ 1 × 10 12 cfu), but may be contained at a weight of, for example, 1 ppm to 1000 ppm or more, 10 ppm to 1000 ppm, 30 ppm to 500 ppm, 50 ppm to 300 ppm, etc., and in some cases 1000 ppm to 2000 ppm, or 2000 ppm or more.

[0038] To increase α-tocopherol in meat, for example, when Calsporin (registered trademark) is contained at 50 ppm or more, the desired effect of increasing α-tocopherol can be achieved regardless of the addition of vitamin E. Furthermore, even greater effects can be expected when vitamin E is not added, since α-tocopherol decreases without the addition of vitamin E. On the other hand, to increase amino acids, for example, Calsporin (registered trademark) preferably contains 50 ppm or more, and the effect is expected to be further enhanced by containing 100 ppm or more.

[0039] When the feed additive is, for example, Calsporin (registered trademark), feed containing, for example, 10 to 1000 ppm or more, or 50 to 100 ppm or more of calsporin can be fed to chickens, for example, from 0 days of age until shipment, or, for example, from 15 to 20 days of age until shipment.

[0040] 3. Rearing Method In a sixth aspect, the present invention provides a method for improving the flavor (e.g., palatability) and / or maintaining the freshness of chicken meat, comprising feeding chickens the feed described in Section 2 above.

[0041] In one embodiment, the method of the present invention further comprises obtaining the chicken meat for consumption.

[0042] A free-range chicken coop has multiple sections, and each section can house approximately 10 to 20 chickens. Each section is covered with bedding, which is replaced regularly.

[0043] As described in Section 2 above, the feed additives may be added to early and late-stage chicken feed, and the early-stage feed may be fed from day one (0 days of age) to about 20 days of age, and the late-stage feed may be fed from about 21 days of age until shipment. Alternatively, the feed additives may be added to late-stage chicken feed, and the early-stage feed (not containing the feed additives) may be fed from day one (0 days of age) to about 20 days of age, and the late-stage feed may be fed from about 21 days of age until shipment.

[0044] Chicken meat raised and produced by the method of the present invention has improved flavor (e.g., deliciousness, preferably umami, rich umami, bitterness, off-flavors, etc.), an increased concentration of α-tocopherol, which contributes to inhibiting peroxidation, and can maintain freshness.

[0045] Specifically, the feed additives described in Section 1 above (e.g., Calsporin (registered trademark); approximately 1 x 10 per gram of Calsporin) 10 In the meat of chickens fed diets containing, for example, 50 ppm to 100 ppm (50 to 100 g per ton of feed) of Bacillus subtilis C-3102 (including cfu of Bacillus subtilis C-3102 strain), the contents of many types of amino acids, including glutamic acid, increase in a concentration-dependent manner depending on the feed additive, and the smell (odor) of the chicken meat tends to decrease. Evaluation of the chicken meat extract using a taste sensor reveals that the rich umami flavor, umami taste, and bitter and unpleasant flavors can be enhanced.

[0046] Furthermore, when chickens are fed a diet containing the feed additives described in Section 1 above (e.g., Calsporin (registered trademark)), regardless of whether vitamin E is added to the feed, the α-tocopherol content in the chicken meat increases compared to chickens fed a diet without the feed additive (control).

[0047] Furthermore, when slaughtered chicken meat is refrigerated for three days, frozen, and then thawed, the K value (a constant for determining freshness: the lower the better) decreases depending on the concentration of the feed additives described in Section 1 above (e.g., Calsporin (registered trademark)).

[0048] The method of the present invention increases many amino acids compared to the control, and the quantitative combination of these amino acids is thought to bring out the flavor (e.g., deliciousness) of chicken meat, such as umami, umami richness, and a harmony of bitter and unpleasant flavors. As used herein, "umami" refers to an initial taste, such as the umami of amino acids and nucleic acids, while "umami richness" refers to an aftertaste, a persistent richness exhibited by umami substances. It is generally known that, with regard to amino acids, umami and sourness are caused by glutamic acid and aspartic acid, sweetness is caused by glycine, alanine, threonine, serine, glutamine, proline, and asparagine, and bitterness is caused by amino acids such as tryptophan and phenylalanine (Kawai M et al., Amino Acids, 43:2349-58, 2012). Furthermore, an increase in α-tocopherol in chicken meat improves antioxidant activity or inhibits peroxidation, and also maintains freshness, which is thought to have a positive effect on the taste or palatability of chicken meat, such as its umami and rich umami flavor. [Example]

