CN1297056A - Process for preparing monascorubin with higher yield - Google Patents

Abstract

A process for preparing monascorubrin with increased yield includes slant culture of Monascusanka (CGMCC No.0424) in culture medium, liquid seed culture, fermenting in fermenting liquid, centrifugal separation of thallus from bacterial liquid, and processing the thallus and bacterial liquid. The compound amino acids are added to said fermenting liquid. The obtained monascorubrin features high optical and thermal stability and not decolouring under light radiation for 10 days and at 130 deg.C for 10 hr.

Description

A kind of plain production method of red red colouring agent for food, also used as a Chinese medicine that improves the monascorubin yield

The invention belongs to the processing method of utilizing the Production by Microorganism Fermentation monascorubin, is that a kind of chemical substance that adds during the fermentation is to improve the new processing method of monascorubin yield.

Because developing rapidly of foodstuffs industry, the demand of food color also increase day by day.Food dye has synthetic colour and natural pigment two big classes.The color and luster of synthetic colour is stable, and is with low cost, easy to use, but majority is by the coal tar synthetic; Because its chemical property may directly damage HUMAN HEALTH, or produces objectionable impurities in the human body metabolism, thus might be carcinogenic with cause all difficult and complicated illness, the user is worried.National governments also limit its usage quantity gradually or ban use of, and for example the U.S. just banned use of amaranth in 1976.China also only allows limited use now.Natural pigment can be by extracting in the animal and plant body, and is general nontoxic edible, do not damage HUMAN HEALTH, become the glad safe pigment that uses of people in recent years.Monascorubin is a kind of pigment that monascus ruber produces through fermentation, and it produces restrictions such as not being subjected to time place weather condition, the not only harmless also nutritious and pharmacological action of product.

China produces monascorubin with Monascus anka Nakazawa et sato and has traditional history.The Yuan Dynasty Wu Duan " daily book on Chinese herbal medicine " (1329) book in the record of existing " the broken capable medicine gesture of blood of red colouring agent for food, also used as a Chinese medicine wine brewing ", this shows that red colouring agent for food, also used as a Chinese medicine is in making among the people at that time; Its making method is answered under the outstanding Exploitation of the Works of Nature of star just on the books Song of the Ming Dynasty.The starter making method and the above-mentioned part that is very different of record in the works Compendium of Material Medica paddy portion the 25th of Qing Dynasty's initial stage LI Shi-Zhen, the cultural method of the approximate monascus ruber pure strain of bacterial classification that the Qing Dynasty uses when producing red colouring agent for food, also used as a Chinese medicine in Fujian Province latter stage.These traditional red colouring agent for food, also used as a Chinese medicine production technique exist that operation is cumbersome, labour intensity is big, floor surface cultivate floor space big, yield poorly problems such as technical difficulty is big, quality instability.

By traditional zymotechnique differentiation modern production technology till now.The method of producing monascorubin now is not outer in following technology: open production method in as Japanese Patent Publication 52-72890, though its technology is simple, fermentation level is lower, and the look valency is 68.4U/ml only, and cost is higher; The special clear 57-190050 of the Japan Technology introduced is that to adopt maltose and magnesium aminosuccinate be substratum, though its fermented liquid look valency increases, reaches 248U/ml, used culture medium raw material price height.The manufacture method (its authorization notification number is CN85103584B) on May 13rd, 1985 again at the monascorubin polysaccharide sheet of Patent Office of the People's Republic of China application, be a kind ofly to pass through Monascus color aspergillus tubigensis mutagenic fungi (Monascus anka Nakazawaet Sato M130) that a plant height produces monascorubin by rice 5%, NaNO ₃0.17%, ferment in the substratum of yellow seriflux constant volume composition (its pH value is 3.5～4), the gained ferment filtrate is added protein, collect protein monascorubin precipitation and make the short method of obstructing mould polysaccharide sheet of monascorubin in iso-electric point, though the monascorubin through above-mentioned production technique gained increases (total look valency of its fermented liquid can reach 287U/ml), color and luster and photo and thermal stability are not ideal.And for example application number is 94115547.1 in the manufacture method of disclosed high colour-value water-soluble red pigment of red rice on the 29th May in 1996, it is to produce solid fermentation Monascus color Aspergillus strain by a plant height, add the solubility animal proteinum during the fermentation, vegetable-protein, cobalt chloride and SODIUMNITRATE are made water-soluble monascus rice (4000U/g) by fermentation again through adjusting its PH, make monascorubin (10000U/g), it comprises the purebred cultivation in inclined-plane, strain cultivation, the Red kojic rice production of solubility and from Red kojic rice, extract monascorubin four parts; It is a kind of processing method of producing Red kojic rice and extracting monascorubin from Red kojic rice, and the productive labor intensity of Red kojic rice is big, and microbiological contamination look valency is low, and is water insoluble, and because it adopts albumen mould when producing, its technology is complicated, and the look valency improves not too obvious.Application number is the manufacture method of 93120954.4 water-soluble monascus pigment for another example, though added MnSO in its fermentating formula ₄, but its fermented liquid look valency is lower by (170～250U/ml).Also having application number is the preparation method etc. of 95103021.3 monascorubin.No matter be which kind of above-mentioned technology, it is relatively low that the monascorubin that they were produced removes its fermentation look valency, outside tone is bad, also all has the less-than-ideal defective of photo and thermal stability.The monascorubin that these methods are produced generally just fades about one day in illumination; Its heat resistanceheat resistant temperature limit and just faded under 110 ℃ high temperature about 110 ℃ in about 2 hours.

