KR20030082859A - Method of producing meat flavor from cultures of pleuotus ostreatus mycelia - Google Patents

Abstract

The present invention relates to a method for producing a natural meat flavor component by liquid culture of Pleurotus eryngii mycelium, inoculate and incubate the mycelia of Pleuotus ostreatus in a liquid medium containing cysteine and ribose (ribose) According to the production method of the meat flavor of the present invention, comprising the step of heating the culture solution, by using the oyster mushroom can produce a meat flavor having excellent natural flavor, to produce a large amount of natural meat flavor It can be used to provide a foundation to enable industrial process development.

Description

Method of production of meat flavor from oyster mushroom mycelium culture {METHOD OF PRODUCING MEAT FLAVOR FROM CULTURES OF PLEUOTUS OSTREATUS MYCELIA}

The present invention relates to a method for producing natural meat flavor components by liquid culture of Pleurotus mycelia.

Biotechnology has a wide range of applications for biotechnology, as well as the convergence of technologies between sectors. Therefore, as the interest and demand for human welfare and environment increase, the scope of medicine, environment, chemistry, food, energy, It is expanding to agriculture and the ocean. As a result, fewer than four companies participated in biotechnology prior to 1981, but the number has been increasing since 1982, and the government has been promoting the biotechnology industry.

Mushrooms are composed of fruiting bodies with nutritional mycelium and spores with propagation organs, which correspond to the roots, stems and leaves of higher plants, and fruiting bodies correspond to flowers (Park, HM, Shin, HD, Oh, DC, Yun). , KS, and Lee, CK Fundamentals of the Fungi.The World Science.Seuol. Korea., 2000). The fruiting period of the mushrooms is short and for the most part of the year, downy fine thread-like mycelium grows in organic matter such as humus or old trees. Spores formed in the fruiting body germinate to become the first mycelia, and the compatible hyphae are cytoplasm fused with each other to become secondary mycelia. If the secondary hyphae are infested with sufficient nutrients, the hyphae will be organized under proper conditions and form the tertiary hyphae to produce fruiting bodies (Sung, JM, Yu, YB, and Cha, DL Mushroom Science.Kyohak.Seuol. , 1998).

Mushrooms are carbohydrates (Hong, J. S., and Kim, T. Y. Composition of organic sugar and sugar alcohol inPleurotus ostreatus, Lentinus edodes and Agaricus bisporus.Kor. J. Food Sci. Tech. 20: 459-462, 1988; Hwang, S. H., Kim, J. I., and Sung, C. J. Analysis of dietary fiber content of some vegetables, mushrooms, fruits and seaweeds.J. Kor. Soc. Food nutr. 29: 89-96, 1996), proteins (Hong, JS, Kim, YH, Lee, KR, Kim, MK, Cho, JS, Park, KH, Choi, YH, and Lee, JB Composition of organic acid and fatty acid inPleurotus ostreatus, Lentinus edodes and Agaricus bisporus.Kor. J. Food Sci. Tech. 20: 100-105, 1989) Kwon, Y. J., and Uhm, T. B. A study on the lipid components in oyster mushroom (Pleurotus florida).J. Kor. Soc. Food Nutr. 13: 175-180, 1984), minerals and vitamins (Bano, Z., and Rajarathnam, S.Pleurotusmushroom. Part III. Chemical composition, nutritional value, postharvest physiology, preservation, and role as human food.CRC Reviews in Food Sci. Nutri.27: 87-158, 1988), as well as nutrients such as Jung, S. T., and Hong, J. S. Volatile components of oyster mushroom (Pleurotus sp.) cultivated in korea. Kor. J. Mycol.19: 299-305, 1991) and is a health food that is recognized as a low-calorie and pollution-free food. In particular, anti-cancer activity, biofunction control and prevention and improvement of geriatric diseases such as stroke and heart disease have been reported (Jung, IC, Park, S., Park, KS, and Ha, CH Antioxidative effect of fruit body and mycelia extracts) ofPleurotus ostreatus. Kor. J. Food Sci. Tech. 28: 464-469, 1996; Kim, G. J., Kim, H. S., and Chung, S. Y. Effects of varied mushroom on lipid compositions in dietary hypercholes trolemic rats.J. Kor. Soc. Food nutr. 21: 131-135, 1992), growing interest in mushrooms.

There have been many studies on the scent of mushrooms. The scent of Lentinus edodes has the slightest difference depending on the harvest time, but 1-octen-3-ol accounts for 51-52%. Next is n-hexanal (2.6%), cis -2-octenol (2.3%), etc. (Flores J. Medierranean vs northera european meat products.Processing technologies and main differences.Food Chem. 59: 505-510, 1997). In addition, the important fragrance components of Agaricus bisporus are reported as 3-octanone, 3-octenone, 1-octen-3-ol, benzaldehyde, 1-octenone, 2-octen-1-ol, and the like. (Picardi, SM, and Issenberg, P. Investigation of some volatile constituents of mushroom ( Agaricus bisporus ); Change which occur during heating.J . Agric.Food Chem. 21: 959-962, 1973), as well as fragrances in the genus Agaricus. Much research has been done regarding ingredients (Cronin, DA, and Ward, MK The characterization of some mushroom volatile. J. Sci. Fd. Agric. 22: 447-480, 1971; Mega, JA Mushroom flavor.J. Agric Food Chem. 29: 1-4, 1981; Tressl, R., Bahri, D., and Engel, KH Formation of eight-carbon components in mushroom ( Agaricus bisporus ) .J. Agric.Food Chem. 30: 89- 93, 1982; Chen, CC, and Wu, CM Volatile components of mushroom ( Agaricus subrufecens ). J. Food Sci. 49: 1208-1210, 1984).

