KR100634867B1 - Method for crushing of cell wall and extrcting a pigment thereof - Google Patents

Abstract

The present invention relates to a method for pulverizing a pigment containing microorganisms and a method for extracting a pigment, the method comprising: culturing a microorganism containing a pigment; Putting the cultured microorganisms in a drier and drying them; And pulverizing the dried microorganisms in a mill and extracting the microorganisms pulverized by the method with a solvent. And it provides a method for extracting the pigment comprising the step of drying the extract is used as a functional material of food and food additives or feed or feed additives.

The present invention is a simple and effective destruction of the cell wall of the microorganisms containing natural pigments, it is possible to efficiently extract the natural pigments, by using the method as a feed and feed additives or food storage and food additives and beneficial to the human body As it can be used as a functional material and can be applied inexpensively and easily, the ripple effect is expected to be great when entering the industry.

Pigment, microorganism, grinding, extraction, cell wall

Description

Method for crushing of cell wall and extrcting a pigment according}

1 is a graph showing the content of astaxanthin extracted with ethanol and dimethyl sulfoxide solvent by drying the yeast according to the number of treatments on a laboratory scale (A) and a plant scale (B),

Figure 2 is a SEM photograph showing the dried particles of papia rhododima dried by drying scale according to the present invention,

3 is a SEM photograph showing the cell wall destruction effect of the yeast according to the control method (a), spray dryer group (b), drum dryer group (c), drying after spray drying (d),

Figure 4 is added to the feed of the dried yeast of the present invention fed to chickens and after slaughter, a picture showing the degree of accumulation of astaxanthin in the chicken leg portion.

The present invention relates to a method for pulverizing microorganisms containing pigments and a method for extracting pigments, and more specifically, by simply using a spray dryer or a drum dryer, and then pulverizing with a mill to efficiently destroy the cell walls of microorganisms. And a method for increasing the extraction effect of the functional pigment.

In general, the cartenoid pigments (algae), microorganisms (microorganism), shellfish, etc. are commonly known as pigments having an antioxidant effect.

Among these, astaxanthin is a pigment that has a scarlet color such as shrimp or lobster shellfish, seaweed belonging to Haematococcus sp., Microorganism belonging to Phaffia sp., Bird's beak, egg yolk, etc. Enhancement, flavor, immune function, vitamin A precursor, antioxidant effect, aging and anti-cancer effects are known.

However, astaxanthin pigment extraction from crustaceans, seaweeds and microorganisms has not been commercialized due to its low pigment content and complex extraction process.

This is because pigments of astaxanthin are mainly deposited in cells, and the cell wall must be efficiently destroyed in order to extract astaxanthin effectively.

For example, Phaffia rhodozyma (a pseudonym xanthophyllomyces dendrorhous) containing astaxanthin is prepared by adding it to a feed and feeding it to animals, which does not effectively utilize astaxanthin in vivo. It is not fresh enough to be stored in the shells and eggs of many animals and thus does not function as a feed additive (Johnson, EA et al., 1977).

In order to solve this problem, various methods of breaking the cell wall have been developed in the past, and as a chemical treatment method, a method of extracting astaxanthin by crushing and neutralizing the cell wall of yeast through acid hydrolysis is known [Bacteriol. Rev., 39. 197-231 (1975); US Patent No. 5,210,186; European Patent EP 0 553 814], the physical method of breaking the cell wall using a machine such as a French press (French pressure), Homogenizer (Braun homogeniger), a homogenizer (Microfluidiger) and then extracted with a solvent There is a method [Enzyme Microb. Technol., 8. 194-203 (1986); J. Appl. Bacteriology 70. 181-191 (1991)], as a biological treatment method is known to be treated using cellulase, hemiserulase, pectinase and the like enzymes that break down the cell wall [Appl. and Environ. Microbiol. 35 (6). 1155-1159 (1978).

However, among the above methods, the chemical treatment method is low in efficiency because it destroys the pigment to be extracted along with cell wall destruction, and the mechanical method requires a lot of treatment costs such as equipment cost and labor compared to the pigment extraction rate. Has been a problem that is difficult to apply to commercialization for mass production, such as higher processing costs such as reagent and operating costs as well as equipment costs.