[0049] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. [Example 1] <Enhancing the umami and richness of chicken meat by feeding chickens feed containing Calsporin (registered trademark) (containing Bacillus subtilis C-3102 strain)> [Test method] Day-old broiler chickens (male, UK Ross; Matsumoto Keien Zao Hatchery, Miyagi, Japan) were raised to 14 days of age and then divided into three groups (5 birds per group). From 15 days of age, the chickens were fed three different diets: (i) control group (fed with normal feed), (ii) experimental group 1 (fed with normal feed + Calsporin (registered trademark; Asahi Biocycle Co., Ltd.) (50 ppm)), and (iii) experimental group 2 (fed with normal feed + Calsporin (100 ppm)). Each group was fed eight replicates of these diets until 40 days. The viable cell count per gram of calsporin was 1 x 10 10 The animals were then bled and slaughtered, and the superficial pectoral muscle (so-called breast meat) was collected from the carcass and subjected to the following tests.

[0050] (1) Measurement of free amino acids and their derivatives (A) Sample preparation 0.4 g of tissue was mixed with 1 ml of 0.5 mol / L trichloroacetic acid and homogenized using a bead mill (2,500 rpm, 15 seconds, cooling setting 5). The homogenized material was centrifuged at 10,000 × g for 3 minutes at 4°C. 600 μl of the supernatant was transferred to a 1.5 ml tube, and 600 μl of hexane was added and vigorously stirred. 150 μl of the aqueous solution was collected for each of the following (a) and (b). For (a), 50 μl of 0.3 mol / L sodium hydroxide and 800 μl of sodium citrate buffer (pH 2.2) were added. For (b), 50 μl of 0.3 mol / L lithium hydroxide and 800 μl of lithium citrate buffer (pH 2.2) were added and stirred. The solution was filtered through a 0.45 μm membrane filter and used as a sample.

[0051] (B) Protein constituent amino acid analysis [Separation conditions] Column: Shim-pack Amino-Na (100 mm × 6.0 mm ID), Mobile phase: Shimadzu Amino Acid Mobile Phase Kit Na (Shimadzu Corporation, Kyoto, Japan; product code 228-21195-94), Flow rate: 0.4–0.6 ml / min (using the gradient end conditions recommended by Shimadzu), Temperature: 60°C, Injection volume: 10 μl. [Detection conditions] Shimadzu Amino Acid Analysis Kit OPA Reagent (Shimadzu Corporation, product code 228-2119593), reaction reagent flow rate 0.2 ml / min, temperature: 60°C, detection wavelength: excitation 350 nm / emission 450 nm.

[0052] (C) Analysis of free amino acids including dipeptides [Separation conditions] Column: Shim-pack Amino-Li (100 ml x 6.0 mm, ID), mobile phase: Shimadzu Amino Acid Mobile Phase Kit Li Type (Shimadzu Corporation, product code 228-21195-95), flow rate: 0.6 ml / min (using the gradient end conditions recommended by Shimadzu), temperature: 39°C, injection volume: 10 μl. [Detection conditions] Shimadzu Amino Acid Analysis Kit OPA Reagent (same as above), reaction reagent flow rate 0.2 ml / min, temperature: 39°C, detection wavelength: excitation 350 nm / emission 450 nm.

[0053] (2) Odor analysis The odor analysis was outsourced to Shimadzu Techno Research, and was performed using the Shimadzu FF-2020S system (FF-2020, FAS-1, FDL-1).

[0054] (3) Evaluation of chicken meat extract using a taste sensor (3.1.) Preparation of chicken soup (i) 15 g of superficial pectoral muscle was collected from broilers in the control group and experimental group 1, placed in a reagent bottle containing 110 ml of distilled water, and incubated in a boiling water bath for 1 hour while stirring every 10 minutes. (ii) After incubation, the mixture was returned to room temperature, and the mince soup was filtered through absorbent cotton and then filter paper (ADVANTEC Toyo 5A), and then stored frozen until analysis.

[0055] (3.2.) Evaluation using a taste sensor (i) The taste sensor TS-5000Z manufactured by Intelligent Technology Corporation was used to evaluate bitterness, off-flavors, umami, and umami richness. Bitterness and off-flavors are derived from bitter substances, and at low concentrations correspond to richness or secret flavors. Umami is the umami taste of amino acids, nucleic acids, etc. Umami richness is the persistent richness exhibited by umami substances.