On the other hand, Pleurotus ostreatus ( Pleuotus ostreatus ) is an edible mushroom that grows on softwoods such as cottonwood and cut stumps, and has a good taste, aroma, high nutritional value, and anti-cancer and blood pressure-lowering ingredients. It is a health food with increasing production and consumption worldwide. The oyster mushroom belongs to mycelial basidiomycete basidiomycete subclass statement dansil agaricales songyigwa of biological classification system haksang fungus (Ahn, JS, and Lee, KH Studies on the volatile aroma components of edible mushroom (Pleurotus ostreatus) of Korea. J. Kor. Soc.Food Nutr. 15: 258-262, 1986; Mottram, DS Flavor formation in meat and meat products: a review.Food Chem. 62: 415-424, 1998). Dark grey, with stand white. However, most of the recently grown circular elders, summer elders, and four-legged elders dominate the funnel with a concave central part of the lampshade (Hong, JS Studies on the physio-chemical properties and the cultivation of oyster mushroom ( Pleurotus ostreatus ) J. Kor. Agric. Chem. Soc. 21: 150-184, 1978).

Pleurotus oyster mushroom is highly preferred as a food ingredient due to its unique flavor and texture. It is known that 3-octanol, 3-octanone, 1-octen-3-ol, isobutyl alcohol, and isoamyl alcohol account for more than 89% of the volatile aroma components that have a significant effect on the flavor of oyster mushroom. Sensory and chemistry analysis of cooked porcine meat patties in relation to warmed-over flavor and pre-slaughter stress.Meat Sci . 59: 229-249, 2001. However, the research on the fragrance component of the culture solution liquid cultured oyster mushroom mycelium is not known.

On the other hand, when looking at the compounds that produce the meat flavor (meat flavor), most of the compounds produced when the beef is heated, typical of 3-mercapto-2-methyl-4,5-di in furans (furans) Hydrofuran, 4-mercapto-2-methylfuran, 3-mercapto-2-methylthiophene, 3-mercapto-2-methyl-2,3-dihydrothiophene in thiophenes , 3-mercapto-2-methyl-4,5-dihydrothiophene, 4-mercapto-2-methyl-4,5-dihydrothiophene. Others include thiazols, pyrazines, oxazoles, and other heterocyclic sulfur compounds (Ko, SN, Yoon, SH, Yoon, SK, and Kim, WJ Development of meat-like) flavor by maillard reaction of model system with amino acids sugars.Kor . J. Food.Tech . 29: 827-838, 1997; Farmer, LJ, Ronald, LS, and Patterson.Compounds contributing to meat flavor.Chem., 40: 201-205, 1991; Hackerman, RH Studies on chitin 1.Enzyme degradation of chitin and chitin esters.J. Biol. Sci. 7: 168-170, 1954; Hong, JS, Lee, JY, Kim, HK, Kim, MK, Jung, GT, and Lee, KR Studies on the volatile aroma components of Pleurotus ostreatus.Kor . J. Mycol. 14; 31-36, 1986).

An object of the present invention is to produce natural meat flavor using mushrooms that are widely cultivated for food, by establishing a medium composition and culture conditions for producing meat flavor using liquid culture method of mushroom mycelium, optimal culture conditions To provide a method for producing meat flavor components by liquid culture of oyster mushroom mycelium.

1 is a graph showing the pH change of the culture solution during the culture period of each mushroom mycelium,

2 is a GC chromatogram of volatile aroma components isolated from the basal medium,

3 is a GC chromatogram of volatile scent components isolated from the basal medium containing Cys (1%) and Rib (2%),

4 is a GC chromatogram of volatile odorant components isolated from the culture medium incubated PO in the basal medium,

5 is a GC chromatogram of volatile aroma components isolated from the culture medium in which PO was added to the basal medium to which Cys (1%) and Rib (2%) were added.

6 is a GC chromatogram of volatile aroma components isolated from a culture medium heated with addition of Cys (1%) and Rib (1%) after incubating PO in a basal medium,

7 is the MS spectrum of 2-formyl-5-methylthiophene,

8 is an MS spectrum of dimethylformylthiophene.

Meat production method of the present invention for achieving the above object,

It is characterized by comprising the step of inoculating the mycelium ( Pleuotus ostreatus ) mycelium in a liquid medium containing cysteine (ristein) and ribose (ribose), and heating the culture solution.

Here, the liquid medium is 10% (w / v) soybean meal, 2.7% (w / v) per sulfur white, 0.5% (w / v) KH ₂ PO _{4 and} 0.5% (w / v) MgSO ₄ · 7H ₂ O Is dissolved in distilled water and sterilized, and cysteine and ribose are preferably added to the medium at concentrations of 1% and 2%, respectively.

In addition, the culture of oyster mushroom mycelium is preferably incubated for 7 days at a shaking speed of 120 rpm at 25 ° C. with a pH of the liquid medium at 5.5, and the culture solution is preferably heated at 121 ° C. for 15 minutes.

As such, by inoculating and incubating the mycelia mushroom mycelium in a liquid medium containing cysteine and ribose, it is produced 2-furancarboxaldehyde and furfuryl alcohol, which are meat precursors, and then the meat is heated by heating the culture solution. The flavor components 2-formyl-5-methylthiophene and dimethylformyl-thiophene are produced.

In the present invention, the cultivation conditions and medium to produce a natural meat flavor component through the mycelial culture of oyster mushroom useful as an edible mushroom with good taste and aroma, high nutritional value and anticancer action, blood pressure lowering effect The composition was specified.

That is, by adding cysteine and ribose to the base medium containing soybean meal, sulfur white sugar, KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O, and inoculating the mycelia mushroom mycelium and culturing at optimum conditions, meat flavor It was possible to produce 2-furancarboxaldehyde and furfuryl alcohol, which are considered precursors of the fragrance component of. Subsequently, when the culture medium was heated, 2-formyl-5-methylthiophene and dimethylformyl-thiophene, which are important components for the production of meat flavor, were produced as the thiophene compound produced by heating the beef. It was confirmed.

