CN110540952B - Prokaryotic microorganism for producing lutein and application thereof in production of lutein - Google Patents
Prokaryotic microorganism for producing lutein and application thereof in production of lutein Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, relates to a xanthophyll-producing prokaryotic microorganism and application thereof in production of xanthophyll, and particularly relates to sphingomonas and a method for producing xanthophyll by using the same. The prokaryotic microorganism Nocardia sphingomonas NBD5 for producing lutein not only has fast growth speed, but also has lutein content as high as 2.00 mg/g-cell dry weight at the temperature of 30 ℃. In addition, the cell wall of the sphingomonas mainly takes the glycosphingolipids, and the xanthophyll is easier to extract and more efficient than that extracted from microalgae and marigold, so that the production of the xanthophyll by using the sphingomonas has important application prospect and value.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a xanthophyll-producing prokaryotic microorganism and application thereof in production of xanthophyll, and particularly relates to sphingomonas and a method for producing xanthophyll by using the same.
Background
The lutein is an important carotenoid pigment, belongs to derivatives of alpha-carotene, and has the advantages of bright color, strong tinting strength, safety, no toxicity, strong oxidation resistance and the like. Xanthophylls are widely present in vegetables, flowers, fruits and certain microalgae organisms. The research finds that the lutein mainly has the following five aspects of medical value: 1) protecting vision, preventing arteriosclerosis of eyeball, delaying and relieving symptoms of presbyopia, and preventing and treating cataract and senile macular degeneration; 2) has excellent antioxidation effect, is helpful for preventing a series of diseases caused by aging of human organs and enhancing the immunity of the organism; 3) has anticancer effect and unique biological effect in inhibiting tumor growth; 4) the early arteriosclerosis is delayed, and the oxidation of cholesterol can be obviously reduced; 5) experiments in dogs and cats have demonstrated that lutein enhances both cell-mediated and humoral immune responses. In addition, xanthophylls are used as food pigments and also for coloring pharmaceutical and cosmetic products. At present, the global lutein price is increased at a rate of 6.3%, and the market scale is estimated to reach $ 3.5 billion, however, lutein has about 100 million tons of gaps in the global market, and the lutein far meets the needs of domestic and foreign markets, so that the lutein has wide market potential.
At present, the chemical synthesis technology of lutein is not successful, so that lutein can only be extracted and purified from marigold and other plants. The lutein production process comprises four steps: firstly, preserving fresh marigold flowers by an ensiling method, dehydrating, drying, crushing and briquetting the flowers after ensiling to prepare marigold particles to obtain a crude lutein product, and extracting and purifying the crude lutein product by an organic solvent to obtain high-purity lutein. However, the marigold has long growth period and is easily influenced by seasons, so that the application of the marigold in large-scale industrial production is limited. Microalgae are a new biological source of potential production of lutein that has received attention in recent years, and microalgae that can produce lutein include chlorella, and the like, and have a lutein content in their cells that is substantially equivalent to that of marigold. Although microalgae have a short growth cycle and can be cultured in bioreactors, microalgae have a tougher cell wall and are more difficult to extract lutein than marigold, thus consuming more solvent and cost for extraction during the extraction stage, reducing economic benefits.
Therefore, research and development of other biological resources with fast growth speed, high lutein content and easy lutein extraction has become a very important research target.
Disclosure of Invention
One of the purposes of the invention is to provide a prokaryotic microorganism for producing lutein, which has the advantages of fast growth and high lutein content, and lays a foundation for meeting the requirements of more efficient and larger-scale lutein industrialized production.
The second purpose of the invention is to provide an application of the prokaryotic microorganism for producing lutein.
The invention also aims to provide sphingomonas, which has the advantages of fast growth and high lutein content and can meet the requirements of high-efficiency and large-scale lutein industrial production.
The fourth purpose of the invention is to provide a method for producing lutein by using the sphingomonas, which has simple operation, short production period and high lutein yield.
To this end, the present invention provides, in a first aspect, a xanthophyll-producing prokaryotic microorganism containing xanthophyll in its cells.
According to the invention, the prokaryotic microorganism comprises Sphingomonas, the cells of which contain lutein.
In some preferred embodiments of the invention, the dry powder of the bacterial cells of Sphingomonas contains 0.5-2mg/g lutein.