[0056] (4) Measurement of α-tocopherol content in superficial pectoral muscle [Sample preparation] 0.2 g of tissue was homogenized in 1 ml of 1% (w / v) sodium chloride solution. 0.2 ml of the homogenate was transferred to a 10 ml test tube, and 1 ml of 3% (w / v) pyrogallol in ethanol was added. After mixing, the homogenate was heated at 70°C for 2 minutes. 0.1 ml of 60% (w / v) potassium hydroxide solution was added, and the mixture was again heated at 70°C for 2 minutes. After cooling on ice, 2 ml of 1% (w / v) sodium chloride solution and 1.5 ml of ethyl acetate / n-hexane (1:9, v / v) solution were added. The mixture was vigorously stirred for 5 minutes, then centrifuged at 3,000 rpm for 5 minutes at room temperature (20-25°C), and 1.3 ml of the upper layer was transferred to a test tube. 1 ml of n-decane / n-hexane (1:20, v / v) solution was added, and the solvent was evaporated by blowing nitrogen gas on a 30°C heat block. After cooling, 100 μl of isopropyl alcohol was added to dissolve the residue, and the solution was filtered through a 0.45 μm membrane filter and used as a sample. [Separation / measurement conditions] Column: Shim-pack CLC-ODS (6 mm x 150 mm), mobile phase: HPLC-grade methanol, temperature: 40°C, injection volume: 20 μl, flow rate: 1 ml / min, detection wavelength: excitation 296 nm / emission 325 nm.

[0057] (5) K value (5.1.) Sample preparation The superficial pectoral muscles from broilers in the control group and experimental group 1 were processed in the following two ways. (i) The superficial pectoral muscles were frozen immediately after slaughter and then thawed immediately before the test. (ii) The superficial pectoral muscles were refrigerated for 3 days after slaughter, then frozen and thawed immediately before the test.

[0058] (5.2.) Measurement of K value [Sample preparation] 0.4 g of tissue was homogenized with 1 ml of 10% (v / v) perchloric acid solution. The homogenized tissue was centrifuged at 4,500 rpm for 10 minutes at 4°C, and the supernatant was collected. 10 N potassium hydroxide solution was added to the supernatant, and the pH was adjusted to 6.8. The sample was used for testing. [Separation / measurement conditions] Column: STR ODS-II (150 mmL x 4.6 mmI.D., Shimadzu Corporation), mobile phase: buffer solution containing 100 mM phosphate and 225 mM trimethylammonium / acetonitrile (100 / 1), temperature: 40°C, injection volume: 10 μl, flow rate: 0.8 ml / min, detection wavelength: 260 nm. [Calculation] (Hypoxanthine + Inosine) / (ATP + ADP + AMP + Inosinic acid + Inosine + Hypoxanthine) (%)

[0059] [result] (1) Free amino acids and their derivatives The free amino acid levels in the superficial pectoral muscles of chickens fed CS-supplemented diets in the control group (calsporin (CS) 0 ppm), experimental group 1 (CS 50 ppm), and experimental group 2 (CS 100 ppm) were measured, and the results are shown in Figures 1A, 1B, and 1C. Each column in the graph shows the average concentration of each amino acid (n = 10–12), and the error bars show the standard error (SE). When chickens were fed calsporin (CS)-added feed, the amino acids and their derivatives in the chicken meat were measured, and the following was found from Figures 1A to 1C. (i) In the 50 ppm and 100 ppm CS treatments, the amounts of glutamic acid, glutamine, taurine, and alanine increased in a CS concentration-dependent manner, and the amounts of threonine, tyrosine, serine, and arginine also increased (Figure 1A). (ii) The amounts of essential amino acids methionine, lysine, leucine, isoleucine, valine, phenylalanine, and histidine were higher in the 50 ppm and 100 ppm CS groups than in the control group (Figure 1B). (iii) The amounts of o-phosphoethanolamine, hydroxyproline, and 1-methylhistidine increased depending on the concentration of CS added (Figure 1C).

[0060] (2) Odor analysis results The results of the odor analysis are shown in Figure 2. As shown in Figure 2, the odor index value decreased in the CS (100 ppm) treatment area. Furthermore, the contribution of each component to odor was reduced in the CS treatment area for hydrogen sulfide (smells like hot springs or rotten eggs), sulfur compounds (smells like rotten cabbage), amine compounds (smells like rotten fish), organic acids (a pungent sour smell), aldehydes (a pungent sweet and sour burnt smell), esters (smells like glue), aromatic compounds (smells like gasoline), and hydrocarbons (a faint waxy smell). However, no difference was observed in ammonia (smells like human waste).