Hereinafter, a method of producing meat flavor (hereinafter abbreviated as MF) according to the present invention will be described in more detail with reference to the following examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

As a raw mushroom used in the present embodiment, strains such as Pleurotus ostreatus ( hereinafter PO), spirit mushrooms ( Agaricus blazai; abbreviated AZ), shiitake mushrooms ( Lentinus edodes; abbreviated LE) ) Was used by HK Biotech (Chinju, Korea). Soybean meal was purchased in the Pacific (Seoul, Korea), sulfur white sugar was Cheil-Jedang (Seoul, Korea), KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O and Na ₂ SO ₄ from Shinyo (Shinyo, Osaka, Japan). The amino acids used in the experiments were cysteine (Gys), glutamine (Gln), methionine (Met), and fructose (Fru), galactose (Gal), glucose (Glu), maltose (Mal), ribose (Rib), and sugar Suc and Xyl were purchased from Sigma (Sigm, St. Louis. MO, USA), and organic acids such as acetic acid were purchased from Aldrich, Milwarfe. WI, USA. Diethyl ether, ethanol, hexane are the second. tea. HPLC grade was purchased from JT Baker, Phillipsurg. NJ, USA and other reagents used were reagent grade or higher.

Example 1 Liquid Culture of Mushroom Mycelium

The basic medium used for the cultivation of mushroom mycelium was 10% (w / v) soybean meal, 2.7% (w / v) per white bean, 0.5% (w / v) KH ₂ PO _4, 0.5% MgSO ₄ · 7H ₂ O ( w / v) was dissolved in distilled water and sterilized before use.

The culture conditions were inoculated PO, AZ, LE in the basal medium (mushroom liquid mycelium, 25 ml) and the temperature 20, 25 and 30 ℃, pH 4.5, 5.5, 6.5 and 7.5, shaking speed 60, 120 and 220 rpm, Incubation periods were 3, 7 and 10 days, respectively. The pH of the medium before and after mushroom mycelium culture was measured. The mushroom liquid mycelium in 100 ml of the mushroom mycelium culture solution was vacuum filtered on a filter paper (Whatman No. 2), dried at 80 ° C. for 5 hours, and weighed.

① The following Table 1 shows the dry weight of the mushroom mycelium according to the culture temperature, which is the result of culturing for 7 days at 120 rpm shaking in the basic medium.

Temperature (℃) Mycelium dry weight (g / 100 ml) PO AZ LE 20 5.67 ± 0.2 5.50 ± 1.0 4.08 ± 0.5 25 6.08 ± 0.1 5.78 ± 1.5 4.58 ± 0.4 30 5.68 ± 0.3 5.20 ± 0.2 4.25 ± 0.2

As shown in the table above, the dry weight of each mycelium ranged from 5.67 to 6.68 g / 100 ml of PO, 5.20 to 5.78 g / 100 ml of AZ, and 4.08 to 4.58 g / 100 ml of LE. It can be seen that it grows.

② The following Table 2 shows the dry weight of the mushroom mycelium according to the culture pH, the result of incubation for 7 days at 25 ℃, shaking speed 120 rpm in a basic medium.

pH Mycelium dry weight (g / 100 ml) PO AZ LE 4.5 4.75 ± 0.5 5.08 ± 0.1 3.94 ± 0.1 5.5 6.28 ± 0.4 6.54 ± 0.5 4.58 ± 0.5 6.5 6.07 ± 0.2 6.12 ± 0.5 4.04 ± 0.2 7.5 5.04 ± 0.5 5.15 ± 0.3 3.05 ± 0.3

In the above table, the dry weight of each mycelium is 4.75∼6.28 g / 100ml of PO, AZ of 5.08∼6.54g / 100ml, and LE of 3.05-44.5g / 100ml. Was found to grow most actively.

③ The following Table 3 shows the dry weight of the mushroom mycelium according to the shaking speed, which is the result of incubating for 7 days at 25 ℃ in the basal medium adjusted to pH 5.5.

Shaking speed (rpm) Mycelium dry weight (g / 100 ml) PO AZ LE 60 4.05 ± 0.1` 4.80 ± 0.2 4.15 ± 0.1 120 6.58 ± 0.2 5.87 ± 0.3 4.98 ± 0.5 220 1.05 ± 0.2 1.20 ± 0.1 1.32 ± 0.5

In the above table, the dry weight of each mycelium was 1.05 to 6.58 g / 100 ml for PO, 1.20 to 5.87 g / 100 ml for AZ, and 1.32 to 4.98 g / 100 ml for LE. You can see that this is the most active. At the shaking speeds of 60 rpm and 220 rpm, the mycelial growth was lower than that of 120 rpm. In particular, mycelial growth was reduced by about 70-80% compared to 120 rpm. These results indicate that the shaking (contact with air) is less suitable for growth of mycelial pellets at less than 120 rpm, and the hyphae is stimulated by severe shaking at 120 rpm and above, which inhibits mycelial growth. It was thought that the optimum shaking speed was 120 rpm.

④ The following Table 4 shows the dry weight of the mushroom mycelium according to the culture period, the result of incubation at 25 ℃, 120 rpm in the basal medium pH is adjusted to 5.5.

Incubation period (days) Mycelium dry weight (g / 100 ml) PO AZ LE 3 4.12 ± 0.1 4.13 ± 0.1 3.10 ± 0.2 7 6.85 ± 0.1 6.87 ± 0.1 4.58 ± 0.4 10 12.21 ± 0.1 13.05 ± 0.1 10.52 ± 0.2

In the table above, the dry weight of each mycelium is 4.12-12.21 g / 100 ml of PO, 4.13-13.05 g / 100 ml of AZ, and 3.10-10.52 g / 100 ml of LE, and when the culture period is 3 days, The growth was low, and the mycelia were overgrown at 10 days so that MF could not be obtained. Therefore, the optimal incubation period to produce the maximum MF was determined to be 7 days.

On the other hand, Figure 1 is a graph showing the pH change of the culture medium during the culture period of each mushroom mycelium. From this it can be seen that the change in pH of the culture medium according to the culture period is not large.