In a second aspect, the present invention provides the use of a prokaryotic microorganism according to the first aspect of the present invention for the production of lutein.
In some preferred embodiments of the invention, the use comprises culturing a strain corresponding to said Sphingomonas in said prokaryotic microorganism of the first aspect of the invention by aerobic heterotrophic fermentation to obtain bacterial cells of said Sphingomonas.
In a third aspect, the invention provides a sphingomonas, wherein the bacterial cells contain xanthophyll.
In some embodiments of the invention, the dry cell powder of Sphingomonas bacteria contains 0.5-2mg/g lutein.
In some further embodiments of the invention, the bacterial cells of Sphingomonas are obtained from the corresponding species of Sphingomonas by aerobic heterotrophic fermentation.
In some particularly preferred embodiments of the invention, the Sphingomonas bacterium is the strain Nonam Sphingomonas mobilis NBD5(Sphingomonas morindae strain NBD5) with a accession number of CGMCC No. 18380.
In a fourth aspect of the present invention, there is provided a process for producing xanthophyll using Sphingomonas bacterium according to the third aspect of the present invention which comprises:
step A, inoculating a fermentation strain into a fermentation culture medium for fermentation culture to obtain a fermentation culture of sphingosine monad;
step B, carrying out centrifugal separation treatment on the fermentation culture of the sphingomonas, and harvesting bacterial cells of the sphingomonas;
step C, extracting lutein from bacterial cells of sphingomonas, and separating and purifying the obtained extracting solution to obtain a pure product of the lutein;
wherein the fermentation strain is obtained by seed culture of corresponding strains;
the corresponding strain of the fermentation strain is a strain having at least 90% homology with the 16S rDNA of the strain of Sphingomonas according to the third aspect of the invention; preferably the corresponding strain of the fermentation broth is a strain having at least 95% homology to the 16S rDNA of the strain of Sphingomonas according to the third aspect of the invention; it is further preferred that the corresponding strain of the Zymobacter strain is a strain of Sphingomonas according to the third aspect of the invention (i.e.the strain Novosphingomonas NBD 5).
In some preferred embodiments of the present invention, in step C, the bacterial cells of sphingomonas are dried to obtain dry powder of the bacterial cells of sphingomonas, and lutein is extracted therefrom, and the obtained extract is separated and purified to obtain pure lutein.
In some specific embodiments of the invention, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
in some particularly preferred embodiments of the invention, the pH of the fermentation medium is 6.8 to 7.2.
In some embodiments of the invention, in step A, the temperature of the fermentation culture is 18-38 ℃, preferably 30-38 ℃.
The invention provides a prokaryotic microorganism for producing lutein, which comprises sphingomonas, wherein the bacterial cells of the prokaryotic microorganism contain the lutein; the prokaryotic microorganism has fast growth and high lutein content, lays a foundation for meeting the requirement of more efficient and larger-scale industrialized production of lutein, and has important application value and prospect in the aspect of producing the important life active substance lutein by adopting the prokaryotic microorganism.
The invention further provides a Sphingomonas, which is a norsphingomonas NBD5 strain (Sphingomonas morindae strain NBD 5); the Novosphingomonas noni NBD5 has high growth speed, and the lutein content in the cells of the Novosphingomonas is basically equivalent to that in marigold and microalgae, so the Novosphingomonas noni NBD5 has important application value and prospect in the aspect of quickly producing the lutein with high yield by adopting prokaryotic microorganisms.
Drawings
For the present invention to be readily understood, the following description is made with reference to the accompanying drawings.
FIG. 1 shows a morphological photograph of a monoclonal colony (A) of the Nocardia bacterium NBD5 of the present invention and a microscopic (Olympus-cx41) with 1000-fold magnification of the cells (B).
FIG. 2 shows the 16s rDNA molecular clade of the Nocardia bacterium NBD5 of the present invention.
FIG. 3 shows liquid chromatography peaks and scanning patterns of lutein in the lutein standard (A) and the cell extract of Nocardia bacterium NBD5 (B) of the present invention.
FIG. 4 shows the effect of different temperatures on growth (A) and lutein content (B) of Sphingomonas norgesticulatus NBD 5.
