CN101041848A - Method for improving carotenoid output by adding glycerol in ferment process - Google Patents

Abstract

The invention discloses a method to add glycerin to improve output of carotenoid in invoice method, which is characterized by the following: including culture and extraction; adopting rhodotorulic strain (Rhodotorule glutinis) CGMCC No.2.703 as zymogeneous bacteria strain; proceeding slant face culture and seed culture; seeding in liquid state and solid state fermentation medium; proceeding microbiological ferment; generating carotenoid; adding glycerin in the fermentation medium to increase the output of carotenoid; adding 1-15% glycerin of culture medium according to mass ratio into liquid state fermentation medium; adding 1-20% glycerin of culture medium according to mass ratio.

Description

In fermentation method, add the method that glycerine improves carotenoid output

Technical field

The invention belongs to the extracting method of foodstuff additive, be specifically related to the method that rhodotorula bacterium liquid state and solid state fermentation improve carotenoid output.

Background technology

Carotenoid (Carotenoids) is that a class is yellow, orange red or red Polyenes, is important liposoluble vitamin and the natural antioxidants that has extensive physiological function in the human body.At present, carotenoid is regarded as category-A nutrition pigment by international organizations such as FAO and WHO, and gets permission more than 50 countries and regions as the foodstuff additive of nutrition, painted dual function and be widely used in protective foods and medicine and cosmetic industry.Aspect food-processing, pink group carotene can be used as the tinting material of food such as lactic drink, ice-creams; Aspect the aquatic feeds goods, the carotenoid additive can improve the Animal nutrition situation effectively, improves the color and luster of yolk, poultry meat, can improve the colour of skin of fish and shrimp; Aspect cosmetic product, because the effect of carotenoid uvioresistant and pleasant color and luster can be used as the emulsifying agent in astringent, nourishing cream and the makeup; Aspect pharmaceutical use, carotenoid can be anticancer and be prevented and treated heart trouble, can strengthen specificity and non-specific immune function and strengthen the immunity of tumour, also can treat photosensitive diseases, cardiovascular disorder and cataract.

The method of producing carotenoid now mainly is a microbe fermentation method, utilize rhodotorula fermentative production carotenoid to have that nutritional requirement is simple, growth cycle is short and many advantages such as thalline is nutritious, and belong to the microorganism fodder that China's feedstuff industry is confirmed, have great practical value and DEVELOPMENT PROSPECT.

The step that liquid state fermentation rhodotorula bacterial strain produces carotenoid is as follows: rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703 is transferred on the slant activation substratum from preserving substratum, after cultivating in the biochemical incubator, connect 2 and encircle and in seed culture medium, be cultured to logarithmic phase, get seed culture fluid and be inoculated on the liquid state fermentation substratum and cultivate.

The production stage of solid state fermentation rhodotorula bacterial strain production carotenoid is as follows: rhodotorula bacterial strain (Rhodotoruleglutinis) CGMCC No.2.703 is transferred on the slant activation substratum from preserving substratum, after cultivating in the biochemical incubator, connecing 2 encircles be cultured to logarithmic phase in seed culture medium, get seed culture fluid and be inoculated in solid-state fermentation culture medium, cultivate in the incubator.

Summary of the invention

The purpose of this invention is to provide a kind of method that glycerine improves carotenoid output of adding in fermentation method, used glycerine is cheap, and consumption is few, has significantly improved the output of carotenoid.

Technical scheme of the present invention is: a kind of method of in fermentation method, adding glycerine raising carotenoid output, and it comprises cultivates and extracts;

Culturing process: adopting rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703 is fermentation strain, after slant culture and seed culture, is seeded in respectively in liquid and the solid-state fermentation culture medium and produces carotenoid through microbial fermentation;

It is characterized in that:, improve carotenoid output by in fermention medium, adding glycerine;

In the liquid state fermentation substratum, press the glycerine that mass ratio adds the 1-15% of substratum; In solid-state fermentation culture medium, press the glycerine that mass ratio adds the 1-20% of substratum.

Main process of the present invention is: rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703 is transferred on the slant activation substratum from preserving substratum, after cultivating in the biochemical incubator, connect 2 and encircle and in seed culture medium, be cultured to logarithmic phase, get seed culture fluid and be inoculated on the liquid state fermentation substratum and cultivate; Or get seed culture fluid and be inoculated in solid-state fermentation culture medium, cultivate in the incubator.Its key is: when fermention medium disposes, add the glycerine of liquid state fermentation substratum 1-15% (w/w mass ratio), add the glycerine of solid-state fermentation culture medium 1-20% (w/w mass ratio).