Example 2: Meat Flavor (MF) Production Medium

In order to investigate the medium composition suitable for MF production, 1% (w / v) of amino acids (Cys, Gln, Met) were added to the basal medium (250 ml), and 2% (w / v) of sugar (Fru, Gal, Rib, Xyl). v) addition and (3) A medium in which 1% (w / v) of amino acid and 2% (w / v) of sugar were mixed and added.

Each treatment was sterilized and inoculated with PO, AZ, and LE mushroom liquid mycelium (25 ml), followed by incubation at 25 ° C. and 120 rpm for 7 days according to the method of Example 1 above, and the culture was heated and sensory tested. The characteristics and intensity of each fragrance are indicated by + (weak), +++ (medium), +++++ (strong).

① The results of culturing PO by adding amino acids (Cys, Gln, Met) to the basal medium showed that there was a pungent odor of sulfur in the culture medium with Cys, sweet smell in the culture medium with Gln, and the addition of Met The culture was unscented. As a result of the culture of AZ, the irritating odor of sulfur was observed in the culture medium containing Cys, and the mildew smell was observed in the culture medium containing Gln and Met. As a result of culturing LE, the culture medium containing Cys had a pungent odor of sulfur, and the culture medium containing Gln had a mild smell. The culture medium containing Met was unscented. These results are shown in Table 5 below.

Mushroom type Amino Acid Added Odor characteristics Meat flavor mellowness Dirt smell Fungus Sulfur stimulation Charcoal odor PO Control CysGlnMet ++++++ + + +++++ AZ Control CysGlnMet +++++++++ +++++ LE Control CysGlnMet + +++++ ++++ +++++

As a result of the above table, the culture of mushroom mycelium with amino acid added to the basal medium showed little difference from the control without amino acid. Therefore, the addition of amino acid alone did not significantly affect the production of MF. Appeared.

In addition, the results of observing the pH change during the growth of mushroom mycelium in the basal medium to which various amino acids were added are shown in Table 6 below.

Amino acid or sugar treatment PO AZ LE PH before culture PH after incubation PH before culture PH after incubation PH before culture PH after incubation amino acid CysGlnMet 5.525.565.57 5.506.335.62 5.515.585.54 5.545.845.52 5.525.565.55 5.325.675.27 Party FruGalRibXyl 5.685.545.545.22 5.955.975.725.81 5.625.565.545.28 5.685.545.655.68 5.585.525.565.34 5.545.525.455.25

As shown in the above table, when the amino acid was added to the basal medium, the pH was not significantly changed compared to the basal medium, but after incubating the mushroom mycelium, the pH was 0.1-0.3 or more higher than before the culture. The pH rose to 6 or more.

② Next, sugar (Fru, Gal, Rib, Xyl) was added to the basal medium (250 ml) at a concentration of 2% (w / v) to incubate PO, AZ and LE, respectively (25 ° C., 120 rpm, 7 After the work, the sensor was heated and the sensory results showed that in the case of PO, all the sugar had a sweet odor. In the case of AZ culture, the fungus smelled in the culture medium containing Fru and Xyl, and the earth smelled in the culture medium in which Gal was added, and the sweet smell was observed in the culture solution in which Rib was added. In one culture, the sweet scent, the addition of Gal had a mildew odor, and the addition of Rib and Xyl was unscented. These results are shown in Table 7 below.

Mushroom type Party Odor characteristics Meat flavor mellowness Dirt smell Fungus Sulfur stimulation Charcoal odor PO ControlFruGalRibXyl +++++++++++++++ +++ AZ ControlFruGalRibXyl +++ ++++++ +++++++++++ LE ControlFruGalRibXyl ++++++ ++++ +++++

As shown in the table above, the mushroom mycelium cultured with sugar in the basal medium showed little difference from the control without sugar. Therefore, the addition of sugar alone did not significantly affect the production of MF. .

In addition, in Table 6, which shows the results of observing the pH change during the growth of the mushroom mycelium in the basic medium containing various sugars, there was no significant change in the pH before and after mushroom mycelium culture. Afterwards, the pH is about 0.1-0.2 higher than before the culture, and in particular, the culture medium of PO can be seen to have a higher pH of 0.5 or more than the culture medium of other mycelia.

③ Add 1% amino acid (Cys, Gln, Met) and 2% sugar (Fru, Gal, Rib, Xyl) to the basal medium (250 ml) to culture the PO (25 ° C, 120 rpm, 7 days). The results of sensory evaluation by heating showed that the culture medium containing Cys, Fru, Gal, and Xyl had an irritating odor of sulfur, and the culture medium containing Cys and Rib had strong meat flavor. The added culture was unscented. Sweet flavours were found in the cultures containing Gln and Rib, mildew odors in the cultures containing Met, Fru, and Gal, and sweet odors in the cultures containing Met and Rib, and earthy odors in the cultures containing Met and Xyl. These results are shown in Table 8 below.

amino acid Party Odor characteristics Meat flavor mellowness Dirt smell Fungus Sulfur stimulation Charcoal odor Cys FruGalRibXyl +++++ ++ +++ +++++++++ Gln FruGalRibXyl ++++++ ++++ + Met FruGalRibXyl +++ +++ ++++++ +

From the above results, it can be seen that the strong MF is produced when PO is cultured in the basal medium to which Cys and Rib are added.