Strain preservation
Noni Sphingomonas (Sphingomonas morindae), which is separated and identified by Beijing university of science and technology, has been preserved in China general microbiological culture Collection center (CGMCC for short); address: north west road No.1 institute No. 3 institute of china academy of sciences, north kyo, chaoyang district, etc.) for storage, the storage date is as follows: 12/8/2019, deposit number: CGMCC No. 18380. The strain of the present invention was named as Novosphingomonas nori NBD5 strain (Sphingomonas morindae strain NBD 5).
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Term (I)
"Water" used in the medium or fermentation culture process of the present invention means, unless otherwise specified, sterile pure water obtained by filtration through a 0.22 μ filter.
In the invention, the centrifugal separation treatment refers to a process of putting an object to be separated into a centrifugal tube, performing centrifugal separation at a certain rotating speed, and then separating supernatant from precipitate.
The term "crude wet cells of Sphingomonas" as used herein means that the cells of Sphingomonas are obtained by concentrating and separating a fermentation culture, i.e., by subjecting the fermentation culture to a centrifugal separation treatment.
The term "sphingomonas bacterium wet cell" as used herein refers to a sphingomonas bacterium cell obtained by washing and separating a precipitate (i.e., sphingomonas crude wet cell) obtained by subjecting a fermentation culture to centrifugal separation, i.e., a precipitate (i.e., sphingomonas crude wet cell) obtained by subjecting a fermentation culture to centrifugal separation is resuspended and washed with a physiological saline solution, and then centrifuged to obtain a sphingomonas bacterium (i.e., sphingomonas cell).
Embodiments II
As mentioned above, the chemical synthesis technology of lutein has not been successful, but the extraction and purification of lutein from marigold and other plants has limited its application in large-scale industrial production due to the long growth period of marigold and the influence of seasons. Microalgae are a new biological source for producing lutein, and although the growth cycle is short and can be cultured in a bioreactor, the wall breaking of microalgae is tougher and the extraction of lutein is more difficult, thus consuming more solvent and extraction cost in the extraction stage, resulting in a reduction in the overall economic benefit. In view of this, the present inventors have conducted extensive studies on a method for producing lutein.
The inventor considers and searches a prokaryotic microorganism with fast growth and high lutein content through research. The inventor further studies and firstly discovers that the sphingomonas can produce the lutein, and the content of the lutein in the bacterial cells is basically equivalent to that of marigold and microalgae. The inventor also finds that the growth cycle of sphingomonas is shorter than that of microalgae as a prokaryotic microorganism, and a large amount of biomass can be obtained by culturing in a fermentation tank. In addition, the cell wall of the sphingomonas mainly takes the glycosphingolipids, and the xanthophyll is easier to extract and more efficient than that extracted from microalgae and marigold, so that the production of the xanthophyll by using the sphingomonas has important application value. The present invention has been made based on the above findings.
Accordingly, the present invention relates in a first aspect to a xanthophyll-producing prokaryotic microorganism, the cells of which contain xanthophyll.
Preferably, the prokaryotic microorganism is sphingomonas, and the bacterial cells of the prokaryotic microorganism contain lutein.
Further, the dry powder (bacterial powder) of the bacterial cells of the sphingomonas contains 0.5-2mg/g of lutein.
The second aspect of the present invention relates to the use of a prokaryotic microorganism according to the first aspect of the present invention for the production of lutein, which may be understood as a method for producing lutein using a prokaryotic microorganism according to the first aspect of the present invention.
In some preferred embodiments of the present invention, the method comprises culturing the corresponding species of sphingomonas in the prokaryotic microorganism according to the first aspect of the present invention by aerobic heterotrophic fermentation to obtain the bacterial cells of sphingomonas.
The third aspect of the invention relates to sphingomonas, and the bacterial cells of the sphingomonas contain lutein.
The inventor successfully screens out a prokaryotic endophyte from noni in san city, Hainan province, and a monoclonal colony of the prokaryotic endophyte is yellow, and the shape of a cell (thallus) is a short rod, as shown in figure 1. Genomic DNA was extracted, and identified as Sphingomonas moringa (Sphingomonas morindae sp.) by PCR amplification and molecular identification based on 16s rDNA sequencing, and this strain was identified and designated as Nonainterest Sphingomonas NBD5 strain (Sphingomonas morindae strain NBD5) based on the above (see FIG. 2). The strain is preserved in China general microbiological culture Collection center (CGMCC for short), and the preservation number is as follows: CGMCC No. 18380.