[0061] (3) Evaluation of chicken meat extract using a taste sensor Chicken soup was prepared (as described above) from the superficial pectoral muscles (breast meat) of the control group, experimental group 1 (CS 50 ppm), and experimental group 2 (CS 100 ppm), and the taste components of the chicken soup were analyzed using a taste sensor (product name TS-5000Z) from Intelligent Sensor Technology (Kanagawa, Japan). The results of the evaluation of "umami," "rich umami flavor," and "bitterness and off-flavors" of the superficial pectoral muscle extract of chickens fed a diet supplemented with calsporin (CS) are shown in Figure 3.

[0062] As shown in FIG. 3, CS 50 ppm and CS 100 ppm enhanced umami, rich umami flavor, and bitterness and off-flavors compared to the control.

[0063] "Umami" in taste sensors indicates umami components such as amino acids and nucleic acids, which supports the fact that free amino acids are increasing. "Umami richness" is the persistent richness exhibited by umami substances, and is effective in soups and dipping sauces. Bitterness and impurities correspond to richness and secret flavors.

[0064] As shown in (1) above, by feeding chickens CS-added feed, many amino acids increase compared to the control group, and it is thought that the quantitative combination of these amino acids brings out a balance of umami, rich umami flavor, and bitter and unpleasant flavors.

[0065] (4) α-Tocopherol content in chicken The effects of calsporin (CS)-supplemented diet on α-tocopherol concentrations in the superficial pectoral and gastrocnemius muscles of chickens (broilers) were investigated, and the results are shown in Table 1. The CS levels were 0 ppm (control group), 50 ppm (test group 1), and 100 ppm (test group 2). α-Tocopherol concentrations were measured in the following cases: when no antibiotics were added to the feed but vitamin E was added ("no antibiotics"), and when neither antibiotics nor vitamin E were added ("no antibiotics, no vitamin E preparation"). The results are shown in Table 1.

[0066] [Table 1] As shown in Table 1, the α-tocopherol concentrations in the superficial pectoral muscle and gastrocnemius muscle were higher in both the CS50 ppm and CS100 ppm groups than in the control group, and tended to plateau.

[0067] α-Tocopherol is a component of vitamin E and has the strongest antioxidant effect among vitamin E components. Therefore, it enhances the effect of inhibiting oxidation of meat (suppressing peroxidation), contributing to improving its flavor.

[0068] (5) K value (freshness determination constant) Table 2 shows the results of an investigation into the effect of calsporin (CS)-supplemented feed on the K value of chicken (broiler) superficial pectoral muscle after freezing and rethawing. The CS levels were 0 ppm (control), 50 ppm (test group 1), and 100 ppm (test group 2). The K value was measured for superficial pectoral muscle (breast meat) when it was obtained and frozen immediately ("immediately frozen"), and when it was frozen three days later ("frozen after three days"). The lower the K value, the better the freshness.

[0069] [Table 2] As is clear from Table 2, the K value of the breast meat from chickens fed CS-added feed was lower than that of the control group, whether it was frozen immediately or after 3 days, indicating that it maintained its freshness.

[0070] (6) Summary of results The results of the above examples of feeding calsporin to chickens can be summarized as follows: (1) Feeding calsporin to chickens improves the concentrations of biochemical indicators related to the palatability of chicken meat (e.g., among amino acids, glutamic acid, which contributes to umami and sourness; alanine, threonine, and serine, which contribute to sweetness; alanine and serine, which contribute to sweetness and umami at high concentrations; and phenylalanine, tyrosine, arginine, isoleucine, leucine, valine, methionine, lysine, and histidine, which contribute to bitterness and complex flavors). Furthermore, the concentration of α-tocopherol, which contributes to inhibiting peroxidation, increases, maintaining freshness. Specifically, chicken fed diets containing, for example, 50 to 100 ppm of calsporin exhibited increases in various amino acids, including glutamic acid, in a concentration-dependent manner. Furthermore, odor analysis revealed a decrease in the odor index value in the calsporin-fed group. Evaluation of chicken soup using a taste sensor revealed enhanced umami richness, umami, and bitter impurity component values. (2) When chickens were fed a diet containing calsporin, regardless of whether vitamin E was added to the diet, the amount of α-tocopherol in the meat increased compared to chickens fed a diet without calsporin. (3) When chicken meat was refrigerated for three days after slaughter, frozen, and then thawed, the K value (a constant for determining freshness: the lower the better) decreased depending on the concentration of added calsporin. [Industrial Applicability]