However, in recent years, as ethanol extract of astaxanthin from the yeast strain papia rhodoma has been approved as a natural pigment in food additives, it is necessary to increase the content of astaxanthin in the bacteria itself when added to feed or applied to food additives. As well as research on how to effectively break down the cell wall involved in the extraction method is being actively conducted.

Accordingly, the present inventors have attempted to develop a cell wall destruction method and a method of extracting the pigment from the pigment-containing microorganisms, which are simple and inexpensive to treat, and the present invention has been completed by using a drying method and a rolling drum mill.

Therefore, an object of the present invention is a microorganism comprising culturing a microorganism containing a pigment, putting the cultured microorganisms in a dryer and drying them to destroy cell walls, and then pulverizing the dried microorganisms in a mill. It is to provide a grinding method.

In addition, an object of the present invention is to provide a feed and feed additives or food and food additives containing the microorganisms pulverized from the grinding method and the extract extracted with the solvent.

In order to achieve the above object, the present invention comprises the steps of culturing a microorganism containing a pigment; Putting the cultured microorganisms in a drier and drying them; And it provides a microbial grinding method comprising the step of grinding the dried microorganisms (mill).

In addition, to achieve another object of the present invention, the present invention comprises the steps of extracting the microorganisms pulverized by the method with a solvent; And it provides a method for extracting the microbial pigment comprising the step of drying the extract.

In addition, in order to achieve another object of the present invention, the present invention provides a microorganism ground by the grinding method.

In addition, to achieve another object of the present invention, the present invention provides a feed and feed additive containing the microorganism.

Furthermore, the present invention provides a pigment extract extracted by the extraction method.

Furthermore, the present invention provides a food and food additive containing the pigment extract.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

The present invention, in order to extract the carotenoid pigment containing astaxanthin, mass culture of the microorganism producing the pigment, and put the cultured microorganism in a dryer to dry and destroy the cell wall, roller roller drying the microorganism is destroyed Grinding in a drum drum (mill), to produce a microbial powder that can efficiently extract the pigment using a solvent.

At this time, the microorganism in the present invention is preferably using at least one strain of the genus ( Paffia sp.) Selected from the group consisting of Phaffia rhodozyma and its mutant strain, which is fast growing in culture This is because the strain is poorly contaminated.

And, in the present invention, the dryer used to break the cell wall, it is preferable to use a spray dryer (spray dryer) or a drum dryer (drum dryer).

The spray dryer and drum dryer may be used as long as the spray dryer and the drum dryer are commonly used in the field of the present invention.

In addition, it is preferable that the roller drum mill used by this invention uses 1 type of mill selected from the group which consists of a ball mill, a rod mill, and a roller mill.

At this time, the wheat has the following characteristics depending on the type, but in the present invention, since it can effectively destroy the cell wall using the above characteristics, it may be used in the present invention.

(1) ball mill

Most representatively, it is widely used for fine grinding to ultrafine grinding of ceramic raw materials, chemical industrial raw materials, etc. as well as mass grinding of ore, cement raw material and cement clinker. A ball mill is a cylindrical container rotating around a horizontal axis with an amount of iron balls filled about one third of its volume. Rotating the shell raises and falls the ball accordingly. As a result of the movement of the balls, the raw material particles supplied to the shell are subjected to a pulverizing action, which in turn becomes fine.

(2) rod mill

Instead of a ball, it is a mill using a rod of special or carbon steel shorter than the length of a cylindrical shell as a grinding medium. The diameter of the rod is generally around 75 mm. Rod mills are more suitable for rough grinding than ball mills.

(3) roller mill

Several rollers are pressed into a rotating table or bowl-shaped crushing container by gravity, centrifugal force, elastic force, etc., and are compressed in the raw material particles. Crushed fine particles are discharged by the airflow, which is mainly used to grind raw materials in cement plants or coal in power plants.

In the present invention, a pigment is extracted by adding a solvent to the dried microorganisms which destroy the cell wall, and the extracted pigment is dried to prepare a pigment extract.