The liquid chromatography analysis of the cell extract of the sphingomonas noni NBD5 and the comparison analysis of the peak time and scanning pattern of the lutein standard (see figure 3) confirm that the sphingomonas noni NBD5 can produce lutein.
In the invention, the bacterial cells of the sphingomonas are obtained by aerobic heterotrophic fermentation culture of corresponding strains of the sphingomonas, and research shows that the cell dry powder of the noni sphingomonas NBD5 contains 0.5-2mg/g of lutein.
In a fourth aspect of the present invention, there is provided a method for producing xanthophyll using the Sphingomonas bacterium of the third aspect of the present invention, which comprises:
step A, inoculating a fermentation strain into a fermentation culture medium, and performing fermentation culture for 3-5 days at 18-38 ℃, preferably 30-38 ℃ and at the rotating speed of a shaking table of 100-;
step B, carrying out centrifugal separation treatment on the fermentation culture of the sphingomonas, and harvesting bacterial cells of the sphingomonas;
step C, extracting lutein from bacterial cells of sphingomonas, and separating and purifying the obtained extracting solution to obtain a pure product of the lutein;
wherein the fermentation strain is obtained by seed culture of corresponding strains.
As known to those skilled in the art, 16SrRNA is commonly used internationally for molecular identification of bacteria, and thus 16SrRNA can be used for comparison of similarity to obtain homology. Therefore, the fermentation strain used in the present invention is not limited to the field isolate used in the present invention, and 16S rDNA is a DNA sequence corresponding to the coding rRNA on the chromosome of the bacterium and exists in the chromosomal genome of all bacteria. FIG. 2 shows that the Sphingomonas of the present invention is a 16S rDNA-based molecular phylogenetic tree of the strain NBD5, Sphingomonas noni.
Thus, in the present invention, the corresponding strain of the fermentation species is a strain having at least 90% homology to the 16S rDNA of the strain of sphingomonas according to the third aspect of the invention; preferably the corresponding strain of the fermentation broth is a strain having at least 95% homology to the 16S rDNA of the strain of Sphingomonas according to the third aspect of the invention; it is further preferred that the corresponding strain of the Zymobacter strain is a strain of Sphingomonas according to the third aspect of the invention (i.e.the strain Novosphingomonas NBD 5). That is, on the premise of not changing the 16S rDNA of the Nocardia sphingomonas NBD5 strain, a person skilled in the art can obtain a strain highly homologous to the 16S rDNA of the Nocardia sphingomonas NBD5 strain of the invention and a strain having the same or similar lutein-producing function by simply screening or mutagenizing the Nocardia sphingomonas NBD5 strain of the invention.
In the step B, the centrifugal separation treatment includes resuspending and washing the precipitate (i.e., crude and wet sphingomonas bacterium) obtained by the centrifugal separation treatment of the fermentation culture with physiological saline, and then performing centrifugal separation treatment to obtain sphingomonas cell (i.e., wet sphingomonas bacterium).
In the step B, the precipitate (i.e., crude wet sphingomonas cell) obtained by the centrifugal separation of the fermentation culture is generally subjected to at least 1 washing and separating treatment, preferably the precipitate (i.e., crude wet sphingomonas cell) obtained by the centrifugal separation of the fermentation culture is subjected to at least 2 washing and separating treatments, and more preferably the precipitate (i.e., crude wet sphingomonas cell) obtained by the centrifugal separation of the fermentation culture is subjected to 3 washing and separating treatments to obtain sphingomonas cells (i.e., wet sphingomonas cell).
The conditions for the centrifugation in the step B are not particularly limited in the present invention, and in some embodiments of the present invention, the substance to be separated may be centrifuged for 10min at 8000-.
The inventor researches and discovers that the lutein can be directly extracted from the bacterial cells of the sphingomonas and can also be extracted from the bacterial cell dry powder of the sphingomonas by using an organic solvent, and the lutein is extracted from the bacterial cell dry powder of the sphingomonas, so that the product yield is high, the used organic solvent is less, and the product purity is higher. Therefore, in some preferred embodiments of the present invention, in step C, the bacterial cells of sphingomonas are dried to obtain dry powder of sphingomonas cells, and lutein is extracted therefrom, and the obtained extract is separated and purified to obtain pure lutein.
