Xanthophyll-producing flavobacterium strain

Abstract

The invention relates to a lutein-producing flavobacterium strain and belongs to the technical field of biology, the lutein-producing flavobacterium strain can produce lutein, the yield is 71.08 + / -1.33 mu g / g FW, in addition, the invention discloses a lutein microbial fermentation production method, and the method comprises the steps of bacterial liquid preparation, lutein production and product extraction and chemical detection. Compared with extraction of xanthophyll from marigold and microalgae, the flavobacterium strain has the advantages that the growth cycle is 40-48 hours, the production cycle of the flavobacterium strain is short, cultivated land can be saved, the content of pigment impurities is low, the cost can be reduced, and the flavobacterium strain has the potential of industrial application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a lutein-producing Flavobacterium. Background Art

[0002] Lutein is a carotenoid component naturally present in many plants, with a distinct yellow pigment characteristic. Its molecular formula is C 40 H 56 O2. As a natural plant pigment with multiple biological activities, lutein has been widely used in food, medicine, and cosmetics. Its unique molecular structure, consisting of two ionone rings and an oxygen-containing polyene chain, gives lutein its remarkable antioxidant properties. In the human body, lutein is primarily concentrated in the retina and macula of the eye, playing a vital role in protecting the eye from light damage and preventing eye diseases such as age-related macular degeneration. Lutein also exhibits potential anti-inflammatory and anti-cancer effects. Furthermore, lutein is a natural pigment with no side effects and can be used to color egg yolks, poultry, and chicken feed. Because its safety and nutritional properties meet market demand, it is widely used in feed coloring in various countries. Therefore, lutein is also known as "plant gold."

[0003] Currently, the most researched lutein production process is primarily through extraction from marigolds. However, with technological advancements, research results have also been achieved in extracting lutein from microalgae. The choice of raw materials determines lutein yield, yield, and production process, while also influencing the accompanying separation methods and, consequently, the overall production cost.

[0004] Marigold has a relatively long growth cycle of about 100 days, and can only be used to extract lutein after it matures. Large-scale cultivation of marigolds occupies a large amount of arable land resources. Microalgae have a shorter growth cycle than marigolds, but generally take about 20 days. At the same time, marigolds and microalgae have a rich variety of pigments. In addition to lutein, marigold pigments also contain chlorophyll, anthocyanins, and other carotenoid members. Microalgae pigments also contain chlorophyll, other carotenoid members, as well as phycocyanin and phycoerythrin. Therefore, whether extracting lutein from marigolds or from microalgae, there is the problem of containing many other pigment impurities. In addition to the extraction operation, impurity removal treatment is also required, which makes the lutein extraction process complicated and increases production costs.

[0005] Furthermore, the marigold extraction process is complex, involving multiple steps, including harvesting, preservation and fermentation, drying and dehydration, crushing and granulation, cryogenic extraction, and concentration. The subcritical low-temperature extraction technique used requires precise control of the solvent's boiling point, which is technically demanding and can affect extraction results if not handled properly. Microalgae have thick cell walls, resulting in low lutein extraction efficiency, and industrial production technology is immature. Summary of the Invention

[0006] To overcome the problems existing in the background technology, the present invention discloses a lutein-producing Flavobacterium, which is capable of producing lutein with a yield of 71.08±1.33μg / g FW. In addition, the present invention discloses a method for the microbial fermentation production of lutein, which includes the preparation of a bacterial liquid, the production of lutein, and the extraction and chemical detection of the product. Compared with the extraction of lutein from marigolds and microalgae, the growth cycle of this strain of Flavobacterium is 40-48h. The production cycle of this strain of Flavobacterium is short, which can save arable land, and the content of pigment impurities is low, which helps to reduce costs and has the potential for industrial application.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions: The Flavobacterium Flavobacterium luteinifaciens , used to produce lutein; The Flavobacterium is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC NO: 32333 and the deposit date being October 24, 2024.