During the fermentation, add an amount of glycerine and can play the microbial fermentation promoter action, help the growth of rhodotorula bacterium, promote the synthetic of carotenoid: the glycerine that in the liquid state fermentation substratum, adds the 1-15% (w/w) of fermented liquid, improved carotenoid output, fermenting at the end, carotenoid output is 3.35-5.52 μ g/ml in the fermented liquid, is 2.9 μ g/ml when not adding, and improves 15.5-90.3%.Add the glycerine of the 1-20% (w/w) of substratum in solid-state fermentation culture medium, improved carotenoid output, carotenoid output is a 10.4-14.4 μ g/g butt in the fermented liquid that ferments at the end, is 7.5 μ g/g when not adding, and improves 38.7-92.0%.

The invention has the beneficial effects as follows: solved existing microbial fermentation rhodotorula bacterium and produced the lower problem of output in the fermentative carotenoid process, used glycerine is cheap, consumption is few, has significantly improved the output of carotenoid, and the experimental level of fermentation culture reaches leading domestic level.

Embodiment

One, the mode of prior art is routinely prepared bacterial classification, is prepared fermention medium by the present invention

Bacterial classification

Rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703 is hidden the center preservation and is provided by Hubei Province's industrial microorganism key lab of Hubei University Of Technology bacterium.

Substratum

Wort slant medium (g/L): wort 50, glucose 20, yeast extract paste 20, agar 20, pH7.0.

Seed culture medium (g/L): glucose 20, peptone 20, yeast extract paste 10, natural pH.

Liquid state fermentation substratum: sucrose 30g, (NH ₄) ₂SO ₄10g, yeast extract paste 3g, MgSO ₄0.3g, add the water constant volume to 1L, add glycerine 10g-150g nature pH again.

Solid-state fermentation culture medium is formed: brewer's grains: dregs of beans: wheat bran is 1: 3: 2, and every kilogram of solid-state fermentation culture medium contains K ₂HPO ₄0.5g, MgSO ₄1.0g the total water content that adds behind the water is 60%, adds glycerine 10-200g again, natural pH.

Two, cultural method

Rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703 is transferred on the slant activation substratum from preserving substratum, cultivate after the 48h under 28 ℃ of conditions in the biochemical incubator, connecing 2 encircles in seed culture medium, 28 ℃ of vibrations (200r/min) are cultivated 18h to logarithmic phase, get seed culture fluid, inoculum size by 5% is inoculated on the liquid state fermentation substratum of the glycerine that has added 1-15% (w/w), and 96h is cultivated in 28 ℃ of vibrations (200r/min).Inoculum size by 10% is inoculated on the solid-state fermentation culture medium of the glycerine that has added 1-20% (w/w), cultivates 96h in 28 ℃ of fixed temperature and humidity incubators.

Three, the rhodotorula microbial fermentation produces the extraction and the measuring method of carotenoid

From liquid state fermentation liquid, take out 4ml and carry out centrifugal (3500r/min, 15min) obtain wet thallus, directly add 6ml hydrochloric acid (3mol/L), 1h is soaked in vibration under the room temperature, in boiling water bath, boil 4min, cooling rapidly, the centrifugal 15min of 3500r/min abandons supernatant liquid, and precipitation adds 3ml acetone with 2-3 back of distilled water wash, vibration is with lixiviate carotenoid under the room temperature, be the centrifugal 15min of 3500rpm then, get supernatant and be the carotenoid extracting solution, as still having pigment in the cell debris, add acetone and further extract, united extraction liquid.The extracting solution that will contain carotenoid scans in ultraviolet spectrophotometer, obtains maximum absorption wavelength.