Specifically, when the lutein is directly extracted from the bacterial cells of the sphingomonas, the step C comprises mixing the bacterial cells of the sphingomonas with an organic solvent, carrying out ultrasonic oscillation, carrying out low-temperature freeze-thaw treatment, carrying out centrifugal separation treatment, repeating the operation for at least three times, and carrying out membrane separation treatment on the obtained supernatant to obtain a pure product of the lutein.
When the lutein is extracted from the sphingomonas cell dry powder, the step C comprises drying the sphingomonas cell to obtain sphingomonas cell dry powder; mixing dry powder of sphingosine monad bacteria cells with an organic solvent, carrying out ultrasonic oscillation, low-temperature freeze thawing treatment, carrying out centrifugal separation treatment, repeating the operation for at least three times, and carrying out membrane separation treatment on the obtained supernatant to obtain a pure lutein product.
Preferably, in the step C, the collected sphingomonas cells are freeze-dried for 24 hours by a vacuum freeze-dryer to obtain the powder of norsphingomonas NBD5 (i.e., the dry powder of sphingomonas cells).
Further specifically, in the above step C, the conditions for extracting and separating and purifying lutein are as follows:
(1) the organic solvent is a mixed solvent of n-hexane and alcohol, the volume ratio of n-hexane to alcohol is 6.7:1, and preferably, the alcohol comprises methanol and/or ethanol.
(2) The weight-volume ratio of the sphingomonas cell dry powder to the organic solvent is 1:50 (g/mL).
(3) Placing the mixture of the sphingosine monad cell dry powder and the organic solvent into a centrifuge tube, and shaking for 1 minute in an ultrasonic cleaning machine.
(4) And (3) refrigerating the mixture of the sphingomonas cell dry powder subjected to ultrasonic oscillation and the organic solvent in a refrigerator at the temperature of-20 ℃ for 1 h.
(5) Centrifuging the substance for 10min under the conditions of 8000-.
(6) The supernatant was subjected to membrane separation with a 0.22. mu. organic filter.
The inventors studied the effect of different temperatures on the growth and production of lutein by sphingomonas noni NBD5 and found that not only did the growth of noni sphingomonas NBD5 be fast but the lutein content in the cells was high at a temperature of 30 ℃ (fig. 4).
According to the method, the fermentation culture is shaking table or fermentation tank fermentation culture of strains, and the fermentation strains are inoculated to a fermentation culture medium in the form of seed liquid. The inoculation amount of the seed liquid is 0.1-1% (v/v); preferably, the inoculation amount of the seed liquid is 0.2-0.5% (v/v); further preferably, the amount of the seed liquid to be inoculated is 0.5% (v/v).
Specifically, the fermentation medium comprises the following components in 1L of water in terms of 1L of water:
preferably, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
more preferably, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
in some embodiments of the invention, a 40% (wt/v) sodium hydroxide solution and a 36% (v/v) hydrochloric acid solution are used to adjust the initial pH of the fermentation medium, which has a pH of 6.8-7.2; further preferably, the pH of the fermentation medium is 7.2.
According to some embodiments of the present invention, the method for preparing lutein further comprises the step of seed culture before step a: the monoclonal colony of the Nocardia sphingomonas NBD5 strain provided by the invention is selected and inoculated into 100mL of fermentation liquid culture medium, and after shake cultivation is carried out for 3d at the temperature of 30 ℃ and the rotating speed of 200r/min, the fermentation strain (seed solution) is prepared.
The present inventors studied the effect of different temperatures on the growth and production of lutein by sphingomonas noni NBD5, and found that not only did the growth of noni sphingomonas NBD5 be fast but also the lutein content in the cells was high at a temperature of 30 ℃ (see fig. 4).
In some embodiments of the present invention, the preparation of lutein using the strain NBD5 (CGMCC No.18380) of Sphingomonas noni comprises the following steps:
(1) preparing a fermentation medium according to the following steps of: 20, yeast powder: 15, peptone: 8, sodium chloride: 5 formula of culture medium (g.L)-1) 500mL of the liquid medium was prepared and each of the prepared liquid medium was put into a 500-mL Erlenmeyer flask in an amount of 100mL per flask. Sealing the prepared and bottled culture medium with a sealing film capable of being ventilated, sterilizing at 121 ℃ for 20 minutes under high temperature and high pressure, and then sterilizing in a clean bench for 20 minutes by ultraviolet rays.