At this time, the solvent that can be used for the pigment extract is preferably at least one organic solvent selected from ethanol, methanol, acetone, dimethyl sulfoxide and a mixed solvent thereof, more preferably food based on the Food and Drug Administration And ethanol for use in food additives.

In addition, the pigment extract or dried microorganism according to the present invention is used as food and food additives or feed and feed additives.

At this time, the pigment extract of the present invention is controlled without limiting the amount of additives, if necessary, in food and food additives, edible oil products, dressing products, mayonnaise products, cream products, confectionery products such as chocolate and potato chips, beverage products, It is used in capsule products, tablet products, powder products, and bakery products such as bread and cookies.

In addition, the pigment extract of the present invention is used in addition to the health supplements, such as solid, powder, particles, capsules, pills, tablet form or liquid form, such as dispersion, emulsification.

In addition, it is used to improve storage stability, palatability and emulsification stability by using in storage food (sausage or canned products), and it is general purpose without special use concentration for improving stability, palatability and emulsification stability of processed foods and animals. Use as.

In addition, the pigment extract of the present invention and dried microorganisms are mixed with materials such as organic nutrients and inorganic nutrients for the expected effect of the pigment itself and for the storage stability of meat or meat products for poultry, pig farming, dairy farming, It is a material for animal feed and feed additives for cattle, and it is used universally without any specific concentration.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited to these examples.

Experimental Example

1. Determination of total cartenoid content

1 ml of the cultured microorganism was taken out and centrifuged. The supernatant was separated, washed with water by adding water to the pellet, and the water was removed as much as possible, followed by 1 ml of dimethyl sulfoxide (DMSO). , 55 ° C.) or ethanol and uniformly vortex with a homogenizer, and then add 1 ml of acetone, petroleum ether, and 20% (w / v) NaCl solution in order and mix Cartenoids were accumulated in the ether layer.

Then, the petroleum ether layer was separated, and the absorbance was measured at 474 nm using a spectrophotometer to quantitatively analyze the total content of carotenoids.

2. Analysis of carotenoids

Samples containing carotenoid pigments were separated by thin layer chromatography, and then quantitatively analyzed by measuring absorbance at inherent wavelengths for each of the separated carotenoids.

At this time, the thin layer chromatography was separated from the silica gel TLC plate (Silica gel 60, 5 × 20 cm , 0.25 mm thickness; E. Merck, Darmstadt, Germany), the pigment layer was scraped off and the 1 ml of acetone Diluted to use as sample.

In addition, the mobile phase solvent when using TLC was mixed with acetone: petroleum ether at 20:80 ( v / v ), and each carotenoid content (%) was `` each carotenoid amount × total carotenoid amount (mg / g yeast). The carotenoid% was calculated based on the sum of the absorbances of all the carotenoids and the ratio of the carotenoid absorbance of astaxanthin.

3. Astaxanthin content measurement

Ethyl alcohol was added to the dried microorganisms, and astaxanthin was extracted in the same manner as in Experiment 1, and the astaxanthin sample extracted in ethanol was quantitatively analyzed by measuring absorbance at 476 nm using a spectrophotometer. It was.

4. Measurement of residual ethanol content of dried astaxanthin

The residual ethanol content extracted from ethanol and remaining in dry astaxanthin was prepared by first preparing 0.1 M potassium phosphate buffer (pH 9.0), and NAD solution (nicotinamide adenine dinucleotide, sigma-aldrich, USA) And alcohol dehydrogenase (ADH, sigma-aldrich, USA) solution was prepared, and then mixed with 100 μl of NAD solution, 20 μl of ADH and potassium phosphate buffer solution to make a total volume of 10 ml. 1 ml of the solution was added to the cuvette, ΔAbs (absorbance parameter) was measured using kinetic, and quantitative analysis was performed using a standard curve.

5. Cell mass measurement of strains

 The cell mass of the yeast strain was weighed by drying a 10 ml solution containing yeast in a vacuum oven at 80 ℃.

