TWI760892B - Process for producing monacolin k and/or chlorogenic acid and cultured product obtained thereform - Google Patents

Abstract

Disclosed herein is a method for producing Monacolin K and/or chlorogenic acid, comprising: culturing a Monascus ruberstrain in a medium containing a coffee fruit material, wherein the coffee fruit material is free of coffee bean.

Description

Method for producing MONACOLINK and/or chlorogenic acid and culture product obtained by the method

The present invention relates to a method for producing Monacolin K and/or chlorogenic acid and a culture product obtained by the method.

Rhododendron is a group of red molds belonging to the genus Monascus , including Monascus purpureus , Monascus pilosus and Monascus ruber , etc. They have been widely It is widely used to ferment food materials (such as rice, meat) to make various fermented foods (such as red yeast rice, red glutinous rice, red glutinous rice wine, etc.). In recent years, these fermented foods have been found to have a variety of physiological activities, and a variety of active ingredients have been isolated and identified, including, for example, Monacolin K, Monascidin A, monascorubramine, monascorubin, Red yeast yeast yellow pigment (monascin) and so on. Among them, the cholesterol-lowering activity has attracted special attention, and the corresponding active ingredient is Monacolin K.

Therefore, how to improve the production of Monacolin K has become the goal of related researchers in this field. For example, CN 102911977 A discloses a method for fermenting and producing Monacolin K by using a solid substrate as a carrier of red yeast rice, which mainly includes mixing a pretreated solid substrate carrier with a nutrient solution and fermenting the red yeast yeast. In different examples of this mainland patent case, bagasse, a mixture of corncob and bagasse, tea stems, puffed barley, boiled millet and green coffee beans are used as solid substrate carriers, and the nutrient solutions used are all A combination of glucose, soy hydrolyzate, glycerin, salts, and corn steep liquor, the strain of the rhododendron is Monascus anka CGMCC 0517. The results obtained are collated in Table 1 below. Table 1. Production of Monacolin K obtained by the example of CN 102911977 A Example solid matrix carrier Monacolin K (mg/g) 1 bagasse 11.31 2 Corn Cob and Bagasse 10.42 3 tea stalk 7.76 4 Puffed barley 9.32 5 steamed millet 13.21 6 green coffee beans 4.54

It can be seen that the yield of Monacolin K obtained by using green coffee beans is significantly lower than that obtained by other solid matrix carriers, and even only about one-third of that obtained by cooking millet. Moreover, coffee beans have become the most economically valuable crop and the second most traded commodity in the world after oil, making it uneconomical to use them to produce low-yield Monacolin K.

Summary of Invention

In the present invention, Applicants have unexpectedly discovered that coffee pulp, a by-product of the coffee bean production process, can be used to produce Monacolin K, and compared Monascus pilosus ) and purpureus ( Monascus purpureus )], using Monascus ruber to ferment them to produce a large amount of Monacolin K. In addition, it was also found that the content of an active ingredient in coffee berries - chlorogenic acid - was also greatly increased during the cultivation process.

Thus, in a first aspect, the present invention provides a method for the production of Monacolin K and/or chlorogenic acid, comprising: culturing a Rhodotorula rubrum strain in a medium containing a coffee berry material, wherein the The coffee berry material does not contain coffee beans.

In a second aspect, the present invention provides a culture product prepared by a method as described above.

In a third aspect, the present invention provides a food product comprising a cultured product as described above.

The above and other objects, features and advantages of the present invention will become apparent upon reference to the following detailed description and preferred embodiments.

Detailed description of the invention

It is to be understood that if any antecedent publication is cited herein, that antecedent publication does not constitute an admission that, in Taiwan or any other country, the antecedent publication forms a common general practice in the art part of knowledge.

For the purposes of this specification, it will be clearly understood that the word "comprising" means "including but not limited to" and that the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Of course, the present invention is in no way limited by the methods and materials described.

The present invention provides a method for producing Monacolin K and/or chlorogenic acid, which comprises: culturing a strain of Rhodotorula rubrum in a medium containing a coffee berry material, wherein the coffee berry material does not contain coffee beans.