On the other hand, the results of observing the pH change during the growth of mushroom mycelium in the basic medium mixed with various amino acids and sugars are shown in Table 9 below. As seen here, the pH was not significantly changed before and after the cultivation, but was increased by about 0.3 to 0.5 in the culture solution added with Gln.

amino acid Party PO AZ LE PH before culture PH after incubation PH before culture PH after incubation PH before culture PH after incubation Cys FruGalRibXyl 5.605.535.555.37 5.735.745.615.66 5.575.545.535.40 5.655.525.465.59 5.555.525.435.43 5.435.425.295.29 Gln FruGalRibXyl 5.625.555.555.39 6.146.156.036.07 5.65.555.565.43 5.485.415.465.48 5.575.545.505.45 5.615.65.565.46 Met FruGalRibXyl 5.635.565.565.40 5.705.585.675.63 5.585.555.545.41 5.605.535.595.60 5.575.545.565.45 5.535.405.495.39

Next, AZ was incubated (25 ° C., 120 rpm, 7 days) by adding 1% of amino acids (Cys, Gln, Met) and 2% of sugars (Fru, Gal, Rib, Xyl) to the basal medium (250 mL). The sensory results of heating the cultures showed that irritating odor of sulfur was observed in the culture medium containing Cys, Fru, Gal, and Xyl, and the culture medium containing Cys and Rib was unscented. In the culture medium containing Gln and Fru, there was a smell of soil, and the culture medium containing Gln and Gal had a mildew smell. In addition, the culture solution containing Gln, Rib, and Xyl had a sweet smell. In the culture medium containing Met, Fru, Rib, and Xyl, the mildew smell was strong, and the culture medium containing Met and Gal was unscented. These results are shown in Table 10 below.

amino acid Party Odor characteristics Meat flavor mellowness Dirt smell Fungus Sulfur stimulation Charcoal odor Cys FruGalRibXyl ++ ++ +++++++++ + Gln FruGalRibXyl ++++++ +++++ +++++++ + Met FruGalRibXyl + ++++ ++++++++++

In view of the above results, the culture medium in which AZ was cultured in the basal medium to which amino acids and sugars were added showed almost no difference in the characteristics of the flavor. Therefore, AZ was judged to be unsuitable for producing MF.

On the other hand, as shown in Table 9, the pH was not changed before and after the culture.

Next, 1% of amino acids (Cys, Gln, Met) and 2% of sugars (Fru, Gal, Rib, Xyl) were added to the basal medium (250 mL) to incubate LE (25 ° C, 120 rpm, 7 days). The sensory results of heating the cultures showed that irritating odor of sulfur was observed in the culture medium containing Cys, Fru, Gal, and Xyl, and the culture medium containing Cys and Rib was unscented. Gln, Rib, and Xyl were added in the sweet scent. Gln and Fru were added in the odorless culture. Gln and Gal were added in the odor. The culture medium containing Met, Rib, and Xyl had an earthy odor. The culture medium containing Met and Fru had a mildew odor. The culture medium containing Met and Gal was unscented. These results are shown in Table 11 below.

amino acid Party Odor characteristics Meat flavor mellowness Dirt smell Fungus Sulfur stimulation Charcoal odor Cys FruGalRibXyl + +++++++++ Gln FruGalRibXyl ++++++ + +++ Met FruGalRibXyl + ++++++ +++++ +

In view of the above results, the culture medium of LE cultured in the basal medium to which amino acids and sugars were added showed almost no difference in the characteristics of the flavor. Therefore, LE was also judged to be unsuitable for producing MF.

On the other hand, as shown in Table 9, the pH was not changed before and after the culture.

Example 3: Meat flavor (MF) production culture conditions

The results of the above experiments showed that strong MFs were produced when PO was incubated in basal medium containing Cys and Rib. Accordingly, in order to study the effect of adding Cys and Rib to the medium in the culture of PO affect the MF production, ① basal medium, ② basal medium added with 1% Cys and 2% Rib, and ③ basal medium PO was incubated, ④ POs were cultured in a basal medium containing ④ Cys 1% and 2% Rib, and ⑤ POs were incubated in the basal medium, and then Cys 1% and Rib 2% were added.

Culture of each treatment was incubated in the same manner as in Example 1. The culture was subjected to vacuum filtration with a filter paper (Whatman No. 2), and the heat was not sensory tested. The heated culture was extracted with ether and analyzed by GC-MS.

① sensory test

In order to know the characteristics of the produced fragrance, 7 to 10 panelists who were trained in advance were subjected to sensory evaluation of the characteristics of the fragrance.

First, the sweet odor was weakly detected from the liquid heating the basal medium, and the soil was smelled from the culture medium in which PO was cultured in the basal medium. Flavors with the irritating characteristics of sulfur were detected from the solution with or without heating the basal medium containing Cys and Rib. Sensory examination of the culture solution by incubating PO in basal medium containing Cys and Rib showed a scent of sulfur with irritant characteristics. Strong MF was produced by heating the culture solution. The fragrance with the irritating characteristic of sulfur was detected from the solution heated by adding Cys and Rib to the culture medium in which PO was cultured in the basal medium. The above result is shown in following Table 12.

process Odor characteristics Meat flavor mellowness Dirt smell Fungus Sulfur stimulation Charcoal odor heating Basic Medium (1) Cys + Rib (2) PO (3) Cys + Rib + PO (4) PO + Cys + Rib (5) +++++ ++++ +++++++ +++ No heating Basic Badges Cys + RibPOPO + Cys + Rib ++ +++ + ++++++++

(1) Sterile medium containing soybean meal, sulfur white sugar, KH ₂ PO ₄ and MgSO ₄ · 7H ₂ O in distilled water.

(2) basal medium with Cys (1%) and Rib (2%) added.

(3) Incubate PO in basal medium.

(4) PO incubated in basal medium to which Cys (1%) and Rib (2%) were added.

(5) After culturing PO in basal medium, add Cys (1%) and Rib (2%).

From the above results, it was confirmed that MF was produced by incubating PO in the basal medium to which Cys and Rib were added, and then heating the culture solution. Therefore, it can be seen that four conditions of PO, Cys, Rib, and heating are essential for MF generation.

② volatile fragrance

The following pretreatment was carried out to analyze the volatile aroma components: 50 ml (20% of the total volume) of the sample was extracted with 20 ml of ether for 2 minutes, left at room temperature for 5 minutes, and then separated from the ether and water layers. The dehydration was carried out by adding a fixed amount of Na ₂ SO ₄ to the ether layer. The aroma component extracted in the ether layer was concentrated in a concentration tube under N ₂ gas and analyzed using GC under the conditions of the following Table 13. GC-MS was analyzed under the same conditions as GC. The carrier gas was helium (2 ml / min), the ionization voltage was 70 eV, and the ion source temperature was 250 ° C. Aromatic compounds analyzed by GC-MS were identified using a database of WILEG D6 & NIST.