[0008] Preferably, the breeding and fermentation method of Flavobacterium comprises the following steps: 1) Sampling and Isolating the Strain: River samples from Yunnan's natural environment were diluted, spread onto TSA medium, and yellow single colonies were selected. Pure strains were obtained by three-zone purification. 2) Strain identification: The strain from step 1) was subjected to 16S rRNA gene and genome sequencing. The sequencing results were compared and analyzed to determine the taxonomic status. The strain was identified as a new species of the genus Flavobacterium. 3) Prepare seed solution: Dilute a pure strain of Flavobacterium and spread it on a plate. Pick a single colony and place it in a shake flask filled with TSB medium. Culture it in a shaker to obtain the seed solution of Flavobacterium. 4) Fermentation culture: Take the prepared Flavobacterium seed liquid and add it to the fermentation medium for shake flask culture to obtain the Flavobacterium bacterial liquid; 5) Product collection: Centrifugation is used to collect the bacteria from the Flavobacterium bacterial solution, and then extraction, separation, and purification are performed to obtain lutein; 6) Identification of lutein products.

[0009] Preferably, in step 3) of preparing the seed solution, the TSB culture medium comprises per liter: 13-17 g tryptone, 3-4 g sodium chloride, 4-6 g soy peptone, 2-4 g glucose, and 2-3 g dipotassium hydrogen phosphate; The volume of the culture medium bottle is 100-150 ml, the culture conditions are 28-30 ° C temperature, the shaker speed is 180-200 rpm, the culture is carried out for 24-28 hours, and the bacterial solution OD 600 When it is 0.8-1.0, it can be used as seed liquid for inoculation.

[0010] Preferably, in step 4) the fermentation culture, each liter of the fermentation medium comprises: 13-17 g of tryptone, 2-4 g of sodium chloride, 4-6 g of soy peptone, 1-3 g of sucrose, 2-3 g of dipotassium hydrogen phosphate, and 2.5-3.5 ml of tomato juice; the pH of the fermentation medium is 7-7.8, and the fermentation medium is sterilized at 121° C. for 30 min; the fermentation culture temperature is 28-30° C., the volume of each fermentation medium bottle is 100-150 ml, the inoculum size is 5-10%, the shaker speed is 180-200 rpm, and the culture time is 40-48 h.

[0011] Preferably, in step 5), the collection operation is to use a centrifuge to collect the bacteria, the centrifuge speed is 9000-10000 r / min, and the centrifugation time is 8-10 min.

[0012] Preferably, in step 5), the extraction operation comprises the following steps: (1) Weigh the bacterial cells, dilute them with phosphate buffer solution (pH 7-8), and transfer the mixture to a homogenizer for 5 minutes. (2) The homogenized sample was diluted again with pH 7-8 phosphate buffer solution and mixed thoroughly; (3) Add ethyl acetate and extract by ultrasonic for 5-10 min. Repeat 5-7 times.

[0013] Preferably, in step 5), the purification method comprises the following steps: (1) After the extraction operation is completed, the remaining extract is evaporated at 35-40 degrees Celsius to remove the organic phase; (2) Methanol is added to the extract after the organic phase is removed to dissolve it, and then filtered through a 0.45 μm filter head; (3) The filtered extract was separated using a high performance liquid chromatography (HPLC) instrument equipped with an Agilent ZORBAX SBC-18 9.4*150mm column and an isocratic elution of methanol:water 95:5 to extract the lutein in the extract.

[0014] Preferably, the methanol solution is obtained by mixing methanol and water in a ratio of 95:5.

[0015] Preferably, in step 6) lutein product identification, the extracted sample is subjected to ECD spectrum detection, optical rotation detection, nuclear magnetic resonance detection, and mass spectrometry detection.

[0016] The beneficial effects of the present invention are as follows: the lutein-producing Flavobacterium can produce lutein with a yield of 71.08±1.33 μg / g FW. In addition, the present invention discloses a method for microbial fermentation production of lutein, which includes the preparation of a bacterial liquid, the production of lutein, and the extraction and chemical detection of the product. Compared with the extraction of lutein from marigold and microalgae, the growth cycle of this strain of Flavobacterium is 40-48 hours. The production cycle of this strain of Flavobacterium is short, which can save arable land, and the content of pigment impurities is low, which helps to reduce costs and has the potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the TLC test results; Figure 2 This is the absorption peak diagram of the bacterial extract sample; Figure 3 Schematic diagram of HPLC detection results of lutein standard sample; Figure 4 The HPLC test results of bacterial extract samples are shown below; Figure 5 This is a colony diagram of a Flavobacterium strain; Figure 6 This is the growth curve of Flavobacterium; Figure 7 This is the HPLC standard curve of lutein; Figure 8 is the 13C-NMR spectrum (100 MHz) of the prepared compound; Figure 9 HPLC spectrum of the spiked experiment; Figure 10 is the UV absorption curve of lutein standard; Figure 11 is the UV absorption curve of the absorption peak of the spiked experiment; Figure 12 This is the ECD spectrum of the separated compound and lutein standard. DETAILED DESCRIPTION