[0071] According to the present invention, by feeding chickens calsporin (a probiotic agent with Bacillus subtilis C-3102 strain as the active ingredient), useful effects such as an increase in free amino acids in the meat, an increase in α-tocopherol, a reduction in odor, and maintenance of the freshness of the meat can be achieved, thereby improving the flavor (e.g., palatability) of the chicken meat, improving peroxidation inhibition, and maintaining the freshness of the meat. [Accession number]

[0072] Bacillus subtilis strain C-3102 (international accession number FERM BP-1096) was originally deposited on December 25, 1985, at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (at the time of deposit: Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry), 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (at the time of deposit: 1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki, Japan), under accession number FERM BP-8584, and was transferred to international deposition at the same institution under accession number FERM BP-1096 (at the time of transfer: Fermentation Research Institute Accession No. 1096) on June 28, 1986. This strain is currently preserved at the Patent Microorganism Deposit Center of the Biotechnology Center of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, postal code 292-0818).

[0073] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An agent for increasing free amino acids, derivatives thereof and / or α-tocopherol in chicken meat, characterized in that the agent contains as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or a derivative, bred strain or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and is administered to chickens.

2. 2. The enhancer of claim 1, wherein the Bacillus subtilis bacteria is in the form of a spore.

3. The Bacillus subtilis bacteria were 1 x 10 per gram of the enhancer ５ cfu ~ 1 x 10 １３ 3. The agent according to claim 1 or 2, wherein the agent is a microbial colony containing 1000 or more cfu.

4. A flavor-improving agent for chicken meat, comprising as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis C-3102 strain (FERM BP-1096) or a derivative, bred strain, or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and the flavor-improving agent is administered to chickens, wherein the flavor improvement of chicken meat is an increase in free amino acids in the chicken meat.

5. The chicken meat flavor improving agent according to claim 4, wherein the chicken meat has a delicious flavor.

6. The chicken meat flavor improving agent according to claim 4 or 5, wherein the improvement of chicken meat flavor is achieved by enhancing umami and rich umami flavor or bitter impurities.

7. The chicken meat flavor improving agent according to any one of claims 4 to 6, wherein the Bacillus subtilis bacterium is in a spore state.

8. The Bacillus subtilis bacteria was 1 x 10 per 1 g of the flavor improving agent. ５ cfu ~ 1 x 10 １３ The chicken meat flavor improving agent according to any one of claims 4 to 7, wherein the chicken meat flavor improving agent is 0.01 cfu or more.

9. An agent for maintaining the freshness of chicken meat, comprising as an active ingredient at least one Bacillus subtilis bacterium selected from the group consisting of Bacillus subtilis strain C-3102 (FERM BP-1096) or a derivative, bred strain, or mutant strain thereof, and other Bacillus subtilis strains having properties equivalent to those of the C-3102 strain, and the agent comprises administering the agent to chickens, wherein the freshness of the chicken meat is maintained by increasing the α-tocopherol concentration in the chicken meat.

10. The agent for maintaining freshness of chicken meat according to claim 9, wherein the maintaining of freshness of chicken meat is achieved by lowering the K value (a constant for determining freshness) when the chicken meat is thawed after freezing.

11. The agent for maintaining freshness of chicken meat according to claim 9 or 10, wherein the Bacillus subtilis bacterium is in the form of a spore.

12. The Bacillus subtilis bacteria is 1 x 10 per 1 g of the freshness-maintaining agent. ５ cfu ~ 1 x 10 １３ The agent for maintaining freshness of chicken meat according to any one of claims 9 to 11, wherein the freshness of chicken meat is 0.01 cfu or more.

13. A feed additive for improving the flavor and / or maintaining the freshness of chicken meat, comprising the increasing agent, flavor improving agent, or freshness maintaining agent according to any one of claims 1 to 12.

14. A feed for improving flavor and / or maintaining freshness of chicken meat, comprising the feed additive according to claim 13 in an amount of 1 ppm to 1000 ppm or more by weight.

15. The feed according to claim 14, which is an early feed or a late feed.

16. A method for improving the flavor and / or maintaining the freshness of chicken meat, comprising feeding chickens the feed according to claim 14 or 15 and raising them.

17. 17. The method of claim 16, further comprising obtaining the chicken for consumption.

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