As used herein, the terms "culturing,""cultivation," and "fermentation" are used interchangeably. Preferably, the culture of the Rhododendron erythraea strain is solid-state culture. The operating procedures and parameter conditions for the cultivation of the red yeast yeast fall within the professional quality and routine technical scope of those who are familiar with this technology. In this regard, reference may be made to, for example, Chen F. and Hu X. (2005), Int. J. Food Microbiol. , 103:331-337; Jirasatid S. et al . (2013), J. Microbiol. Biotechnol. , 23:364-374; and Kanpiengjai A. et al . (2018), BioTechnologia , 99:109-118.

It can be understood that the operating conditions related to the cultivation will be further changed according to factors such as the strain of Rhodotorula rubrum used and the dosage ratio of the medium to the medium, so as to achieve the best production effect. The selection of these operating conditions is routinely determined by those skilled in the art.

As used herein, the term " Monascus ruber strains" is intended to encompass those strains that are readily available to those skilled in the art (eg, available from domestic or foreign depositories), or Strains isolated and purified from natural sources using microbial isolation methods commonly used in the art. Preferably, the Rhododendron rhodochrous strain is at least one selected from the following: Rhododendron rhodochrous BCRC 31523, BCRC 31529, BCRC 31532, BCRC 31533, BCRC 31534, BCRC 31535, BCRC 31538, BCRC 33303, BCRC 33314 , BCRC 33323, BCRC 33324, BCRC 33326, BCRC 33329, and BCRC 33448. In a preferred embodiment of the present invention, the Rhododendron erythraea strain is Rhododendron erythraea BCRC 31535.

According to the present invention, based on the total weight of the medium, the R. rubra strain can be cultured using an inoculum of about 1 to 50 wt %. In a preferred embodiment of the present invention. The Rhododendron rhodozyma strain was cultured using an inoculum of 5 wt%.

As used herein, the terms "coffee fruit", "coffee berry" and "coffee cherry" are used interchangeably.

According to the present invention, the coffee fruit material comprises coffee pulp [also known as mesocarp]. Preferably, the coffee berry material further comprises a coffee outer skin [also known as exocarp]. Alternatively, the coffee fruit material may further comprise a parchment [also known as endocarp].

According to the present invention, the coffee berry material can be derived from different coffee species. Preferably, the coffee species may be selected from the group consisting of: Coffea liberica (also known as Liberia), Coffea canephora (also known as coffee) Canifora), Coffea arabica (also known as Aracabi), and high-yielding coffee ( Coffea dewevrei ). In a preferred embodiment of the present invention, the coffee species is Caffodil.

According to the present invention, the coffee cherry material can optionally be homogenized to produce a homogenate prior to addition to the medium.

According to the present invention, the coffee berry material may have a content of about 0.1 to 99 wt% based on the total weight of the medium. Preferably, the coffee berry material has a content of about 50 to 67 wt%.

According to the present invention, the medium may further contain a starch-based substrate selected from the group consisting of rice, taro, sweet potato, buckwheat, pumpkin, potato, and combinations thereof. Preferably, the medium further contains a rice selected from the group consisting of: white rice, brown rice, purple rice, black rice, glutinous rice (waxy rice), mixed grain rice, and combinations thereof. In a preferred embodiment of the present invention, the medium further contains white rice.

According to the present invention, the weight ratio of the coffee berry material to the starch-based substrate may be between 1:0 and 1:4. In a preferred embodiment of the present invention, the weight ratio of the coffee berry material to the starch base is 1:2.

According to the present invention, the medium may contain about 0 to 10 wt % of carbohydrates based on the total weight of the medium. Preferably, the saccharide is selected from the group consisting of fructose, glucose, sucrose, lactose, galactose, maltose, and combinations thereof. In a preferred embodiment of the present invention, the saccharide is fructose.

According to the present invention, the medium may contain about 0 to 10 wt% salts based on the total weight of the medium. Preferably, the salts are selected from the group consisting of sea salt, table salt, rose salt, and combinations thereof.