6. Measurement of Coloration of Pigment

 After the experiment was completed, the chickens were bleeding, hair loss, and the head was removed by cutting between the upper and lower skulls of the first cervical vertebrae, and cutting the lower tibia and metatarsal joints. After removal of the leg, the esophagus, trachea, lungs, liver and viscera were removed (the above methods are known as poultry practices at the Institute of Animal Science and Technology). The coloration of the skin in the state and the coloration of the poultry was measured after measuring the coloration of the skin and then measured the coloration of the chicken in the state of removing the skin.

At this time, the degree of coloring is a chroma (Chroma meter; Minolta Co. Cr 301) brightness ( L ; lightness), redness ( a ; redness), yellowness ( b ; yellowness) was measured by the International Lighting Commission's CIE (Commision Internationale de Leclairage) values, and the standard plate used white tiles of Y = 92.40, x = 0.3136, y = 0.3196.

7. Carotenoid Pigment Concentration in Blood

 Blood collected from 4 ml of subcutaneous vein of the experimentally completed chicken was collected in a blood collection bottle containing EDTA (disodium salt of ethylenediaminetetraacetic acid), stored in a freezer, and then carotenoids using HPLC (high pressure liquid chromatography). The content of was analyzed.

In this case, the column (MetaChem; Nucleosil 100Å, 5micron), the detector (Detector) was used M730D (UV / VIS Detector, Younglin equipment), the mobile phase (tBME: Hexane: IPA: MeOH The flow rate was 1.5 ml / min, the wavelength was 476 nm, and the sample injection was 20 mu l.

8. TBA measurement for measuring antioxidant activity on skin of sample

About 2.5 g of the sample was collected, and 6 ml of TCA (Trichloroacetic acid) at 4 ° C. was added thereto, followed by uniform mixing (Vortex mixing), and then titrated so that the total volume of the solution was 12.5 ml with distilled water (DW). .

After filtering the solution with Filter paper (Whatman No. 1), 3 ml of TBA (Thiobarbituric acid) was added to 3 ml of the collected filtrate, sealed with a lid, and mixed uniformly in a cow. It was left to stand at room temperature for 15 hours.

The sample was then measured for absorbance at 530 nm to calculate the TBA value.

Example 1 Strain and Culture Experiment

In this embodiment To produce astaxanthin pigments, mutants of wild type papia rhododima (nickname: Xanthophyllomyces dendrorhous ) 67-385 were mutated to catenoids by induction of antimycin. A yeast strain of Phaffia rhodozyma strain 2A2N capable of producing more than cartenoids was used.

Then, the yeast strains were seeded in a 300 ml flask containing 30 ml YM medium, and the seed cultures were incubated in a 2 L fermentor, which was then supplied to a mass incubator. Incubated at 20 ° C. for 3-5 days.

At this time, the impeller (Impeller) speed was adjusted to 200 ~ 600 rpm so that dissolved oxygen is supplied steadily to the air circulation.

Example 2 Drying and Crushing of Cultured Microorganisms

In this embodiment, to determine the effect of the grinding method of the present invention, after drying and pulverizing the cultured microorganisms, the cell wall grinding effect of the microorganisms by the grinding method according to the present invention, the total content of carotenoids, component analysis of carotenoids , Electron microscopy and astaxanthin contents were measured and analyzed.

First, papia rodojima (Xanthophyllomyces dendrose; hereinafter referred to as yeast) cultured in Example 1 was dried using a spray dryer and a drum dryer.

At this time, when the drying conditions of the spray dryer is a laboratory scale (pilot scale) by spraying the spray dryer of Eyela Co. Japan (inlet temperature is 180 ℃, outlet temperature is 80 ℃ 1 Liter was dried, and in the case of plant scale, the spray dryer of Samyoung Chemical was sprayed at 200 liters per hour with an inlet temperature of 190 ° C. and an outlet temperature of 90 ° C. to dry a total of 30 tons.

In the case of drum drying, the drum dryer was dried at 150 ° C. at a rotation speed of 210 rpm per minute using the single drum dryer.