[0018] In order to achieve the above-mentioned purpose, the technical scheme and beneficial effects are more clearly defined, and the present invention is specifically described below with reference to the embodiments; the following embodiments can specifically illustrate the present invention, but do not limit the present invention in any way; in the following embodiments: Lutein standard: Lutein, HPLC ≥ 90%, Shanghai Yuanye Biotechnology Co., Ltd.; TLC silica gel plate: Huanghai brand, model HSGF254, size 5x10 cm.

[0019] The seed liquid culture medium includes: 13-17 g of tryptone, 3-4 g of sodium chloride, 4-6 g of soy peptone, 2-4 g of glucose, 2-3 g 2.5 g of dipotassium hydrogen phosphate, pH = 7-7.8, and sterilization at 121°C for 30 min.

[0020] TSA culture medium includes: 15 g tryptone, 5 g sodium chloride, 5 g soy peptone, 2.5 g glucose, 2.5 g potassium hydrogen phosphate, 15 g agar, pH = 7-7.8, sterilized at 121°C for 30 min.

[0021] The fermentation medium includes: 13-17 g of tryptone, 2-4 g of sodium chloride, 4-6 g of soy peptone, 1-3 g of sucrose, 2-3 g of dipotassium hydrogen phosphate, 2.5-3.5 ml of tomato juice, pH=7-7.8, and sterilization at 121°C for 30 min.

[0022] Example 1: Screening process of lutein-producing bacteria.

[0023] 1. Identification of lutein-producing strains: (1) Sampling and strain isolation and culture: River samples from Yunnan's natural environment were diluted and spread on TSA medium. After incubation at 28°C for two days, yellow single colonies were selected, purified by three-zone line drawing, and then sequenced and analyzed.

[0024] (2) 16S rRNA gene sequencing: Total bacterial DNA was extracted using the Chelex-100 method. Prepare a 5% Chelex-100 aqueous solution, take 100µL and place it in a PCR tube. Use a sterile bamboo stick to pick up a small amount of bacteria and place it in the tube. Vortex and mix thoroughly. Incubate in a PCR instrument at 99°C for 20 minutes. After centrifugation, the supernatant was used as a DNA template. PCR amplification was performed using universal bacterial primers (27F, 1492R): 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' 1492R:5'-GGTTACCTTGTTACGACTT-3' The PCR system is: The PCR operation program is: Finally, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rRNA gene sequencing.

[0025] (3) Genome sequencing. The bacteria were sent to Shanghai Meiji Biotechnology Co., Ltd. for genome sequencing.

[0026] The 16S rRNA gene sequencing results were compared and identified on EzBioCloud.net|Search about Bacteria or Archaea. Based on the 16S rRNA gene and genome sequence analysis, Flavobacterium ( Flavobacterium ) The species was published and the identification results are shown in Table 1.

[0027] Table 1 Identification results of strain CGMCC 32333 As shown in the identification results, the 16SrRNA gene of strain CGMCC 32333 is similar to Flavobacterium The valid published species were <98.65%, DNA-DNA hybridization value DDH (%) <70%, average nucleotide identity (ANI) (%) <95%, and identified as Flavobacterium The new species was named as Xanthobacterium luteinogenum because of its lutein-producing characteristics. Flavobacterium luteinifaciens (lu.te.i.ni.fa'ci.ens.NLneut.n.luteinum,lutein;L.pres.part.faciens,making;NLpart.adj.luteinifaciens, luteinproducing).

