TWI470078B - Isolated galactomyces geotrichum strain and use thereof for decomposing organic acids - Google Patents

Description

Application of Geotrichum candidum isolate for decomposing organic acids

The present invention relates to an isolated strain of Geotrichum candidum and its use, and in particular to a strain of the Geotrichum candidum strain, Galactomyces geotrichum THUFS, which can be used to decompose organic acids, and uses thereof.

Noni contains phenolic compounds such as sputum, scutellaria, rutin, and flavonoids, which have antibacterial, anti-cancer, anti-oxidant, immune-modulating, analgesic and cardiovascular effects. Noni juice has become a high-priced health food, but the mature noni fruit will emit a pungent odor. Therefore, improving the bad flavor of Noni juice is an important issue to improve product quality.

There are about 51 volatile compounds isolated from Noni, including caproic acid, caprylic acid, decanoic acid, scutellarin and 3-methyl-3-buten-1-ol (3-methyl-3). -buten-1-ol) is a component with a high content, accounting for about 85% of the total. After the innocene is ripened, its main volatile components, butyric acid, caproic acid and caprylic acid, produce irritating rancid odour, which greatly reduces the acceptance of noni fruit.

Therefore, in order to remove the bad odor, the commercially available Noni juice is usually mixed with the fermentation, esterification, deacidification, volatilization, acid-base neutralization, etc. The method reduces the concentration of these organic acids. For example, in recent years, some studies have been conducted to remove the undesirable flavor of noni fruit, such as the use of falling film decompression and concentration technology to remove butanoic acid and 2-methylbutyric acid; 3-methyl-3-buten-1-ol (3-methyl-3-buten-1-ol) was almost completely removed by a vacuum concentration technique; ethanol was added for esterification, and the results showed that this method can be used to reduce Free form of butanoic acid, 2-methylbutyric acid, hexanoic acid, and octanoic acid, and produces some fruity flavored esters; added calcium hydroxide It can reduce the concentration of butanoic acid, 2-methylbutyric acid, hexanoic acid and octanoic acid. Although the above methods can reduce the unpleasant flavor of Noni juice, the nutrient components of noni juice, ruthenium and scutellarin, are also significantly reduced, and the processing cost of Noni juice is greatly increased, so that other ways to improve Noni The flavor of juice is an important issue to date.

Geotrichum candidum is a yeast that resembles a small filamentous fungus, and its colonies are white fluffy. Geotrichum candidum belongs to the classification of the genus Ascomycetes, Yeast, Candida, Geotrichum, and its sexual name is Galactomyces geotrichum. Some Geotrichum candidum has the fermentation characteristics of yeast; yeast fermentation process will affect the ingredients of the food itself, such as higher alcohols, organic acids, acetaldehyde and pyruvic acid, etc., which also contain many volatile components. The type of yeast and the fermentation temperature will interact with the raw materials to affect the formation of volatile components, resulting in different flavors, which in turn affect food quality.

The organic acid metabolized by the yeast fermentation process is one of the main sources of food flavor, and is also a precursor of esters; pyruvic acid is a metabolite of glycolysis, which is metabolized to alcohol, lactic acid and acetic acid under different conditions. . In addition, yeasts perform β-oxidation on long-chain fatty acids to form organic acids and short-chain fatty acids, and are also precursors of food flavors.

Therefore, if the organic acid which selectively degrades the bad odor can be selectively utilized by microorganisms, the flavor of Noni juice can be greatly improved. In view of the above, the present invention provides a strain of the genus Galicectomyces geotrichum THUFS, which is capable of decomposing organic acids, especially volatile organic acids in noni fruit, and metabolically improving the pungent taste caused by volatile organic acids.

One embodiment of an aspect of the present invention provides a strain of Geotrichum candidum, which is deposited in the Institute of Food Industry Development, and its registration number is BCRC 920079.

According to one embodiment of the present invention, the growth temperature of the aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, may be from 5 ° C to 40 ° C, preferably from 25 ° C to 35 ° C, and more preferably 30 ° C.