Thereafter, the yeast dried with a mill (roller mill, light agricultural machinery) was ground to prepare an average particle diameter of 3 mm or less.

At this time, the mill was repeatedly crushed step by step from 10 to 10 times under the condition of 210rpm, 5HP as a flat roller (flat roller).

Example 3 Crushing Effect of Microorganisms According to Drying Parameters

(1) Effect of Pigment Extraction by Using Spray Dryer

This example was performed in the same manner as in Example 2 to determine the extraction effect of the carotenoid pigments according to the drying scale and the number of times using a spray dryer.

The dried yeast was ground using a flat roller mill, and ethanol (EtOH) and dimethyl sulfoxide (DMSO) as extraction solvents were used for the ground microorganisms.

As a result, as shown in FIG. 1, the content of the pigment extracted at the laboratory scale increased by 1 to 3 times of treatment, after which there was no significant change, while the plant scale increased by 10 times. there was.

(2) Effect of Pigment Extraction by Using Drum Dryer

In this example, to determine the effect of the pigment extraction according to the dryer, the total effect of the carotenoids and the extraction effect of astaxanthin using a spray dryer and a drum dryer was compared / analyzed.

At this time, the culture and grinding and extraction were carried out in the same manner as in Examples 1 and 2, the extraction solvent was used for ethanol.

As a result, as shown in Table 1, the yeast pulverized with a drum dryer was found to have a higher initial pigment extraction than the spray dryer, but the total pigment content was low.

division Carotenoid content [mg / g (yeast)] Astaxanthin [mg / g (yeast)] Spray drying Before treatment 0.02 0.01 After treatment 2.01 1.31 Drum drying Before treatment 0.31 0.18 After treatment 1.26 0.73

This difference is judged to be due to the larger size of the drum dryer due to the hot plate damage on drying.

(3) dry particle size

To determine the size of the dry particles of the yeast pulverized in Examples 3-1 and 3-2, the shape and size were visually measured using a scanning electron microscope (SEM).

As a result, as shown in FIG. 2, the dry particle size was 8.5 to 10 μm, and the plant size was 25 to 30 μm, indicating that there was a difference of 2-3 times depending on the dry size. .

(4) Destructive effect of cell wall by drying method

According to the drying method, the cell wall destruction effect of yeast was visually confirmed through SEM.

As shown in Figure 3 (a) is a control group (scale bar 5㎛), (b) a spray dryer group (scale bar 10㎛), (c) drum dryer group (scale bar 20㎛), (d) Showed the pulverization after spray drying (scale bar 5㎛), and as a result, it was found that pulverization by mill after spray drying has an excellent effect on breaking the cell wall.

Example 4 Substance Use Experiment in Animals of Cell Wall Breaking Yeast

In this embodiment, in order to confirm the bioavailability of the astaxanthin possessed by the microorganisms by feeding the yeasts that cleaved the cell wall in accordance with the method of the present invention, the degree of pigmentation of the laying hens according to the period and the level of addition was measured in vivo. The extent of absorption was compared / analyzed.

Experimental method was carried out as follows.

180 Isa Brown hens (ISA brown hens) were selected as the test subjects, and they were reared for 6 weeks, and the conditions for breeding were housed in a two-stage three-stage iron cage equipped with a nipple on the test axis. They were vegetarian, and the lights were fixed for 17 hours from 4 am to 9 pm.

In addition, the feed was to be fed to the test subject chicken in the same amount randomly in 60 chicken coop (1 per chicken coop), as shown in Table 2, the control of the control (control) to disclose the 225 number of 78-week-old Isa Brown brown laying hens Chicken feed was formulated without astaxanthin at all (no additive group C), and the experimental group was prepared by adding 0.05%, 0.1%, 0.2% and 0.3% astaxanthin, It was performed in portions.

division % Added No addition 0.05 0.1 0.2 0.3 Iterations 3 3 3 3 3 Number of iterations 15 15 15 15 15 Total 45 45 45 45 45

And as shown in Table 3, yellow corn and soybean meal-based blended feed was used, crude protein and energy content of 16% and 2,800 ㎉ / ㎏, respectively.