[0028] 2. Screening of lutein-producing strains: (1) The xanthophyll metabolites of the Flavobacterium identified by sequencing in step 1 were studied. The pure bacteria after fermentation were collected and placed in a centrifuge tube. After dilution with phosphate buffer solution (pH = 7-8), the mixture was transferred to a homogenizer and homogenized for 5 minutes. The homogenized sample was diluted with phosphate buffer solution again and mixed thoroughly. Ethyl acetate was added and ultrasonic extraction was performed for 5-10 minutes, and repeated 5-7 times. The organic phase was removed at 35-40 degrees Celsius, dissolved in methanol, and filtered through a 0.45 μm filter to obtain the extract for subsequent extraction.

[0029] (2) Use methanol to dissolve the lutein standard to prepare the lutein standard sample. The strain extract suspected of containing lutein and the standard sample are subjected to thin layer chromatography (TLC) to determine whether lutein is present. Select a silica gel plate, spot the bacterial extract and the lutein standard sample on the silica gel plate, and use petroleum ether: ethyl acetate = 2:1 as the developing solvent. The final result is as follows: Figure 1 As shown in the figure, the Rf values ​​(ratio shift values) of the standard sample and the extracted sample are basically the same, and the impurity content is low, so the sample is preliminarily determined to be lutein; the TLC detection results are as follows Figure 1 As shown; (3) Perform high performance liquid chromatography (HPLC) and simultaneously test the above bacterial extract samples to observe whether the samples contain suspected lutein components. The Shimadzu LC-20 Series is equipped with an SPD-M20A photodiode array (PDA) detector, and uses an Agilent ZORBAX XDB-C18 column (4.6×250 mm, 5μm) chromatographic column and an Agilent GuardColumn Hardware Kit protective column. The analytical conditions are isocratic elution using a methanol / water system (95:5, v / v). The standard sample detection results have a retention time of 13.870 min; the maximum absorption wavelength is 444 nm, and the bacterial extract sample absorption peak diagram is shown in the figure below. Figure 2 As shown; Lutein standard HPLC test results are as shown Figure 3 As shown; (4) High performance liquid chromatography (HPLC) of bacterial extract samples was performed at a wavelength of 444 nm. The retention time of the test results was 13.930 min, which was almost the same as that of the standard sample. The HPLC test results of bacterial extract samples were as follows: Figure 4 As shown; screening colonies as Figure 5 As shown; Based on the above results, a strain producing lutein was screened out.

[0030] Example 2: Identification of Lutein Yield of Lutein-Producing Bacteria.

[0031] 1. Determination of the yield of lutein-producing bacteria: 1) Growth curve determination: (1) Seed liquid preparation: Pick bacteria from the culture medium and inoculate into 100 ml of TSB liquid medium. Culture at 28°C and 180 rpm for 24 h until the OD600 reaches 1. (2) Inoculate 5% of the above seed solution into 100 ml TSB liquid medium, in parallel, and culture at 28°C, 180 rpm. Take samples every 8 hours for OD600 detection, record the data and draw a growth curve; the growth curve is as follows: Figure 6 As shown; 2) Fermentation broth cell yield: (1) Seed liquid preparation: Pick a single colony from the culture medium and inoculate it into 100 ml of TSB liquid medium. Cultivate at 28°C and 180 rpm for 24 h until the OD600 reaches 1. (2) Inoculate 5% of the above seed solution into 100 ml of fermentation medium. Incubate at 28°C and 180 rpm for 40-48 hours. Then, collect the cells at 9000 rpm for 10 minutes. The cell yield in the fermentation broth is calculated to be 15-20 g / L.

[0032] 2. Determination of lutein yield per gram of lutein-producing bacteria.

[0033] 1) Drawing of the standard curve.

[0034] (1) Weigh a lutein standard (90% purity) and dissolve it in methanol to prepare a methanol solution with a lutein content of 100 μg / mL. Detection was performed using a Shimadzu LC-20 Series equipped with an SPD-M20A photodiodearray (PDA) detector, an Agilent ZORBAXX DB-C18 column (4.6×250 mm, 5 μm), and an Agilent Guard Column Hardware Kit.

[0035] (2) Analytical conditions: Isocratic elution with a methanol / water system (95:5, v / v) was used, flow rate: 1.000 mL / min, and detection wavelength: 444 nm. The injection volumes of the lutein methanol solution were: 2, 4, 6, 8, and 10 μL, respectively. The lutein peak area was integrated and a working curve was plotted.