Solid state fermentation produces the extraction and the measuring method of carotenoid: get 10g solid state fermentation thing, add distilled water 50.0ml washing, get fermented liquid with four layers of filtered through gauze, therefrom take out 4ml and carry out centrifugal (3500r/min, 15min) obtain wet thallus, directly add 6ml hydrochloric acid (3mol/L), 1h is soaked in vibration under the room temperature, in boiling water bath, boil 4min, cooling rapidly, the centrifugal 15min of 3500r/min, abandon supernatant liquid, precipitation adds 3ml acetone with 2-3 back of distilled water wash, and vibration is the centrifugal 15min of 3500rpm then with lixiviate carotenoid under the room temperature, get supernatant liquor and be the carotenoid extracting solution, as still having pigment in the cell debris, add acetone and further extract, united extraction liquid.The extracting solution that will contain carotenoid scans in ultraviolet spectrophotometer, obtains maximum absorption wavelength.

Extracting solution is suitably diluted the back measures absorbancy in the maximum absorption wave strong point, be calculated as follows carotenoid output with 722 type spectrophotometers:

In the formula, A λ _MAXAbsorbancy for carotenoid maximum wavelength place; D is a pigment vat liquor extension rate; V is the cumulative volume (ml) of the used acetone of lixiviate; W is for extracting used fermentating liquid volume (ml); 0.16 be the molar extinction coefficient of carotene.

Four, the present invention improves the principle of carotenoid output

Carotenoid is the secondary metabolite that the rhodotorula bacterial strain is produced, according to 1-deoxy-D-xylulose sugar-5-phosphate synthase (DXS) catalyzing and condensing of glyceraldehyde 3-phosphate and pyruvic acid in the carotenoid biosynthetic pathway by depending on VitB1, change the process of 2-C-methyl D-erythritol into by 1-deoxy-D-xylulose sugar-5-phosphoric acid reduction isomerase (DXR) again, add the biosynthesizing that glycerine helps carotenoid in the fermention medium.Concerning rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703, promote the biosynthesizing of carotenoid in its fermenting process.

Embodiment 1:

In 1L liquid state fermentation liquid culture medium, add the glycerine of 100g, during fermentation ends, the output of carotenoid is 5.52 μ g/ml in the gained fermented liquid, does not add that carotenoid output is 2.9 μ g/ml in the reference examples of glycerine, improves 90.3% than the reference examples of glycerol adding not.

Embodiment 2:

Add the glycerine of 10g in 1L liquid state fermentation liquid culture medium, during fermentation ends, the output of carotenoid is 3.35 μ g/ml in the gained fermented liquid, does not add that carotenoid output is 2.9 μ g/ml in the reference examples of glycerine.

Embodiment 3:

Add the glycerine of 150g in 1L liquid state fermentation liquid culture medium, during fermentation ends, the output of carotenoid is 3.98 μ g/ml in the gained fermented liquid, does not add that carotenoid output is 2.9 μ g/ml in the reference examples of glycerine.

Embodiment 4:

In the 1000g solid-state fermentation culture medium, add the glycerine of 100g, during fermentation ends, add distilled water 50.0ml washing, the output that gets carotenoid in the fermented liquid with four layers of filtered through gauze is 14.4 μ g/g butts, do not add that carotenoid output is 7.5 μ g/g butts in the reference examples of glycerine, improve 92% than the reference examples of glycerol adding not.

Embodiment 5:

In the 1000g solid-state fermentation culture medium, add the glycerine of 10g, during fermentation ends, add distilled water 50.0ml washing, the output that gets carotenoid in the fermented liquid with four layers of filtered through gauze is 10.4 μ g/g butts, does not add that carotenoid output is 7.5 μ g/g butts in the reference examples of glycerine.

Embodiment 6:

In the 1000g solid-state fermentation culture medium, add the glycerine of 200g, during fermentation ends, add distilled water 50.0ml washing, the output that gets carotenoid in the fermented liquid with four layers of filtered through gauze is 13.1 μ g/g butts, does not add that carotenoid output is 7.5 μ g/g butts in the reference examples of glycerine.

Claims (1)

1, a kind of method of in fermentation method, adding glycerine raising carotenoid output, it comprises cultivates and extracts;

Culturing process: adopting rhodotorula bacterial strain (Rhodotorule glutinis) CGMCC No.2.703 is fermentation strain, after slant culture and seed culture, is seeded in respectively in liquid and the solid-state fermentation culture medium and produces carotenoid through microbial fermentation;

It is characterized in that:, improve carotenoid output by in fermention medium, adding glycerine;

In the liquid state fermentation substratum, press the glycerine that mass ratio adds the 1-15% of substratum; In solid-state fermentation culture medium, press the glycerine that mass ratio adds the 1-20% of substratum.