According to one embodiment of the present invention, the aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, has a growth pH of from pH 3 to pH 10, preferably from pH 6 to pH 9, and more preferably from pH 7 to pH 8.

According to one embodiment of the present invention, the growth tolerance of the aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, is from 2 to 8 weight percent of the salt.

One embodiment of the use aspect of the present invention is to provide a use of the Geotrichum candidum strain, Galactomyces geotrichum THUFS, which is applied to the decomposition of organic acids.

According to one embodiment of the use aspect of the present invention, the use of the aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, wherein the organic acid is a volatile organic acid.

According to one embodiment of the use aspect of the present invention, the use of the aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, wherein the volatile organic acid is a volatile organic acid contained in Morinda citrifolia.

According to an embodiment of one embodiment of the use aspect of the present invention, the volatile organic acid is selected from the group consisting of Butanoic acid, Isobutyric acid, and 2-methylbutanoic acid. ), a group consisting of Hexanoic acid, Heptanoic acid, and Octanoic acid.

According to another embodiment of one embodiment of the use aspect of the present invention, the volatile organic acid may comprise Butanoic acid, Isobutyric acid, 2-Methylbutanoic acid. And at least one of Hexanoic acid, Heptanoic acid, and Octanoic acid.

According to still another embodiment of one embodiment of the use aspect of the present invention, the volatile organic acid may comprise Butanoic acid, Isobutyric acid, 2-Methylbutanoic acid. Hexanoic acid, Heptanoic acid, and Octanoic acid.

One embodiment of the method aspect of the present invention provides a method for decomposing a volatile organic acid contained in Morinda citrifolia, comprising transferring the aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, to a noni fruit for fermentation culture. .

According to one embodiment of the method aspect of the present invention, the aforementioned method for decomposing a volatile organic acid contained in Morinda citrifolia, wherein the noni fruit is crushed and the residue is filtered to obtain a noni juice. Then, the Geotrichum candidum strain Galactomyces geotrichum THUFS is inoculated into Noni juice for fermentation culture, and the culture temperature thereof can be 18 ° C to 22 ° C.

One embodiment of the present invention is to provide a composition comprising the former Geotrichum candidum strain, Galactomyces geotrichum THUFS.

The technology and aspects of the present invention will be described in the following, and the present invention will be apparent to those of ordinary skill in the art.

S100‧‧‧ steps

S200‧‧‧ steps

S300‧‧‧ steps

S400‧‧‧Steps

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Morinda citrifolia) a flow chart of a method for containing a volatile organic acid; and Fig. 2 is a photograph of a cell morphology of a Geotrichum toxin strain of the invention: Fig. 3 is a photograph showing the colony appearance of the Geotrichum candidum strain of the present invention; and Fig. 4 is a micrograph of a 200-fold magnification of the Geotrichum candidum strain of the present invention, Galactomyces geotrichum THUFS, which is magnified by iodine liquid; FIG. 5A is a micrograph of the present invention. Growth curve of the Geotrichum candidum strain Galactomyces geotrichum THUFS at 5 ° C, 10 ° C; Figure 5B is the Geotrichum candidum strain of the present invention Galactomyces geotrichum THUFS at 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C Growth curve at temperature; Fig. 6 is a graph showing the relationship between the growth rate and temperature of the Geotrichum candidum strain of the present invention; and Fig. 7 is the growth of the strain of the genus Galicectomyces geotrichum THUFS of the present invention at different pH values. Figure 8 is a graph showing the specific growth rate of the Geotricum fuliginea strain of the present invention, Galctomyces geotrichum THUFS, at different pH values; Figure 9 is a fermentation of the Geotrichum candidum strain, Galactomyces geotrichum THUFS, in vitro, at different salt concentrations. Photo; and Figure 10 is a 50% noni juice before the fermentation of the Geotrichum candidum strain Galactomyces geotrichum THUFS of the present invention After that, a high performance liquid chromatography (HPLC) control map of the changes in volatile organic compounds.