(2) And (3) seed culture, namely selecting a monoclonal colony of the Nocardia sphingomonas NBD5 strain provided by the invention to be inoculated into a fermentation liquid culture medium containing 10mL in a 100-mL triangular flask, and performing shake culture for 3 days at the temperature of 30 ℃ and the rotating speed of 200r/min to obtain a fermentation strain (seed liquid).
(3) Fermenting and culturing, namely inoculating 1mL of the noni sphingosine monad NBD5 seed solution into each bottle of culture medium under the aseptic condition in a clean workbench, then placing the bottle of culture medium into a shaking table for aerobic culture, wherein the rotation speed of the shaking table is 200r/min, and the culture temperature is 30 ℃. After 3 days of culture, a fermentation culture was obtained.
(4) Washing and separating wet thalli, harvesting cells by centrifugation (8000 rpm, 10min), pouring out supernatant, resuspending the cells settled at the bottom of a centrifuge tube with physiological saline, centrifuging again and pouring out supernatant, repeating the washing for 3 times to obtain cells of the noni sphingomonas NBD 5.
(5) And (3) carrying out freeze drying on the collected cells of the Nocardia sphingomonas NBD5 for 24h by a vacuum freeze dryer to obtain the Nocardia sphingomonas NBD5 bacterial powder.
(6) Detecting the content of lutein in the powder of the Nocardia sphingomonas NBD5, weighing 0.1g of the powder of the Nocardia sphingomonas NBD5 into a 10-mL centrifuge tube, and adding 5mL of a mixed solvent of n-hexane and ethanol (the volume ratio of the n-hexane to the ethanol is 6.7: 1). Shaking the centrifugal tube containing the bacterium powder and the extracting solution in an ultrasonic cleaning machine for 1 minute, then placing the centrifugal tube in a refrigerator with the temperature of-20 ℃ for freezing for 1 hour, repeating the operation for three times, then centrifuging the centrifugal tube (8000 rpm, 10min), taking the supernatant fluid to pass through an organic filter membrane with the diameter of 0.22 mu, directly measuring the lutein concentration of the filtrate on a high pressure liquid chromatography, and calculating the lutein content in the Nonaesynomonas NBD5 bacterium powder.
The determination conditions of the xanthophyll content in the powder of the noni sphingomonas NBD5 bacterium are as follows: high performance liquid chromatograph (Shimadzu LC-20ATvp pump, SPDM20Avp diode array detector), Zorbax SB-Aq (4.6mm × 150cm, 5 μm) separation column, mobile phase n-hexane-ethyl acetate (volume ratio 75:25), flow rate of 1mL/min, sample introduction of 40 μ L, and detection wavelength of 446 nm.
To complete the present invention, the present inventors have conducted the following research works:
(1) the noni Sphingomonas NBD5(Sphingomonas morindae sp.) of the present invention was isolated from an endophyte of noni, san city, Hainan, and its colony and cell morphology are shown in FIG. 1. According to 16S rDNA sequence analysis (figure 2), the strain is identified as the Nocardia Sphingomonas NBD5(Sphingomonas morindae strain NBD5), and the preservation number of the strain is CGMCC No. 18380.
(2) The method for preparing the lutein by utilizing the noni sphingomonas NBD5 comprises the following steps: using a culture medium (g.L)-1): glucose: 10-30, yeast powder: 10-20, peptone: 5-10, sodium chloride: 3-8, aerobically culturing the noni sphingomonas NBD53-5 days at the temperature of 18-38 ℃ and the rotating speed of a shaking table of 100-.
(3) The present inventors studied the effect of different temperatures on the growth and production of lutein by sphingomonas noni NBD5, and found that not only did the growth of nonimonas NBD5 be fast but also the lutein content in the cells was high at a temperature of 30 ℃ (fig. 4).