The present invention adds stability and the water miscible new monascorubin production method that aminoacids complex and manganous sulfate improve the monascorubin yield and increase pigment by the excellent species of strain variation and in fermented liquid.

A kind of production method that improves the monascorubin of monascorubin yield of the present invention, be that a kind of monascus is cultivated through slant medium, after inserting the liquid seeds cultivation, insert in the fermented liquid again and ferment, the gained fermented liquid is isolated thalline and bacterium liquid by whizzer, again through handling of thalline and bacterium liquid the production method of monascorubin of high yield.This monascus is the mutagenic fungi Monascus anka (CGMCC NO.0424) of monascus ruber.

Above-mentioned said test tube slant culture medium prescription is:

Zulkovsky starch 4%, maltose 4%, peptone 3%,

Agar 2.5% is transferred PH:5.1～5.5 with lactic acid.

Above-mentioned described liquid seed culture medium prescription is:

Rice meal 3%, peptone 0.5%, corn steep liquor 1%, NaNO ₃0.2%,

MgSO ₄7H ₂O0.1%, KH ₂PO ₄0.25%, lactic acid is transferred PH4.1～4.4.

Above-mentioned described fermentation liquor formulation is as follows:

Rice meal 8%, peptone 0.08%, soya bean 2.5%, proteolytic enzyme 2%,

KH ₂PO ₄0.2％，NaNO ₃0.25％，MgSO ₄·7H ₂O0.08％，

(NH ₄) ₂SO ₄0.08%, MnSO ₄0.1%, aminoacids complex 0.15%,

Lactic acid is transferred PH3.6.

Used bacterial classification is a strain monascorubin superior strain that is obtained through mutagenesis repeatedly by the red colouring agent for food, also used as a Chinese medicine bacterial classification among the present invention, monascus (Monascus anka) B128.This bacterial strain is in China. Beijing. and Zhong Guan-cun, China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, at being numbered of registering on the books in this preservation center: CGMCCNO.0424, the form and the physiological characteristic of this bacterial classification see also table one.