Embodiments of the invention are discussed in more detail below. However, this embodiment can be applied to various inventive concepts and can be embodied in various Within the same specific range. The specific embodiments are for illustrative purposes only and are not limited by the scope of the disclosure.

The present invention provides a Geotrichum candidum strain, Galactomyces geotrichum THUFS, which is deposited in the Institute of Food Industry Development, and its registration number is BCRC 920079. The aforementioned Geotrichum candidum strain, Galactomyces geotrichum THUFS, can be grown at different growth temperatures, for example, 5 ° C to 40 ° C, preferably 25 ° C to 35 ° C, and more preferably 30 ° C. The Geotrichum candidum strain, Galactomyces geotrichum THUFS, of the present invention can be grown in different growth pH environments, such as pH 3 to pH 10, preferably pH 6 to pH 9, and more preferably pH 7 to pH 8. The Geotrichum candidum strain, Galactomyces geotrichum THUFS, of the present invention may also have a growth salt tolerance of 2 to 8 by weight of the salt.

The Geotrichum candidum strain, Galactomyces geotrichum THUFS, of the present invention can be applied to decompose organic acids. Further, the Geotrichum candidum strain, Galactomyces geotrichum THUFS, of the present invention can be applied to decompose volatile organic acids contained in Morinda citrifolia, such as Butanoic acid, Isobutyric acid, 2-Methylbutanoic acid, Hexanoic acid, Heptanoic acid, Octanoic acid, and the like.

Please refer to FIG. 1 , which is a flow chart showing a method for decomposing volatile organic acids contained in Morinda citrifolia according to an embodiment of the method of the present invention. As shown in the figure, the method for decomposing the volatile organic acid contained in the Morinda citrifolia comprises: step S100, cleaning and removing the surface impurities of the fruit of the noni fruit; In step S200, the fruit of the aforementioned noni fruit is dried and the fruit is ripened; in step S300, the fruit of the noni fruit is broken, and centrifuged for 30 minutes at 13,000 revolutions per minute (rpm) to sediment impurities, and then the impurities are filtered. In addition, in order to obtain Noni juice; and in step S400, the Geotrichum candidium strain Galactomyces geotrichum THUFS of the present invention is inoculated into Noni juice at a suitable bacterial concentration for fermentation culture at a culture temperature of 20 °C.

The invention is further illustrated by the following specific test examples. The following test examples are provided by way of illustration only and are not intended to limit the scope of the invention.

[Test Example 1] Characteristics of the Geotrichum candidum strain Galactomyces geotrichum THUFS

The Geotrichum toxin strain geotrichum THUFS of the present invention is isolated from the Chinese sauerkraut by the inventors and further cultured, identified and preserved. When cultivating Galactomyces geotrichum THUFS, it can be cultured using YPD (Yeast Extract Peptone Dextrose Medium) liquid medium, wherein 1 liter (L) of YPD liquid medium contains 20 g (g) of peptone, 10 g of yeast extract Yeast extract, 20 g of glucose, and 1000 ml (mL) of sterile water.

In addition, the Galactomyces geotrichum THUFS can also use YPD (Yeast Extract Peptone Dextrose Medium) plate medium. Cultured, 1 liter (L) of YPD plate medium containing 20 grams (g) of peptone, 10 g of yeast extract, 20 g of glucose, 1000 ml of ml of sterile water And 20 g of agar, and the medium was sterilized at high temperature and high pressure (121 ° C, 15 minutes), cooled to 45 ° C and poured into a sterile Petri dish to be set aside.

After the culture of the Geotrichum candidum strain Galactomyces geotrichum THUFS of the present invention was cultured in the aforementioned medium, its appearance characteristics, colony morphology and other growth characteristics were analyzed.