[0036] 2) Sample pretreatment: Weigh 15.0 g of bacterial cells and dilute them with 10 mL of phosphate buffer solution (pH = 7.4). Transfer the mixture to a homogenizer and homogenize for 5 minutes. Dilute the homogenized sample to 45 mL with phosphate buffer solution and mix thoroughly. The mixed sample is divided into three equal parts, each of which is added with 25 mL of ethyl acetate and ultrasonically extracted for 5 minutes, repeated five times. The organic phase is removed at 40 degrees Celsius, dissolved in 1.000 mL of methanol, and filtered through a 0.45 μm filter to obtain the samples to be tested (samples 1, 2, and 3). The samples to be tested are detected by HPLC, and the lutein content is calculated using the external standard method.

[0037] 3) Test results: (1) The standard curve of lutein standard sample is Y=13653 X-734.1(R 2 =0.9987), lutein HPLC standard curve as shown Figure 7 As shown; (2) The final lutein yield of the lutein-producing bacteria was calculated to be 71.08±1.33μg / g FW.

[0038] Example 3: Identification of lutein-producing bacterial products.

[0039] 1. Collect bacteria for large fermentation: (1) Seed liquid preparation: Streak the strain on a solid plate medium and culture it in a constant temperature incubator at 28°C for 3 days. Use an inoculation loop to pick a single colony and inoculate one loop into 100 mL of seed medium. Culture it at 28°C with shaking at 180 rpm for 24 h. (2) Shake flask fermentation: Prepare 40.5L fermentation medium and use fermentation medium for fermentation. The medium formula is: 13-17g tryptone, 4-6g soy peptone, 2-4g sodium chloride, 1-3g sucrose, 2-3g potassium hydrogen phosphate, 2.5-3.5ml tomato juice, pH = 7-7.8, sterilize at 121℃ for 30min. The bottling volume of each bottle is 100ml, and there are 405 bottles in total. Inoculate the seed liquid in step (1) into the fermentation medium at a ratio of 6%, and culture at 28-30℃, 180-200rmp for 40-48h. (3) Collecting the cells: The fermentation broth obtained in step (2) was centrifuged in a large high-speed centrifuge at 9,000-10,000 rpm for 8-10 minutes. The supernatant was discarded and the cells were collected. A total of approximately 37 L of fermentation broth and approximately 1,124 g of cells were collected, with a cell yield of approximately 30.4 g / L of cells per liter of fermentation broth.

[0040] 2. Product testing: (1) Obtaining the extract: Weigh the cells, dilute with phosphate buffer solution (pH = 7.4), and transfer the mixture to a homogenizer for 5 minutes. The homogenized sample is diluted with phosphate buffer solution again and mixed thoroughly. Ethyl acetate is added and ultrasonic extraction is performed for 5 minutes, which is repeated 5 times. The organic phase is removed at 40 degrees Celsius, dissolved in methanol, and filtered through a 0.45 μm filter to obtain the extract for subsequent extraction. Lutein is extracted from the extract using a high-performance liquid chromatography (HPLC) instrument and concentrated by rotary evaporation to prepare the compound.

[0041] (2) Identification of compound structure: The prepared compound was tested by nuclear magnetic resonance spectrometer to obtain a 13C-NMR spectrum, as shown in Figure 8 As shown, the obtained compound and lutein standard were spiked and detected by HPLC to obtain HPLC chromatogram ( Figure 9 ); The prepared compound and lutein standard were subjected to high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) detection, polarimeter and circular dichroism (ECD) detection to obtain HR-ESI-MS spectrum, optical rotation value and ECD spectrum ( Figure 10 ).