The concrete production method of the present invention is in the purebred access slant medium with the excellent species B128 of the inventor oneself variation, 32～34 ℃ cultivate 10 days stand-by.Slant strains is inserted in the sterilized water disinfect with interior glaze pearl, last shaking table was smashed 1 hour again.In sterilisable chamber, insert in the liquid seed culture medium: dress 50ml liquid seed culture medium in each 500ml triangular flask, each triangle bottle graft 4ml kind liquid (said kind of liquid be bacterial classification B128 after above-mentioned slant medium is cultivated, go up again shaking table smash form), the shaking table cultivation of changeing at 128/ minute was a first-generation kind liquid in 24 hours; The first-generation inserts liquid seed culture medium again: dress 50ml liquid seed culture medium in each 500ml triangular flask, and each triangular flask inserts first-generation kind liquid 4ml, and it is s-generation kind liquid that last shaking table is cultivated 24 hours.S-generation kind liquid inserts the first class seed pot (the sub-substratum of interior dress seed liquor) of the bacterium of having gone out by 4% inoculum size then, cultivated 24 hours at 32 ℃, ventilation is 1:06, the kind liquid of first class seed pot inserts the secondary seed jar (interior dress has been planted liquid nutrient medium) of the bacterium of having gone out again by 4% inoculum size, cultivated 24 hours at 32 ℃, ventilation is usefulness such as 1:06, cultivation.In the fermentor tank of kind liquid by dress fermented liquid in 4% the inoculum size access with the secondary seed jar, ventilation is 1:04～table one: the form and the physiological characteristic of monascus ruber B128 bacterial strain again

Culture condition	Bacterial classification	B128
			Substratum	The wort agar-agar
	Culture temperature	30～32 ℃
			Incubation time	7～10 days
	Morphological specificity	Size			Cultivate 7 days straight every footpath 3.5～4mm of bacterium colony
Colony colour			Be reddle just, gradually become red-purple or red-brown
		Gas silk color		Just be white, gradually become homochromy with bacterium colony
The bacterium colony surface texturing			Many pleats crape, center protrusion, top breach sometimes
		The bacterium colony quality		The epithelium shape
The bacterium colony height
		Colony edge		The edge crenation, relatively more neat
Exudate
		Individual mycelia		Mycelia is 3～5 μ than slightly, multinuclear, tool tabula.
Children mycelia in age is opaque, inclusion granule shape material.
			Old mycelia is transparent, contain oil droplet and cavity,
Most orange red, minority is colourless.
			Asexual spore	Conidium
Shape	Spherical diameter 7.2～10.8 μ pyriform diameters 6.3～7.2 μ
		Color		Orange red or colourless
Decorative pattern	Smooth
		Sexual spore		The quilt device
Shape	Spherical
			Size	Diameter 31～45 μ
Color	Orange red or colourless
			Decorative pattern	Smooth
Thecaspore
			Shape	Oblong
Size	5.4～7.2 * 3.6～3.8
			Color	Colourless
Supervise the cooking and want Te Shengzheng	Saccharogenic power				70 (liquid); 129 (solids)
		Proteolytic enzyme	116 unit/millimeters (liquid); 195 units/gram (solid)

1:06, temperature was cultivated 38 hours for 32 ℃, and this moment, pH value was raised to 6, controlled PH:5.5～6.0 by 50 hours with lactic acid then, and continued to cultivate 60 hours, and PH rises to about 6.5 and puts jar at this moment.At this moment gained fermented liquid look valency can reach 600U～800U/ml.Automatically discharging formula whizzer separates thalline and bacterium liquid again, handles through thalline and bacterium liquid again.The processing of thalline and bacterium liquid is carried out according to the following steps:

1., add the 85% alcohol-pickled of 2 times of volumes in the thalline;

2., bacterium liquid isoelectric precipitation, its supernatant thing of draining, its throw out;

3., the bacterium liquid precipitate thing of gained is poured into and is equipped with the 1. in the filter flask of the alcohol-pickled thalline in step, add 85% alcohol of 1 times of volume (thalline and bacterium liquid) and 1% aminoacids complex again, after soaking 6～8 hours at normal temperatures and pressures, by 260 eye mesh screen suction filtrations, use thin-film evaporator (vacuum tightness 0.6Kg, 80 ℃ of temperature) evaporation concentration to the solid substance that contains 10%～15% again;

4., the gained solid substance separates monascus yellow pigment with pipe examination whizzer, two kinds of parting liquids carry out respectively spraying drying respectively 70% monascorubin and 30% monascus yellow pigment.

Carried out a large amount of experiments according to aforesaid method, its result shows that method proposed by the invention is the best-of-breed technology scheme.

Experiment 1, under the constant situation of above-mentioned test tube slant culture medium prescription and liquid seed culture medium prescription, the compound propylhomoserin ratio in the change fermentation liquor formulation is 5 ‰, the look valency is 315U/ml in the fermented liquid by analysis.