ingredient % Of content corn 68.33 Soy flour 17.82 Corn Gluten Flour 3.60 Soybean oil - Limestone 8.40 0.93 DL-Methionine 50 0.09 L-Lysine 80 0.08 Vitamin-mineral complex 0.50 saline 0.25 total 100 Chemical composition Metabolic Energy (ME), ㎉ / ㎏ 2,800 Crude Protein (CP) 16.00 Calcium (Ca)% 3.40 Useful Phosphoric Acid (P)% 0.275 Methionine% 0.76 Lee Sin % 0.33 Vitamin-mineral complex usage (based on feed 1kg) Vitamin A 1,600,000 IU, Vitamin D ₃ 300,000 IU, Vitamin E 800 IU, Vitamin K3 132 mg, Vitamin B2 1,000 mg, Vitamin B12 1,200 mg, Niacin 2,000 mg, Pantonate calcium 800 mg, Folic acid 60 mg, Choline chloride 35,000 mg, DL Methionine 6,000 mg, iron 4,000 mg, copper 500 mg, magnesium 12,000 mg, zinc 9,000 mg, cobalt 100 mg, BHT 6,000 mg, iodine 250 mg

Example 5 Feed Additive Effect According to the Present Invention

In this embodiment, the feed was added to the yeast dried according to the present invention, and then to determine the effect of astaxanthin by measuring the amount of astaxanthin contained in the blood and skin of the chicken.

At this time, the dried yeast was fed using a drum dryer in the same manner as in Example 3 to 100g per 1kg feed was fed equally for a certain amount for 6 weeks, the control was left unbroken yeast as it is Added feed was used.

Then, after feeding, the amount of astaxanthin contained or deposited in the blood, skin and eggs of the chicken was measured.

At this time, the variance analysis was performed using SPSS 11.0 program for skin coloration and chromophore coloration obtained in this test.Sefte's method was used for the significant difference between treatments. Analysis was performed at the% level.

As a result, when the chicken blood was fed with yeast (hereinafter referred to as control) without damaging the cell wall, the astaxanthin amount was 530ng / ml for 2 weeks, 830ng / ml for 4 weeks, and 1310ng / ml for 6 weeks. In the yeast (hereinafter experimental group) dried with a drum dryer, it was measured as 744ng / ㎖ 2 weeks, 1030ng / ㎖ 4 weeks, 2264ng / ㎖ 6 weeks.

In addition, the pigmentation of the chicken skin was 2 weeks 554 and 1073ng / g, 4 weeks 684 and 1696ng / g, 6 weeks 784 and 1863ng / g in the control group and the experimental group, respectively, as shown in Figure 4 It was found that the coloring degree of was clearly distinguished.

Therefore, the method of pulverizing the microorganism according to the present invention was confirmed to accumulate astaxanthin not only in the skin but also in the lipid part of the body by increasing the extraction effect of the pigment accumulated in the microorganism and the bioavailability which can be used by ingesting it.

As described above, the yeast pulverized according to the method of the present invention is excellent in the degree of destruction of the cell wall, the pigment extraction efficiency is increased accordingly, when used as a feed and feed additives or food and food additives with high bioavailability, It can be used as a functional material because it can effectively use the active ingredient. Accumulation of astaxanthin in fed animals was evident. Astaxanthin accumulated not only in the skin but also in the lipid part of the body.

As described above, the present invention is to simply and effectively destroy the cell wall of the microorganisms containing natural pigments, it is possible to efficiently extract the natural pigments, using the above method as a feed and feed additives or storage of food and food additives And it can be used as a functional material beneficial to the human body, it is expected to have a large ripple effect when entering the industry because it can be applied cheaply and easily.

Claims (10)

delete delete delete delete (1) culturing one or more Papia strains selected from the group consisting of asphaxanthin- containing Phaffia rhodozyma and mutants thereof; (2) drying the cultured strain in a drum dryer; (3) milling the dried strain in a roller mill; (4) extracting the ground strain with ethanol; And (5) drying the extract: Containing, astaxanthin extraction method. delete delete delete delete delete

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