(4) The method for determining the xanthophyll content in the powder of the noni sphingomonas NBD5 comprises the following steps: 0.1g of powder of Sphingomonas noni NBD5 was weighed into a 10mL centrifuge tube, and 5mL of a mixed solvent of n-hexane and ethanol (volume ratio: 6.7:1) was added. Shaking the centrifuge tube containing the bacteria powder and the extracting solution in an ultrasonic cleaning machine for 1 minute, then placing the centrifuge tube in a refrigerator with the temperature of-20 ℃ for refrigerating for 1 hour, repeating the operation for three times, centrifuging the centrifuge tube (8000 rpm, 10 minutes), taking supernate and passing the supernate through an organic filter membrane with the thickness of 0.22 mu, directly measuring the lutein concentration of the filtrate on a high pressure liquid chromatography, and calculating the lutein content in the Nonaesphakia NBD5 bacteria powder (figure 3). The measurement conditions were: high performance liquid chromatograph (Shimadzu LC-20ATvp pump, SPDM20Avp diode array detector), Zorbax SB-Aq (4.6mm × 150cm, 5 μm) separation column, mobile phase n-hexane-ethyl acetate (volume ratio 75:25), flow rate of 1mL/min, sample introduction of 40 μ L, and detection wavelength of 446 nm.
The invention adopts noni sphingosine monad NBD5 to produce lutein fast and efficiently, and lays an important foundation for the more efficient and large-scale industrialized production of lutein.
Example III
The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.
The strain adopted in the experiment of the invention is a strain NBD5 strain (Sphingomonas morindae strain NBD5) with the preservation number of CGMCC No. 18380.
Implementation 1:
(1) according to the weight ratio of glucose: 20, yeast powder: 15, peptone: 8, sodium chloride: 5 formula of culture medium (g.L)-1) 500mL of the liquid medium was prepared and each of the prepared liquid medium was put into a 500-mL Erlenmeyer flask in an amount of 100mL per flask. Sealing the prepared and bottled culture medium with a sealing film capable of being ventilated, sterilizing at 121 ℃ for 20 minutes under high temperature and high pressure, and then sterilizing in a clean bench for 20 minutes by ultraviolet rays.
(2) Under the aseptic condition in a clean workbench, inoculating 1mL of the noni sphingosine monad NBD5 seed solution into each bottle of culture medium, and then placing the bottle of culture medium into a shaking table for aerobic culture, wherein the rotation speed of the shaking table is 200 r/min.
(3) After culturing for 3 days, harvesting cells by centrifugation (8000 rpm, 10min), pouring out supernatant, resuspending the cells settled at the bottom of the centrifuge tube with physiological saline, centrifuging again and pouring out supernatant, repeating the washing for 3 times to obtain cells of the bacterium noni sphingomonas NBD5, wherein the monoclonal colony (A) and the cell microscopic morphology (B) are shown in FIG. 1.
(4) And (3) carrying out freeze drying on the collected cells of the Nocardia sphingomonas NBD5 for 24h by a vacuum freeze dryer to obtain the Nocardia sphingomonas NBD5 bacterial powder.
(5) 0.1g of powder of Sphingomonas noni NBD5 was weighed into a 10mL centrifuge tube, and 5mL of a mixed solvent of n-hexane and ethanol (volume ratio 6.7:1(V/V)) was added. Vibrating a centrifugal tube containing bacteria powder and an extracting solution in an ultrasonic cleaning machine for 1 minute, then placing the centrifugal tube in a refrigerator with the temperature of-20 ℃ for refrigerating for 1 hour, repeating three operations, then centrifuging the centrifugal tube (10000 r/min, 10min), taking supernate and filtering the supernate with an organic filter membrane of 0.22 mu m, directly measuring the lutein concentration of the filtrate on a high pressure liquid chromatography, and calculating the lutein content in the powder of the noni sphingosine monad NBD5 bacteria; the liquid chromatography peak and scanning spectrum of lutein in lutein standard (A) and Nocardia sphingomonas NBD5 cell extract (B) are shown in FIG. 3. The measurement conditions were: high performance liquid chromatograph (Shimadzu LC-20ATvp pump, SPDM20Avp diode array detector), Zorbax SB-Aq (4.6mm × 150cm, 5 μm) separation column, mobile phase n-hexane-ethyl acetate (volume ratio (V/V) 75:25), flow rate of 1mL/min, sample introduction of 40 μ L, and detection wavelength of 446 nm.
(6) The research shows that the xanthophyll content in the powder of the bacterium Novosphingomonas NBD5 is 0.51mg/g when the bacterium is cultured at the culture temperature of 18 ℃.
Implementation 2:
in this example, steps (1) to (5) were the same as example 1, and different from example 1, in step (6), it was found by investigation that the xanthophyll content in the powder of Nocardia Sphingomonas NBD5 was 1.70mg/g when cultured at a culture temperature of 30 ℃.