Experiment 2, in experiment 1, rechanging its aminoacids complex ratio is 1 ‰, analyzes its fermented liquid, its look valency is 460U/ml.

Experiment 3, in experiment 1, rechanging its aminoacids complex ratio is 1.5 ‰, analyzes its fermented liquid, its look valency is 460U/ml.

Experiment 4 changes MnSO on the basis of experiment 1～3 ₄Content, finding does not have bigger variation to look valency in the fermented liquid.

Experiment 5 under the constant situation of above-mentioned test tube slant culture medium prescription and liquid seed culture medium prescription, is removed the aminoacids complex composition in the fermentation liquor formulation, finds that its look valency is lower than 300U/ml.

Know that through above-mentioned experiment the aminoacids complex among the present invention plays a part positive in fermentation liquor formulation, can stimulate the red colouring agent for food, also used as a Chinese medicine growth and improve output.The used aminoacids complex of the present invention is selected existing product on the market for use, and used aminoacids complex is the finished product that Hunan, Hunan Dong Anjisuanchang produces in above-mentioned experiment.

The monascorubin that the present invention produced compared with prior art has following significant advantage:

1, added suitable aminoacids complex in fermentation liquor formulation, increased the stability of pigment and water-soluble, the look valency in the check fermented liquid is 600～800U/ml, compares with more existing koji technology to increase significantly, and its contrast sees also table two.

Table two, the look price ratio is in the fermented liquid in the haematochrome production technology:

Patent documentation	Fermented liquid look valency
		????CN85103584B	Mean value: 160U/ml maximum: 287U/ml
????931209544	????170～250U/ml
		????951030213	Mean value: 320U/ml maximum: 400U/ml
Public clear 57-190050	????248U/ml
		The art of this patent	????600～800U/ml

2, its photo and thermal stability of monascorubin through production method gained of the present invention improves greatly, and its product is colour-fast illumination 20 days, and is colour-fast in following 10 hours of 130 ℃ of temperature; This product and more existing technology comparable situation as shown in Table 3, the monascorubin that therefrom visible the present invention is produced has photo and thermal stability preferably, is far superior to currently available products.Help preservation and application.

Table three, the photo-thermal stability of monascorubin compares:

3, the present invention directly separates monascorubin and monascus yellow pigment, has guaranteed the color and luster purity of product, convenience is provided for the preservation and the application of product.

Claims (2)

1, a kind of production method that improves the monascorubin of monascorubin yield, it is that a kind of monascus is cultivated through slant medium, after inserting the liquid seeds cultivation, insert in the fermented liquid again and ferment, the gained fermented liquid is gone out thalline and bacterium liquid by whizzer, again through handling of thalline and bacterium liquid the production method of monascorubin of high yield, it is characterized in that: said monascus is the mutagenic fungi Monascus anka (CGMCCNO.0424) of monascus ruber; Described fermentation liquor formulation is as follows:

Rice meal 8%, peptone 0.08%, soya bean 2.5%, proteolytic enzyme 2%, KH ₂PO ₄0.2%, NaNO ₃0.25%, MgSO ₄7H ₂O0.08%, (NH ₄) ₂SO ₄0.08%, MnSO ₄0.1%, aminoacids complex 0.15%, lactic acid is transferred PH3.6.

2, monascorubin production method according to claim 1 is characterized in that: the processing of above-mentioned said thalline and bacterium liquid is carried out according to the following steps:

1., add the 85% alcohol-pickled of 2 times of volumes in the thalline;

2., bacterium liquid isoelectric precipitation, its supernatant thing of draining, its throw out;

3., the bacterium liquid precipitate thing of gained is poured into and is equipped with the 1. in the filter flask of the alcohol-pickled thalline in step, add 85% alcohol of 1 times of volume (thalline and bacterium liquid) and 1% aminoacids complex again, after soaking 6～8 hours at normal temperatures and pressures, by 260 eye mesh screen suction filtrations, use thin-film evaporator (vacuum tightness 0.6Kg, 80 ℃ of temperature) evaporation concentration to the solid substance that contains 10%～15% again;

4., the gained solid substance separates monascus yellow pigment with pipe examination whizzer, two kinds of parting liquids carry out respectively spraying drying respectively 70% monascorubin and 30% monascus yellow pigment.