Please refer to FIG. 2, FIG. 3 and FIG. 4, which are respectively the cell morphology of the Geotrichum candidum strain, the appearance of the colony, and the microscopic photograph of 200-fold magnification after staining with iodine solution. The appearance of the Geotrichum candidum strain Galactomyces geotrichum THUFS of the present invention is a column with a circular arc angle, and the colonies thereof are in the form of cotton batt; after the strain is simply stained with iodine solution, the cells can be more clearly observed under the microscope 200 times. It is columnar, the size of the cells is about 10~15 μm, and it has filamentous filaments; the appearance of the colonies spreads out by cotton-like radiation, and the central uplift produces valley-like wrinkles.

In addition, in the growth temperature range of the Geotrichum candidum strain of the present invention, the aforementioned strain is inoculated into 50 ml of YPD liquid medium, and the initial bacterial solution concentration OD _{600 is} controlled to have an absorbance of about 0.01 at different temperatures (5). , 10, 15, 20, 25, 30, 35 and 40 ° C), 150 rpm shaking culture, timed sampling to measure its OD ₆₀₀ absorbance, can draw the growth curve of the Geotrichum candidium strain Galactomyces geotrichum THUFS of the present invention and calculate its Specific growth rate.

Please refer to FIG. 5A and FIG. 5B , which are respectively the growth curves of the Geotrichum candidum strain Galactomyces geotrichum THUFS of the present invention at temperatures of 5, 10 and 15, 20, 25, 30, 35, 40 ° C. The ordinates of Figures 5A and 5B are the logarithm of the OD ₆₀₀ absorbance in nanometers (nm). Please also refer to Fig. 6, which is the relationship between the specific growth rate and the temperature of the Geotrichum candidum strain of the present invention. As shown in the figure, the Geotricum fuliginea strain of the present invention, Galactomyces geotrichum THUFS, has a lag time of about 4 days at a culture temperature of 5 ° C. Therefore, it can be judged that the Geotricum fuliginea strain of the Geotrichum toxin is a Psychrotroph. The maximum growth temperature of this strain is less than 40 ° C, and there is significant growth at 35 ° C. The optimal growth temperature is between 25 and 35 ° C. According to the ratio of specific growth rate to temperature, the growth rate of 30 °C is the largest, reaching 0.71 h ^-1 . The following test examples are all cultured at 30 °C.

The growth pH range of the Geotrichum candidum strain of the present invention is adjusted to 50 ml YPD liquid medium to different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10 and 11), and After inoculating the strain, control the initial bacterial solution concentration OD _{600 to be} about 0.01, shake culture at 30 ° C, 150 rpm, periodically measure the OD ₆₀₀ , and draw the growth of the Geotrichum candidum strain Galictomyces geotrichum THUFS of the present invention at different pH values. Graph and calculate its specific growth rate.

Please refer to FIG. 7 and FIG. 8 , which are respectively a growth curve of the Geotrichum candidum strain Glacitomyces geotrichum THUFS of the present invention at different pH values and a specific growth rate diagram thereof. The results showed that the growth strain of Galactomyces geotrichum THUFS of the present invention has a growth pH of 3-10, an optimum growth pH of 7-8, and a specific growth rate of up to 0.77 h ^-1 .

Further, in the range of the highest salt tolerance of the Geotrichum candidum strain, the Galactomyces geotrichum THUFS of the present invention is in 5 ml of YPD liquid medium, and NaCl is added thereto, wherein the NaCl concentration is 2, 3, respectively. 4, 5, 6, 7, 8, 9 and 10% were inoculated into 50 μl of the bacterial solution at the above-mentioned different NaCl concentrations, cultured at 30 ° C, 150 rpm for 10 days, and the growth was recorded.