Item Hewlett Packard 5890 Condition Oven temperature 50 to 200 ℃ (4 ℃ / min) Initial temperature 50 ℃ Final temperature 200 ℃ Injector temperature 260 ℃ Detector temperature 260 ℃ column Capillary column (Supelcowax-10: 60 m × 0.32 mm, i.d) Detector FID Carrier gas N ₂ (flow rate: 2 ml / min)

Figure 2 is a GC chromatogram of volatile aroma components isolated from the basal medium, with peaks 1-tetradecene, 2 2-furancarboxaldehyde, 3 furfuryl alcohol, and 4 3- (1-ethoxy Ethoxy) -butanol.

FIG. 3 is a GC chromatogram of volatile fragrance components isolated from basal medium containing Cys (1%) and Rib (2%), with peak 1 being 1-tetradecene and 2 being 2-furan-carboxaldehyde, 3 Silver furfuryl alcohol, 4 is 3- (1-ethoxyethoxy) -butanol, 5 is 1,2-benzisothiazole-3- (2H), 6 is thiono [2,3, C] pyridine, 7 represents 1H-4-methyl-2,3,4,5-tetra-hydrobenz [c] azepin-1-one.

Figure 4 is a GC chromatogram of volatile aroma components isolated from the culture medium incubated PO in the basal medium, peak 1 is 1-tetradecene, 2 is 2-furancarboxaldehyde, 3 is furfuryl alcohol, 4 is 1- [13C] -phenylacetamide, 5 represents 2,6-di (t-butyl) -4-hydroxy-4-methyl-2,5-cyclohexadien-1-one.

FIG. 5 is a GC chromatogram of volatile aroma components isolated from a culture culture of PO in a basal medium to which Cys (1%) and Rib (2%) are added. Peak 1 is 1-tetradecene and 2 is 2-furan. Carboxaldehyde, 3 is furfuryl alcohol, 4 is 2-formyl-5-methylthiophene, 5 is 3- (1-ethoxyethoxy) -butanol, 6 is dimethylformylthiophene, 7 is 1,2 Benzisothiazole-3- (2H), 8 is 2,6-di (t-butyl) -4-hydroxy-4-methyl-2,5-cyclohexadien-1-one, 9 is thiono [2,3, C] pyridine, 10 represents formanilide, 11 represents 1H-4-methyl-2,3,4,5-tetrahydrobenz [c] azepine.

FIG. 6 is a GC chromatogram of volatile aroma components isolated from a culture medium heated with addition of Cys (1%) and Rib (1%) after incubating PO in a basal medium, with peak 1 representing 1-tetradecene and 2 2-furancarboxaldehyde, 3 is furfuryl alcohol, 4 is 1- [13C] -phenylacetamide, 5 is dimethylformylthiophene, 6 is 1,2-benzisothiazole-3- (2H), 7 Silver 2,6-di (t-butyl) -4-hydroxy-4-methyl-2,5-cyclohexadien-1-one, 8 is thiono [2,3, C] pyridine, 9 is 1H- 4-methyl-2,3,4,5-tetrahydrobenz [c] azepine is shown.

From the above results, 1-tetradecene and 2-furancarboxaldehyde were produced in all samples, and especially high concentration was present in the sample to which Cys and Rib were added. Furfuryl alcohol was produced when PO was incubated in the basal medium to which Rib was added. Incubating PO in basal medium containing Cys and Rib produced 2-formyl-5-methylthiophene and dimethylformylthiophene. It is considered a fragrance ingredient that plays an important role. Table 14 shows the volatile aroma components of various media identified by GC-MS.

RRT compound process (One) (2) (3) (4) (5) 0.7270.756I.S.1.1831.1931.2431.2981.3811.4171.5011.5181.592 1-tetradecene2-furancarboxaldehydefurfuryl alcohol2-formyl-5-methylthiophen3- (1-ethoxyethoxy) -butanol1- [13C] -phenylacetamidedimethylformylthiophene1, 2-benzisothiazole-3 (2H) -one2,6-di (t-butyl) -hydroxy-4-methyl-2,5-cyclohexadiene-1-onthioo [2,3, C] Pyridineformanilide 1H-4-methyl-2,3,4,5-tetrahydrobenz [c] azino-1-one 0.1870.126trNDtrNDNDNDNDNDNDND 1.1821.988trND0.191NDND0.6830.2140.628ND0.473 0.1350.0830.076NDND1.075NDND4.886NDNDND 2.7197.27812.8020.1750.2150.0890.2178.0260.4840.7100.5300.790 0.7160.8050.042NDND1.040tr0.3863.0890.139ND0.196

In Table 14 above, (1) to (5) are the same as those described in Table 12. In addition, each figure represents the area (%) of the total volatiles in the ether extract shown on the GC chromatogram. I.S. is an internal standard for the calculation of RRT, furfuryl alcohol present in all samples, tr means trace and ND not detected.

The MS spectra of these compounds were nearly identical to the MS spectra of the database as shown in FIGS. 7 and 8. 7 is an MS spectrum of 2-formyl-5-methylthiophene, and FIG. 8 is an MS spectrum of dimethylformylthiophene.

③ fatty acid

Take 50 g (20% of the total volume) of the sample, add 50 ml of hexane: isopropyl alcohol (3: 2, v / v) extraction solvent and 1 mg of internal standard solution, and shake it vigorously for 2 minutes to extract and centrifuge (4,000 rpm, 10 Minutes). After repeating this process twice, the hexane layer was washed three times with water, water was removed with Na ₂ SO ₄ and concentrated to give free fatty acid. 3 mL of 1 NH ₂ SO ₄ / MeOH was added to the extracted fat, followed by methylation (100 ° C., 5 minutes) and injection into GC for analysis. At this time, the analysis conditions are as shown in Table 15 below.