According to the present invention, the culturing of the Rhodotorula rubrum strain may be carried out at a temperature within about 20°C to 40°C for about 7 to 20 days. Preferably, the medium may be supplemented with 10 to 15 wt % of water (based on the total weight of the medium) on one or more of days 1 to 6 after starting the culture.

The present invention also provides a culture product obtained by a method as described above.

According to the present invention, the culture product may contain Monacolin K and/or chlorogenic acid. Preferably, the culture product contains both Monacolin K and chlorogenic acid.

The present invention also provides a food product comprising a cultured product as described above. The cultured product can be used as a food additive, which is added during the preparation of raw materials by conventional methods, or is added during the production process of food, and is formulated with any edible material for human consumption. Food products that are ingested by non-human animals.

According to the present invention, the types of food products include, but are not limited to: milk powder, beverages, confectionery, candies, fermented food, animal feeds, Health foods and dietary supplements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are only for illustration purposes and should not be construed as limitations on the implementation of the present invention. EXAMPLES Example 1. Cultivation of red yeast rice using a medium containing coffee pulp Experimental materials: 1. Red yeast yeast strains:

The Rhododendron strains used in this example were all obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan. (No. 331, Food Road, Hsinchu City, Taiwan, 300), and have been consolidated in Table 2 below, respectively. Table 2. Rhododendron strains used Rhododendron deposit number Red yeast yeast ( Monascus ruber ) BCRC 31535 (corresponds to ATCC 18199) Monascus pilosus BCRC 31502 (corresponds to ATCC 16363) Monascus purpureus BCRC 31615 (corresponds to DSM 1379) 2. Materials of culture medium:

The sources of the various materials of the culture medium used in this example are individually summarized in Table 3 below. Table 3. Sources of Materials for Media Material source Coffee pulp (with rind) Gukeng Coffee Manor white rice Miyoshi rice fructose Sigma-Aldrich soybean oil Taiwan sugar sea salt Jiaxin Sea Water

Before the experiment, coffee pulp homogenate was prepared by homogenizing the coffee pulp with a juicer (Brendtec, model ES3 EZ BLENDER). Experimental methods and results: A. Preparation of inoculum of red yeast rice :

The Rhododendron strain was inoculated onto a potato dextrose agar (PDA) plate (purchased from BD), and cultured in a constant temperature incubator (30°C) for 7 days. sky. Next, the rhododendron mycelium formed on the PDA culture plate was cut into pieces so that it had a volume of 1 cm ³ , and then added to a powder containing 20 g/L white rice, 40 g/L glucose, 10 g/L monosodium glutamate and peptone in 30 mL medium, and cultured in a constant temperature shaking incubator (30 °C, 120 rpm) for 3 sky. Afterwards, the obtained liquid culture was homogenized with a hand-held homogenizer (brand: QIAGEN, model: TissueRuptor), and the formed homogeneous culture was used as an inoculum source for Rhododendron. B. Cultivation of red yeast rice:

The inoculation sources of the 3 kinds of red yeast yeast obtained in the above item A were each divided into 10 groups, and were inoculated with an inoculum amount of 2.5 g (5 wt% of the solid substrate) to each with the following table 4. in the medium of the formula shown, and then cultured in a constant temperature incubator (30°C). The medium was agitated once a day during the culture period, and 5 g of water was supplemented on the 4th, 5th, and 6th days, respectively, and the resulting culture product was finally harvested on the 15th day. Table 4. Recipe of media group Content of various ingredients (g) Coffee pulp homogenate solid starch base carbohydrate grease salt water white rice fructose Soybean oil sea salt C0 0 50 0 0 0 9.5 C1/2 25 25 0 0 0 9.5 F0 0 50 0.5 0 0 9.5 F1/2 25 25 0.5 0 0 9.5 F2/3 33.4 16.6 0.5 0 0 9.5 SB0 0 50 0 0.5 0 9.5 SB1/2 25 25 0 0.5 0 9.5 SB2/3 33.4 16.6 0 0.5 0 9.5 SS0 0 50 0 0 0.5 9.5 SS1/2 25 25 0 0 0.5 9.5 Note: White rice is pre-soaked in reverse osmosis water overnight and drained . C. Preparation of samples to be tested:

Each cultured product obtained in the above item B was subjected to hot air drying at 45 ° C for 3 days, and then a high-speed pulverizer (brand name Rong Cong Precision Technology Co., Ltd., model RT-N04 ) to be ground. 0.2 g of the obtained powder was weighed and 25 mL of methanol was added, followed by ultrasonic vibration treatment at 25° C. for 30 minutes. After that, centrifugation was performed at 3,000 rpm for 10 minutes and the supernatant was collected, thereby obtaining a sample to be tested for the following component analysis. D. Content analysis of Monacolin K :

First, in order to preliminarily analyze whether each culture product contains Monacolin K, referring to the method described in Sun JL et al . (2011), Biol. Res. , 44:377-382, each test sample was subjected to a wavelength of 238 nm. Next, the absorbance value (OD ₂₃₈ ) was read with a spectrophotometer (brand MERCK, model Spectroquant Pharo 300). The OD ₂₃₈ values thus determined are then based on the OD ₂₃₈ values previously made with respect to their own in methanol solutions with different known concentrations (0 to 20 ppm) of Monacolin K (available from Sigma-Aldrich) The correlation curve was converted to Monacolin K concentration. The results showed that the culture products of the three rhododendron strains all contained Monacolin K, among which the highest was obtained from the rhododendron (data not shown).

In order to more accurately analyze the content of Monacolin K in each cultured product, each sample to be tested was filtered with a 0.22 μm filter membrane (purchased from MERCK), and then refer to the “Test method for Monacolin K in food” announced by the Ministry of Health and Welfare. ” for high performance liquid chromatography (HPLC).

The HPLC analytical instruments used are as follows: HITACHI liquid chromatography system (brand HITACHI, model L-2000) and UV detector (brand HITACHI, model L-2455). Operating parameters and conditions are shown in Table 5 below. Table 5. Operating Parameters and Conditions for HPLC Separation column C18 column (MERCK, LiChrospher ^® RP-18) String Specifications 25 cm × 4.6 mm column temperature 25℃ Sample injection volume 10 μL Detection wavelength 238nm mobile phase Acetonitrile/0.1% phosphoric acid in water, 65:35 (v/v) Flow rate (mL/min) 1.5

In addition, for comparison, 20 mg of Monacolin K was dissolved in 10 mL of acetonitrile and then diluted with methanol into methanol solutions with various concentrations (0 to 200 ppm) of Monacolin K as a calibration standard for lactone-type Monacolin K and 1 mL of Monacolin K was mixed with 1 mL of 0.1N NaOH and sonicated at 50°C for 1 hour, then diluted with methanol to have different concentrations (0 to 200 ppm) A methanol solution of Monacolin K was used as the calibration standard for the acid form of Monacolin K.

The results obtained are shown in Table 6 below. Table 6. Monacolin K content (ppm) of each group of cultured products of 3 kinds of red yeast rice group red yeast Rhododendron filiformis Rhododendron C0 15 0 0 C1/2 429 0 0 F0 13 0 0 F1/2 489 0 0 F2/3 881 0 0 SB0 41 0 0 SB1/2 42 0 0 SB2/3 104 0 0 SS0 11 0 0 SS1/2 397 0 0 Note: The content of Monacolin K of lactone type and acid type is calculated by adding up.

It can be seen from Table 6 that, in the absence of coffee pulp, the culture products of the three types of Rhododendron have only a trace amount of Monacolin K or below the detection limit. In the case of using coffee pulp, the presence of Monacolin K was unexpectedly not detected in each group of cultured products of R. rhodozyma and R. purpureus, and only the cultured products of R. K. In particular, the results obtained by using a spectrophotometer to analyze with reference to Sun JL et al . (2011) (same as above) preliminarily show that Rhododendron has a better effect in the production of Monacolin K. Accordingly, the applicant believes that the culture product obtained in this example may contain substances that may interfere with the spectrophotometer, which is not suitable for the analytical method disclosed by Sun JL et al . (2011) (same as above).