Please refer to Fig. 9, which is a photograph of the fermentation of the Geotrichum candidum strain Galactomyces geotrichum THUFS of the present invention in vitro at different salt concentrations. The results showed that the Geotricum fuliginea strain of the present invention, Galactomyces geotrichum THUFS, had a slight growth condition after 10 days of culture in a YPD liquid medium containing 8% NaCl, while the liquid medium containing 9, 10% NaCl had no growth at all, so it was presumed to be the highest. The salt tolerance is about 8%. In summary, the growth conditions of the strain of the genus Galictomyces geotrichum THUFS of the present invention are: a characteristic growth temperature range of 5 to 40 ° C, a growth pH range of 3 to 10, and a maximum salt tolerance of 8%.

[Experiment 2] Analysis of volatile organic acids contained in Noni juice by Galicectomy strain geotrichum THUFS.

One of the uses of the Geotrichum candidum strain, Galactomyces geotrichum THUFS, is used to decompose the volatile organic acid contained in the noni juice, and thus before and after the fermentation of the Geotrichum candidum strain Glacitomyces geotrichum THUFS of the present invention in Noni juice, Analyze changes in volatile organic compounds.

In this test, the changes of volatile organic compounds before and after fermentation in the noni juice were separately extracted by solid phase extraction. The addition of NaCl to saturate the sample, the extraction temperature of 50 ° C, the equilibration time of 15 minutes, the extraction time of 30 minutes and the sample fermentation time of 5 days are the solid phase extraction conditions of this test example.

After the fermentation of Noli juice of the present invention, Galctomyces geotrichum THUFS, the odor has changed significantly, from pungent rot to fruit scent and wine, and the color of the juice is changed from amber to dark green, so it is aimed at volatile organic compounds. Further analysis was performed before and after fermentation.

Please refer to Fig. 10, which is a high performance liquid chromatography (HPLC) control map of the change of volatile organic compounds of 50% Noni juice before and after fermentation of the Geotrichum candidum strain of the present invention. Among them, the upper row is the map before the fermentation of 50% Noni juice, and the lower row is the map after 50% Noni juice is fermented by the Geotricum fuliginea strain of the present invention. The ordinate in the map is abundance, and the abscissa is the retention time (RT) in the column.

As shown in the figure, after the fermentation of Noni juice by the Galicectomyces geotrichum THUFS of the present invention, the types of volatile metabolites were changed, and the difference between before and after the treatment of the juice was analyzed by gas chromatography, and gas chromatography was observed. The spectra were significantly different. Further, gas chromatography/mass spectrometry was used to identify volatile substances before and after fruit juice treatment. The gas chromatography/mass spectrometry showed significant differences.

Please refer to Table 1 and Table 2 below, and at the same time compare Figure 10; Table 1 is the analysis of volatile organic compounds before fermentation of Noni juice, and Table 2 is the juice of Nori juice in the present invention, Galactomyces geotrichum THUFS Analysis of volatile organic compounds after fermentation.

After comparing the data in the database, it was found that the residual amount of the original bad odor after the fermentation of the non-nosed strain Galictomyces geotrichum THUFS of the present invention was 3.94% of butyric acid, 0.63% of hexanoic acid and 4.23% of octanoic acid. - Methyl butyric acid has not been detected, and produces a variety of aroma alcohols, such as Ethanol, Butanol, Isoamylalcohol, Isoprenol, Hexanol (Hexanol) and Octanol, and as a result, it was found that the Geotrichum candidum strain, Galactomyces geotrichum THUFS, of the present invention should remove the carboxyl group of the organic acid and convert it into a hydroxyl group or decompose the short-chain fatty acid into a fusel.

Isobutyric acid, 3-methyl-3-buten-1-ol, Butanoic acid, 2-methylbutyric acid (2) -methyl butyric acid), Hexanoic acid, Heptanoic acid, Octanoic acid, the main source of undesirable flavor of noni fruit is Butanoic acid, 2-methylbutyric acid (Butanoic acid) 2-methyl butyric acid), Hexanoic acid, Octanoic acid. After 50% noni juice is fermented by MRA6, the main volatile substances are ethanol (Ethanol), butanol (Butanol), isoamyl alcohol (Isoamyl Alcohol), Isoprenol, Hexanol, Octanol, Hexanoic acid, Phenethyl alcohol, Octanoic acid, among which hexanol (Hexanol) The content is the most, so that the noni juice after fermentation has an apple-like flavor; while the non-flavored substance of Noni is almost degraded, only part of Hexanoic acid and Octanoic acid remain.