[0042] The prepared compound was an orange-yellow powder. High-resolution mass spectrometry (HR-ESI-MS) showed m / z 591.4178 [M+Na]+(calcd. for C 40 H 56 O2Na+, accurate molecular weight 568.4173 = molar molecular weight 568.871) indicates that its molecular formula is C 40 H 56 O2. Its carbon nuclear magnetic resonance spectrum data is consistent with the lutein data reported in the literature: 13C-NMR (100 MHz, CDCl3): δ C37.1(C-1), 48.4 (C-2), 65.1 (C-3), 42.5(C-4), 126.2(C-5), 138.5(C-6), 125.6(C-7), 138.7(C-8), 135.7 (C-9), 131.3(C-10),124.9(C-11), 137.6(C-12), 136.4(C-13), 132.6 (C-14), 130.1(C-15), 28.7(C-16), 30.3 (C-17), 21.6 (C-18), 12.7(C-19andC-20), 34.0(C-1), 44.6(C-2), The chromatographic peaks of the isolated compounds were basically consistent with those of the lutein standard. The chromatographic peak after spiked has the same UV absorption curve as the lutein standard, indicating that the separated compound has the same planar structure as lutein.

[0043] Optical rotation data of the prepared compounds [α] +66.76°( c 0.100, CHCl3) and the optical rotation data of lutein standard [α] +69.16°( c0.100, CHCl3) is basically the same. At the same time, the prepared compound has the same ECD curve as the lutein standard, so it is judged that the prepared compound has the same absolute configuration as lutein, and therefore, it is judged that lutein is prepared.

[0044] Example 4: Comparison of lutein production by lutein-producing bacteria, marigold, and microalgae.

[0045] 1. Marigold Lutein Extract The lutein in marigold flowers mainly exists in the form of lutein dipalmitate. The data of a paper were analyzed [1]. Dried marigold flower particles with a pigment content of 1.6% were selected. After crushing, petroleum ether was added at a material-liquid ratio of 1:7 (g:mL). The extract was taken at 50℃ twice, each time for 5 hours. The extraction rate was 93.2% and the RSD value was 1.2%. An appropriate amount of extract was dissolved in ethanol, 35% NaOH solution was added, and saponification was carried out at 60℃ for 6 hours. After saponification, the purity of free lutein can reach 98.6%, and the content of all-trans lutein is 96.5%.

[0046] 2. Lutein production by microalgae According to data from a paper [2], under optimal heterotrophic culture conditions, after 7 days of cultivation, Chlorella was harvested, with a final dry weight of 18.90 g / L and a lutein yield of 30.06 mg / L. Lutein from Chlorella was extracted using an organic solvent extraction method, with cell wall breakage and wet extraction. The extraction solvent was methanol / dichloromethane (v / v, 2:1). The extraction conditions were: temperature 42.8°C, solid-liquid ratio 1:94, time 41 minutes, and an extraction rate of 96.8%. (Complete extraction with methanol and repeated extraction until colorless was considered an extraction rate of 100%). (Relative extraction rate (%) = lutein mass extracted by a certain method / lutein mass extracted by the optimal method × 100%) 3. The Flavobacterium for producing lutein Lutein was produced using the Flavobacterium, and the cells were collected after 40 hours of culture. The maximum wet cell weight was approximately 30.40 g / LFW, and the lutein yield was approximately 2.15 mg / L. Using an organic reagent extraction method, the cells were mixed with phosphate buffer (pH = 7.4) and homogenized for 5 minutes, then diluted and mixed. Ultrasonic extraction with ethyl acetate was repeated 5 times, the organic phase was removed, and the extract was dissolved in methanol and filtered to obtain the extract. Lutein was extracted using HPLC and concentrated by rotary evaporation to prepare the compound.

[0047] Compared with marigold, this patented strain occupies less space and has a shorter growth cycle, making it suitable for industrial production. The lutein in the strain exists mainly in monomeric form, and the extraction process does not require saponification, saving time and cost. The strain has low land requirements for lutein production, and the production process is more environmentally friendly. Compared with microalgae, the growth cycle of this patented strain is shorter, the cell wall is thinner, and it is easy to break the cell wall to extract lutein. Compared with microalgae, the lutein extracted by this strain has fewer impurities, does not require complex purification, has high purity, and is suitable for the production of high value-added products. Simplifying the extraction process and reducing purification costs reduce the overall production cost, making it suitable for large-scale industrial production, with high market competitiveness and application prospects; the efficiency comparison of the three methods of producing lutein is shown in Table 2.