The results of HPLC analysis clearly showed that the production of Monacolin K of R. rubrum was significantly increased by using coffee pulp with or without additional added sugars, oils or salts. In particular, the yield improvement of Monacolin K was even more pronounced with the addition of additional sugars.

In order to further understand whether the content of other active ingredients will also be increased, the test samples of the red yeast yeast groups F1/2 and F2/3 were taken for the following analysis. E. Content analysis of chlorogenic acid :

The test samples of groups F1/2 and F2/3 were filtered with a 0.22 μm filter membrane, and then HPLC analysis was carried out with reference to the “Test Methods for Chlorogenic Acids in Capsules and Tablets” announced by the Ministry of Health and Welfare.

The HPLC analytical instrument used was as described above, and the various operating parameters and conditions for the HPLC are shown in Table 7 below. Table 7. Operating Parameters and Conditions for HPLC Separation column C18 column (MERCK, LiChrospher ^® RP-18) String Specifications 25 cm × 4.6 mm column temperature 25℃ Sample injection volume 20 μL Detection wavelength 327nm mobile phase Acetonitrile/1% phosphoric acid in water, 5:95 (v/v) Gradient elution of mobile phase From 0 to 20 minutes, acetonitrile was changed from 5% to 13%; from 20 to 20.1 minutes, acetonitrile was changed from 13% to 5%; from 20.1 to 25 minutes, acetonitrile was maintained at 5%. Flow rate (mL/min) 1

For comparison, the pre-culture medium of groups F1/2 and F2/3 were prepared as test samples and subjected to the same HPLC analysis according to the method described in item C above. In addition, 10 mg of chlorogenic acid (purchased from Sigma-Aldrich) was dissolved in 10 mL of methanol and then diluted with 70% methanol into methanol solutions with different concentrations (0 to 500 ppm) of chlorogenic acid as calibration Standard.

The results obtained are shown in Table 8 below. Table 8. Chlorogenic acid content (ppm) of red yeast rice groups F1/2 and F2/3 before and after cultivation group uncultivated medium culture product F1/2 56.9 144.7 F2/3 48.9 207.3

It can be seen from Table 8 that the red yeast yeast can significantly increase the chlorogenic acid content in coffee pulp (increased by 250% and 423%, respectively), and this increase will also increase significantly with the increase in the amount of coffee pulp.

Based on the above experimental results, it can be seen that the red yeast rice is unexpectedly suitable for culturing in the medium containing coffee pulp to produce Monacolin K and chlorogenic acid in large quantities.

All patents and documents cited in this specification are incorporated by reference in their entirety. In the event of conflict, the detailed description of the case (including definitions) will prevail.

Although the present invention has been described with reference to the specific embodiments above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the scope of the appended claims.

Claims (8)

A method for producing Monacolin K and/or chlorogenic acid, comprising: culturing a strain of Monascus ruber in a medium containing a coffee berry material, wherein the coffee berry material has about 50 to 67wt% content, and contains coffee pulp and does not contain coffee beans. The method of claim 1, wherein the Rhodotorula rubrum strain is Rhododendron erythraea BCRC 31535. The method of claim 1, wherein the medium further comprises a starchy substrate selected from the group consisting of rice, taro, sweet potato, buckwheat, squash, potato, and combinations thereof. The method of claim 3, wherein the starchy substrate is white rice, and the weight ratio of white rice to the coffee berry material is 1:1 to 1:2. The method of claim 1, wherein the medium further contains a carbohydrate selected from the group consisting of fructose, glucose, sucrose, lactose, galactose, maltose, and combinations thereof. The method of claim 1, wherein the medium further contains a salt selected from the group consisting of sea salt, table salt, rose salt, and combinations thereof. A culture product obtained by the method of any one of claims 1 to 6, and comprising Monacolin K and chlorogenic acid. A food product comprising the culture product of claim 7.