Implementation 3:
in this example, the steps (1) to (5) were the same as example 1, and different from example 1, in step (6), it was found by investigation that the xanthophyll content in the powder of Nocardia Sphingomonas NBD5 was 0.49mg/g when cultured at a culture temperature of 38 ℃.
The above results show that the inventors successfully screened and identified a prokaryotic microorganism, Nocardia sp NBD5, from noni, san City, Hainan province, whose monoclonal colony was yellow and cell morphology was short rod-shaped (see FIG. 1). Identified as sphingomonas noni NBD5 by the 16s rDNA molecule (see fig. 2). The liquid chromatography analysis of the cell extract of the sphingomonas noni NBD5 and the comparison analysis of the peak time and scanning pattern of the lutein standard (see figure 3) confirm that the sphingomonas noni NBD5 can produce lutein. It was found by studies of culture at different temperatures (examples 1-3) that 30 ℃ is the optimum control condition for growth and xanthophyll production of Sphingomonas noni NBD5 (see FIG. 4). Experimental results show that the highest lutein content in the powder of the Norem sphingomonas NBD5 can reach about 2.00 mg/g-cell dry weight. The method for producing the lutein by adopting the sphingosine monad with high growth speed and high lutein content can quickly and efficiently realize the industrialized production of the lutein, and has important application prospect and value.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (9)
1. The sphingomonas comprises lutein in bacterial cells, wherein the sphingomonas is a noni sphingomonas NBD5 strain with the preservation number of CGMCC No. 18380;
the dry powder of the bacterial cells of the sphingomonas contains 0.5-2mg/g of lutein;
the bacterial cells of the sphingomonas are obtained by aerobic heterotrophic fermentation culture of corresponding strains of the sphingomonas.
2. A method for producing lutein using the sphingomonas of claim 1, comprising:
step A, inoculating a fermentation strain into a fermentation culture medium for fermentation culture to obtain a fermentation culture of sphingosine monad;
step B, carrying out centrifugal separation treatment on the fermentation culture of the sphingomonas, and harvesting bacterial cells of the sphingomonas;
step C, extracting lutein from bacterial cells of sphingomonas, and separating and purifying the obtained extracting solution to obtain a pure product of the lutein;
wherein the fermentation strain is obtained by seed culture of corresponding strains;
the corresponding strain of a fermentative species is a strain of sphingomonas as claimed in claim 1.
3. The method according to claim 2, wherein in step C, the bacterial cells of Sphingomonas are dried to obtain a dry powder of the bacterial cells of Sphingomonas, and lutein is extracted therefrom, and the obtained extract is separated and purified to obtain a pure product of lutein.
4. The method according to claim 2, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:
the pH value of the fermentation medium is 6.8-7.2.
5. The method according to claim 4, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:
6. the method according to claim 3, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:
the pH value of the fermentation medium is 6.8-7.2.
7. The method according to claim 6, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:
8. the method according to any one of claims 2 to 7, wherein in step A, the temperature of the fermentation culture is 18 to 38 ℃.
9. The method according to claim 8, wherein the temperature of the fermentation culture in step a is 30-38 ℃.
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| WO2004056972A2 (en) * | 2002-12-19 | 2004-07-08 | E.I. Du Pont De Nemours And Company | Parallel chromosomal stacking of traits in bacteria |
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| CN101218352A (en) * | 2005-03-18 | 2008-07-09 | 米克罗比亚精确工程公司 | Production of carotenoids in oleaginous yeast and fungi |
| CN101680015A (en) * | 2007-03-08 | 2010-03-24 | 比奥特伦德生物技术创新工程股份公司 | Cross the selected bacterial strain of product carotenoid or the method for mutant production high purity carotenoid by the fermentation composition |
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| CN1678735A (en) * | 2001-05-04 | 2005-10-05 | 佛罗里达大学研究基金会有限公司 | Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor |
| WO2004056972A2 (en) * | 2002-12-19 | 2004-07-08 | E.I. Du Pont De Nemours And Company | Parallel chromosomal stacking of traits in bacteria |
| CN101218352A (en) * | 2005-03-18 | 2008-07-09 | 米克罗比亚精确工程公司 | Production of carotenoids in oleaginous yeast and fungi |
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