Item Hewlett Packard 5890 Condition Oven temperature 160 ~ 200 ℃ (2 ℃ / min) Initial temperature 160 ℃ Final temperature 200 ℃ Injector temperature 260 ℃ Detector temperature 260 ℃ column Capillary column (Supelcowax-10: 60 m × 0.32 mm, i.d) Detector FID Carrier gas N ₂ (flow rate: 2 ml / min)

Table 16 shows the fatty acid composition of the same sample as in Table 12. C13: 0, C14: 0, C15: 0, C16: 0, C18: 0, C20: 0, C20: 1 and C21: 0 are all contained at similar ppm concentrations in all samples. More than 1.4 ppm of C15: 0 was contained in basal medium and basal medium containing Cys and Rib, and C16: 1 was contained in more than 2 ppm of Cys and Rib solution. In addition, C18: 1 contained at least 2 ppm in the medium in which PO was cultured in the basal medium, and in which the Cys and Rib were added in the basal medium. γ-LN contained 6.27 ppm in the basal medium to which Cys and Rib was added, and 4.02 ppm in the culture culture of PO by adding Cys and Rib to the basal medium.

fatty acid Free fatty acid content (ppm) (One) (2) (3) (4) (5) C13: 0C14: 0C15: 0C15: 1C16: 0C16: 1C18: 0C18: 1γ-LNC20: 0C20: 1C21: 0 0.080.111.400.080.170.960.310.651.890.180.060.24 0.090.081.470.070.112.120.661.986.270.540.060.24 0.070.260.110.010.151.470.564.610.520.430.270.29 0.050.180.040.070.092.180.762.774.020.390.030.24 0.070.310.090.110.191.880.652.261.960.160.140.29

In Table 16 above, (1) to (5) are the same as those described in Table 12. The free fatty acid content (ppm) is sample area / internal standard area × sample weight (50 g) × 10 ⁶ , and the internal standard is heptadecanoic acid (1 mg).

④ Glass sugar

10 g of the sample was taken in a 25 ml volumetric flask, and 5 ml of 99.5% EtOH and 5 ml of 80% EtOH were added and mixed, followed by correction with 80% EtOH at a constant temperature of 20 ° C. 3 ml of the correction solution was first filtered with Sep pak C ₁₈ to remove pigments and polymers, and secondly filtered with a membrane filter (0.2 μm), followed by injection into HPLC, and analyzed under the conditions of the following Table 17. Standard materials were used after mixing arabinose, fructose, galactose, glucose, lactose, maltose, mannose, ribose, sucrose, xylose.

Item Shimaju LC-10A condition column Supelcogel Ca (7.8 mm I.D x 300 mm) Column temperature 80 ℃ Mobile phase Deionized water Detector RID-10A Flow rate 0.5 ml / min

Table 18 shows the free sugar composition of the same sample as in Table 12. As shown here, Fru contained 9.73 ppm in basal medium, 2.09 ppm in PO culture, and 20 ppm or more in samples containing Cys and Rib. Gal and Mal contained similar ppm concentrations in all samples, Glu contained 10.31 ppm in basal medium, 0.94 ppm in PO culture, and 24 ppm in samples containing Cys and Rib. Rib was contained more than 10 ppm only in the ribose added samples, Suc was contained more than 100 ppm in basal medium and PO culture, and more than 30 ppm in the Cys and Rib added samples.

Glass sugar Free sugar content (ppm) (One) (2) (3) (4) (5) FruGalGluMalRibSuc 9.730.7410.310.91ND108.13 32.951.5933.790.1910.6538.58 2.090.800.941.11ND112.76 29.541.2030.322.1410.2338.42 23.681.0824.161.1610.2151.99

In Table 18 above, (1) to (5) are the same as those described in Table 12. Free sugar content (ppm) is (sample area x 100 ppm of standard product) / standard area. In addition, ND means not detected.

⑤ organic acid

5 g of the sample was diluted 5 times with distilled water and filtered first with a Sep pak C ₁₈ to remove pigments and polymers, followed by secondary filtering with a membrane filter (0.2 μm), and then injected into HPLC to obtain the conditions shown in Table 19 below. Analyzed. Standard materials include acetic acid, adipic acid, ascorbic acid, benzoic acid, butyric acid, citric acid, formic acid, fumaric acid, isobutyric acid, isocitric acid, lactic acid, maleic acid, malic acid, malonic acid, oxalic acid, propionic acid, pyroglutamic acid, and succinic acid. It was used after.

Item Shimaju LC-10A condition column Supelcogel C-610H (7.8 mm I.D × 300 mm) (Hydrogen-form resin) Column temperature 30 ℃ Mobile phase 0.1% phosphoric acid Detector SPD-10A, 210 nm Flow rate 0.5 ml / min Injection volume 20 μl

Table 20 shows the organic acid composition of the same sample as in Table 12. Here, ascorbic acid was contained in 1.92 ppm in the basal medium and 2.69 ppm in the PO culture, and benzoic acid, citric acid, isobutyric acid, lactic acid, maleic acid, and malic acid were contained in all samples. Formic acid was contained 27.13 ppm in the culture medium in which PO was cultured in the basal medium to which Cys and Rib was added, and 19.89 ppm in the solution heated by adding Cys and Rib to the culture solution. Fumaric acid was contained 0.41 ppm in the basal medium, 0.65 ppm in the basal medium to which Cys and Rib were added, and malonic acid was contained 9 ppm or more in the sample except the basal medium and the PO culture. Oxalic acid was contained in all samples except PO cultured in basal medium containing Cys and Rib, especially 38.68 ppm of POs in samples containing Cys and Rib.

Organic acid Organic acid content (ppm) (One) (2) (3) (4) (5) Ascorbic acid Benzoic acid Citric acid Formic acid Fumaric acid Isobutyric acid Lactic acid Maleic acid Maleic acid Malonic acid Oxalic acid 1.928.452.27ND0.412.446.340.0912.40ND3.80 ND6.255.10NDND0.2138.130.65173.749.029.22 2.6910.542.58ND0.652.042.810.7942.59ND19.80 ND11.264.2727.13ND0.6882.550.66291.3813.90ND ND6.897.6519.89ND2.5435.282.16251.2313.4438.68

In Table 18 above, (1) to (5) are the same as those described in Table 12. Organic acid content (ppm) is (sample area x 100 ppm of standard product) / standard area. In addition, ND means not detected.