[0048] Table 2 Comparison of the efficiency of three methods of producing lutein marigold microalgae Lutein-producing bacteria Growth cycle 3-4 months 7-14d 40-48h Growing conditions Depends on land and climate conditions, is vulnerable to natural disasters, and requires a large amount of arable land.[3] <![CDATA[Precise control of light intensity, temperature (15–30 °C), and CO2 concentration is required. Outdoor cultivation is susceptible to contamination, and the cost of indoor photobioreactors is high [5]]]> Bacterial culture does not require light and can be carried out in closed fermentation tanks. The control of temperature and pH is more flexible and is less affected by the external environment. The production process does not require agricultural land, has low resource consumption, and is suitable for industrial large-scale production. Production efficiency Low (dependent on planting) long growth cycle, long fermentation cycle of fresh flowers (more than 57 hours), lutein loss rate can reach more than 20% [4] Medium (requires light reactor) with short growth cycle Very high (controlled fermentation) growth rate, short metabolic cycle, can be achieved through high-density culture in fermentation tanks, and has great yield potential Lutein forms Lutein dipalmitate Lutein Lutein Extraction steps Fermentation, drying, and crushing of dried marigold flowers; extraction with an organic solvent; concentration of the extract; saponification, crystallization, and drying to obtain lutein Cultivate and harvest microalgae; disrupt cells; extract with organic solvents; concentrate the extract and purify to obtain lutein[6] The buffer solution is mixed and then mechanically broken, and the extract is concentrated by ultrasonic extraction with ethyl acetate. The HPLC preparation steps are simple, time-consuming and cost-effective. Time consumed Fermentation 2-3 days, extraction 5-10 hours, saponification 1-2 hours Freeze-thaw method: freeze for 48 hours[7] Mechanical cell wall breaking: 5-10min Is saponification required? yes no no impurities Other carotenoids, chlorophyll, etc. Other carotenoids, chlorophyll, etc. Almost no other pigment impurities production costs High (cultivation cost) Medium (photoreactor energy consumption) Lower (fermentation production tank) no need for light References in the table above: [1] Zhang Duoting, Han Jing, Zhang Yue, et al. Extraction and separation of lutein from marigold[J]. Food and Fermentation Industries, 2010, 36(05):167-169+173. DOI:10.13995 / j.cnki.11-1802 / ts.2010.05.009.

[0049] [2] Wang Shuhui. Optimization of culture conditions for lutein production by Chlorella and study on lutein stability[D]. Fujian Agriculture and Forestry University, 2014. DOI: 10.27018 / d.cnki.gfjnu.2014.000037.

[0050] [3] Liu Le, Zhan Junling, Gao Ziyi, et al. Research status of lutein extraction technology from marigold[J]. Green Technology, 2018(20):2.DOI:CNKI:SUN:LVKJ.0.2018-20-077.

[0051] [4] Zhen Lina, Liu Lizhen, Zhu Jinwei, et al. Effect of probiotics on fermentation quality and lutein content of marigold[J]. Chinese Condiments, 2023, 48(6):195-198.DOI:10.3969 / j.issn.1000-9973.2023.06.034.

[0052] [5] Wang Meiqi, Ping Wenxiang, Ge Jingping, et al. Research progress of microalgae in wastewater treatment and biomass recycling and reuse [J]. Acta Microbiologica Sinica, 2022, 62(12):13.

[0053] [6] Shan Mengdi, Zhang Fang, Zhang Yi, et al. Research progress on lutein production from microalgae[J]. Food Research and Development, 2020, 41(6):8. DOI:CNKI:SUN:SPYK.0.2020-06-039.

[0054] [7] Wang Shuhui. Optimization of culture conditions for lutein production by Chlorella and study on lutein stability[D]. Fujian Agriculture and Forestry University, 2014. DOI: 10.27018 / d.cnki.gfjnu.2014.000037.

[0055] In summary, the lutein-producing bacteria is the Flavobacterium disclosed in the present invention. This strain of Flavobacterium can ferment and produce lutein. Compared with the extraction of lutein from marigold and microalgae, the growth cycle of this strain of Flavobacterium is 40-48 hours. This strain of Flavobacterium has a short production cycle, can save arable land, and has a low content of pigment impurities, which helps to reduce costs and has the potential for industrial application.