It can be seen from the above embodiments of the present invention that the application of the present invention has the following advantages: Noni fruit contains various phenolic compounds such as hydrazine, scutellarin, rutin, flavonoids, etc., and has antibacterial, anticancer, antioxidation, immune regulation, and analgesic effects. And improve the efficacy of cardiovascular disease. Noni juice has become a high-priced health food, but the mature noni fruit will emit a pungent odor. Therefore, improving the bad flavor of Noni juice is an important issue to improve product quality. The main ingredients of Noni fruit's bad flavor are volatile butyric acid, caproic acid and caprylic acid. Although the method of removing the pungent odor in Noni juice can reduce the bad flavor of Noni juice, the nutrients of the noni juice, ruthenium and scutellaria, are also significantly reduced, and the processing cost of Noni juice is increased. The invention is isolated from the sauerkraut strain Galicectomyces geotrichum THUFS, which can metabolize various organic acids, alcohols and esters.

The Geotricum fuliginea strain of the present invention, Galactomyces geotrichum THUFS, can degrade the organic acids of the bad odor in Noni juice and strongly improve the flavor of the juice.

The use of the Geotrichum candidum strain, Galactomyces geotrichum THUFS, of the present invention can decompose the characteristics of organic acids, and can not only be applied to decomposition The volatile organic acid of the genus can also ferment and metabolize organic acids such as volatile organic acids in other fruits and vegetables in an environment capable of allowing the growth of the Geotrichum candidum strain of the present invention, Galactomyces geotrichum THUFS. Wait.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

【Biomaterial Storage】

Domestic registration information [please note according to the order of the depository, date, number] Food Industry Development Institute August 14, 2012 BCRC 920079

S100‧‧‧ steps

S200‧‧‧ steps

S300‧‧‧ steps

S400‧‧‧Steps

Claims (8)

The use of a Geotrichum candidum strain, Galactomyces geotrichum THUFS, for decomposing volatile organic acids, wherein the Geotrichum candidum strain, Galactomyces geotrichum THUFS, is deposited in the Food Industry Development Institute of the Corporation, and the accession number is BCRC 920079. The use according to claim 1, wherein the volatile organic acid is selected from the group consisting of Butanoic acid, Isobutyric acid, and 2-methylbutanoic acid. ), a group consisting of Hexanoic acid, Heptanoic acid, and Octanoic acid. The use according to claim 1, wherein the volatile organic acid comprises Butanoic acid, Isobutyric acid, 2-Methylbutanoic acid, and At least one of an acid (Hexanoic acid), a heptanoic acid, and an Octanoic acid. The use according to claim 1, wherein the volatile organic acid comprises Butanoic acid, Isobutyric acid, 2-Methylbutanoic acid, and Acid (Hexanoic acid), heptanoic acid and Octanoic acid. The use according to claim 1, wherein the volatile organic acid is a volatile organic acid contained in Morinda citrifolia. A method for decomposing a volatile organic acid contained in Morinda citrifolia, comprising transferring the Geotricum sinensis strain Galactomyces geotrichum THUFS with accession number BCRC 920079 to a noni fruit for fermentation culture. The method for decomposing a volatile organic acid contained in Morinda citrifolia according to claim 6, wherein the noni fruit is crushed and the residue is filtered to obtain a noni juice, and then the Geotrichum candidum strain Galactomyces geotrichum THUFS was inoculated into the Noni juice for fermentation culture. The method for decomposing a volatile organic acid contained in Morinda citrifolia according to claim 6, wherein the culture temperature of the Geotrichum candidum strain Galactomyces geotrichum THUFS in the noni juice is 18 ° C to 22 ° C.