⑥ free amino acids

In order to remove the protein of the sample, 50 mg of sulfosalicylic acid was added to a conical centrifuge tube and cooled to 4 ° C (1 hour). 1 ml of the sample was added to the tube thus prepared, and then left at 4 ° C. for 1 hour. The precipitate was centrifuged (14,000 rpm, 15 minutes) at 4 ° C. until the supernatant turned completely clear. A certain amount of supernatant was taken and the pH was adjusted to 2.2 using 0.3 M lithium hydroxide. The amount of 0.3 M lithium hydroxide added at this time was about 10-20 microliters with respect to 20 microliters of a sample. In addition, when the pH was measured, the amount of the sample was too small to use a pH meter or a pH test paper, and about 15 μl of 0.3 M lithium hydroxide was added to 20 μl of the sample to adjust to an appropriate pH. The pH-corrected samples were analyzed by 0.2 μm membrane filtering in the amino acid analyzer under the conditions shown in Table 21 below.

Item Hisashia Hirano conditions column Ultrapac 11 cation exchange resin (6 mm x 200 mm) Buffer Speed 45 ml / hour Ninhydrin speed 35 ml / hour Column temperature 50 to 80 ℃ Buffer stage 4 steps Reactor temperature 130 ℃ Buffer pH range 3.2 to 10.0 Injection volume 40 μl

Table 22 shows free amino acid compositions of the same samples as in Table 12. As shown here, phosphoserine, hydroxyproline, threonine, serine, asparagine, glycine, alanine, citrulline, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, δ-aminobutyric acid, ornithine, lysine, arginine are all samples It was contained in. Glutamic acid contained 2.1 µg / g in basal medium and 1.5 µg / g in PO culture, and cystine in 45.5 µg / g, 54.7 µg / g and 41.0 µg / g in Cys-added samples. .

amino acid Amino acid content (㎍ / g) (One) (2) (3) (4) (5) Phosphoserine hydroxyproline threonine serine asparagine glutamic acid glycine alanine citrulline valine cystine methionine isoleucine leucine synthrosine phenylalanine δ-aminoisobutyric acid ornithine lysine arginine 4.47.20.51.82.42.10.41.31.91.0ND1.20.33.75.02.53.52.52.63.1 15.392.71.02.111.5ND0.61.71.21.645.52.21.54.85.13.60.90.53.34.6 4.83.60.90.53.31.54.40.62.02.8ND1.53.05.82.21.54.85.12.82.9 14.855.72.53.83.4ND1.54.40.62.054.72.52.88.86.24.21.20.94.55.3 15.56.12.01.73.8ND0.91.7ND2.541.00.63.22.05.33.42.30.81.51.7

In Table 22 above, (1) to (5) are the same as those described in Table 12. Amino acid content (microgram / g) is area * 10 * M.W. * dilution multiple / 1,000,000. In addition, ND means not detected.

According to the test results of the above examples, the optimum culture conditions of the mushroom mycelium and the characteristics of the MF produced from the liquid culture of PO are as follows:

① As a result of investigating the liquid culture conditions of the mycelia, shiitake mushrooms and shiitake mycelium, it was found that the optimum conditions for all three varieties were inoculated with mushroom liquid mycelium in a basal medium of pH 5.5 and cultured at 25 ° C., 120 rpm for 7 days.

② As a result of investigating the effect on the MF production by varying the medium composition under such optimum culture conditions, the culture medium in which oyster mushrooms were cultured by adding Cys (1%) and Rib (2%) to the basal medium was heated at 121 ° C. for 15 minutes. In one case, MF was produced from the liquid phase, which was unusual but strong and very similar to natural MF.

③ As a result of extracting MF-produced sample with ether and analyzing and identifying by GC-MS, 2-formyl-5-methyl, which is a thiophene compound produced when heating beef, plays an important role in MF production. It was confirmed that thiophene and dimethylformyl-thiophene were produced.

④ The medium cultured with PO by adding Cys and Rib to the basal medium was found to have a high content of 2-furancarboxaldehyde and furfuryl alcohol, which were found in a small amount or absent in other cultures, and these compounds were 2-formyl. It is assumed to be the major precursor of -5-methylthiophene and dimethylformylthiophene.

According to the present invention, using the oyster mushroom can produce a natural meat flavor with excellent aroma, can be utilized to provide a foundation to enable the industrialization process development to produce a large amount of natural meat flavor.

Claims (7)

A method of producing a meat flavor, comprising inoculating and incubating a mycelia of Pleuotus ostreatus in a liquid medium containing cysteine and ribose, and heating the culture solution. The liquid medium of claim 1, wherein the liquid medium comprises 10% (w / v) soybean meal, 2.7% (w / v) per sulfur white, 0.5% (w / v) KH ₂ PO _{4 and} 0.5% (w) MgSO ₄ .7H ₂ O. / v) dissolving in distilled water sterilized production method characterized in that the meat. The method of claim 1, wherein cysteine and ribose are added to the medium at a concentration of 1% and 2%, respectively. The method of claim 1, wherein the culture of oyster mushroom mycelium is characterized in that the pH of the liquid medium to 5.5 and incubated for 7 days at a shaking speed of 120 rpm at 25 ℃. The method of claim 1, wherein the culture solution is heated at 121 ℃ for 15 minutes. [Claim 2] The method of claim 1, wherein the lean mushroom mycelium is cultured to produce 2-furancarboxaldehyde and furfuryl alcohol, which are precursors of meat flavor. The meat flavor production method according to claim 1, wherein 2-formyl-5-methylthiophene and dimethylformyl-thiophene which are meat flavor components are produced by heating the culture solution.