[0056] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A xanthophyll-producing Flavobacterium, characterized in that: The Flavobacterium Flavobacterium luteinifaciens , used to produce lutein; The Flavobacterium was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC NO: 32333.

2. The lutein-producing Flavobacterium according to claim 1, characterized in that: The breeding and fermentation method of Flavobacterium comprises the following steps: 1) Sampling and Isolation of Strains: River samples were collected from the natural environment of Yunnan Province. The samples were diluted and spread on TSA medium. Single yellow colonies were selected and purified by three-zone line drawing to obtain pure strains. 2) Strain identification: The pure strain obtained in step 1) was subjected to 16S rRNA gene and genome sequencing. The sequencing results were compared and analyzed to determine the taxonomic status. The strain was confirmed to be of the genus Flavobacterium. 3) Prepare seed solution: Dilute a pure strain of Flavobacterium and spread it on a plate. Pick a single colony and place it in a shake flask filled with TSB medium. Culture it in a shaker to obtain the seed solution of Flavobacterium. 4) Fermentation culture: Take the prepared Flavobacterium seed liquid and add it to the fermentation medium for shake flask culture to obtain the Flavobacterium bacterial liquid; 5) Product collection: Centrifugation is used to collect the bacteria from the Flavobacterium bacterial solution, and then extraction, separation, and purification are performed to obtain lutein; 6) Identification of lutein products.

3. The lutein-producing Flavobacterium according to claim 2, characterized in that: In step 3) preparing the seed solution, the TSB medium formula per liter includes: 13-17g tryptone, 3-4g sodium chloride, 4-6g soy peptone, 2-4g glucose, and 2-3g dipotassium hydrogen phosphate; The volume of the culture medium bottle is 100-150 ml, the culture conditions are: 28-30 ° C temperature, shaker speed 180-200 rpm, culture for 24-28 hours, the bacterial solution OD 600 When it is 0.8-1.0, it can be used as seed liquid for inoculation.

4. The lutein-producing Flavobacterium according to claim 2, characterized in that: In step 4) of the fermentation culture, each liter of the fermentation medium includes: 13-17 g of tryptone, 2-4 g of sodium chloride, 4-6 g of soy peptone, 1-3 g of sucrose, 2-3 g of dipotassium hydrogen phosphate, and 2.5-3.5 ml of tomato juice; the pH of the fermentation medium is 7-7.8, and the fermentation medium is sterilized at 121° C. for 30 minutes; the fermentation culture temperature is 28-30° C., the volume of each bottle of fermentation medium is 100-150 ml, the inoculum size is 5-10%, the shaker speed is 180-200 rpm, and the culture time is 40-48 hours.

5. The lutein-producing Flavobacterium according to claim 2, characterized in that: The step 5) of collecting the bacteria is performed using a centrifuge with a rotation speed of 9000-10000 r / min and a centrifugation time of 8-10 min.

6. The lutein-producing Flavobacterium according to claim 2, characterized in that: In step 5), the extraction operation includes the following steps: (1) Weigh the bacterial cells, dilute them with a phosphate buffer solution at pH 7-8, and transfer the mixture to a homogenizer for homogenization; (2) The homogenized sample was diluted again with pH 7-8 phosphate buffer solution and mixed thoroughly; (3) Add ethyl acetate and perform ultrasonic extraction for 5-10 min. Repeat the extraction operation 5-7 times.

7. The lutein-producing Flavobacterium according to claim 2, characterized in that: In step 5), the purification method comprises the following steps: (1) After the extraction operation is completed, the remaining extract is evaporated at 35-40°C to remove the organic phase; (2) Add methanol to the extract after removing the organic phase to dissolve it, and then filter it with a filter head; (3) The filtered extract is separated by isocratic elution using a chromatographic column and methanol solution to extract the lutein in the extract.

8. The lutein-producing Flavobacterium according to claim 7, characterized in that: The methanol solution is obtained by mixing methanol and water in a ratio of 95:

5.

9. The lutein-producing Flavobacterium according to claim 2, characterized in that: The step 6) lutein product identification is to perform ECD spectrum detection, optical rotation detection, nuclear magnetic resonance detection, and mass spectrometry detection on the extracted sample.

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