Medium for improving production of astaxanthin by paracoccus and application thereof

Abstract

The application discloses a culture medium for improving production of paracoccus astaxanthin and application thereof. The culture medium comprises the following components according to concentration calculation: yeast extract 4-20 g / L; beef extract 8-20 g / L; bacteriological peptone 4-20 g / L; glucose 10-30 g / L; KH2PO4 0.1-0.2 g / L; Na2HPO4.12H2O 0.1-0.5 g / L; MgSO4.7H2O 0.1-1 g / L; FeSO4.7H2O 0.1-1 g / L; CaCl2.2H2O 0.02-0.1 g / L; and pH 7.2+ / -0.2. In the application, the yeast extract, beef extract and bacteriological peptone are mixed as the nitrogen source, which can promote the accumulation of paracoccus biomass and astaxanthin, and meanwhile, the problem of precipitation after sterilization of the culture medium is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of astaxanthin production by fermentation, in particular to a culture medium for improving the yield of astaxanthin produced by Staphylococcus and its application. BACKGROUND

[0002] Astaxanthin is a carotenoid pigment with the chemical name of 3,3'-dihydroxy-β,β'-carotene-4,4'-dione and the chemical formula of C 40 H 52 O4, which is commonly found in various marine bacteria, microalgae, and crustacean shells, as well as in the flesh of salmon and trout. Natural astaxanthin is composed of a long conjugated double bond carbon chain and a six-membered ring of α-hydroxy ketone at both ends. Its unique structure makes it have stronger antioxidant activity than vitamin E and β-carotene, which can enhance the body's immunity, inhibit cell oxidative damage, and prevent ultraviolet radiation. Astaxanthin has been increasingly used in the fields of medicine, food, feed, health products, and cosmetics due to its strong antioxidant properties and coloring in muscle and egg yolk. It is one of the highest economic and application value carotenoids.

[0003] Commercially available astaxanthin is mainly derived from Haematococcus pluvialis, Xanthophyllomyces dendrorhous (the anamorphic form of Phaffia rhodozyma), and chemical synthesis. However, there are some limitations: the astaxanthin content in Haematococcus pluvialis is 1.5% to 4.0% dry weight, and the configuration is left-handed astaxanthin. However, the long light culture period and harsh growth conditions make astaxanthin expensive. The astaxanthin produced by Xanthophyllomyces dendrorhous is mostly right-handed, with weak antioxidant capacity. The astaxanthin content of wild strains of Phaffia rhodozyma is only 0.05% dry weight, and some mutant strains can reach 1.0% dry weight. Chemically synthesized astaxanthin is a mixed configuration astaxanthin with low antioxidant capacity and poor safety.

[0004] Paracoccus belongs to the phylum of Proteobacteria, the class of Alphaproteobacteria, the order of Rhodobacterales, the family of Rhodobacteraceae and the genus of Paracoccus, and is a non-pathogenic gram-negative bacterium. Some species of Paracoccus can produce carotenoids such as astaxanthin, beta-carotene, canthaxanthin and zeaxanthin, and the nutritional mode is chemotrophic, aerobic and does not require light. Paracoccus has the following advantages as one of the natural astaxanthin sources: strong stress resistance, easy cultivation, large-scale high-density fermentation culture using various carbon and nitrogen sources, fermentation production cycle of 3-10 days, thin cell wall, easy to break, easy to extract pigment, astaxanthin produced in left-handed configuration, strong antioxidant activity and high safety. Therefore, the use of Paracoccus for fermentation production of astaxanthin can greatly reduce the production cost of natural astaxanthin, and has important significance for the commercial application of natural astaxanthin in the future. However, there are few studies on the use of Paracoccus for fermentation production of astaxanthin, and the astaxanthin content and yield of wild-type Paracoccus are low, which is difficult to meet the requirements of commercial production. At present, a culture medium for producing carotenoids Paracoccus carotinifaciens is disclosed in the patent (EP2548968B1, Method of producing astaxanthin by fermentation) of ANCI company under ENEOS company, but it is easy to produce precipitate, which is not conducive to the growth of Paracoccus. Therefore, it is of great practical significance to seek a culture medium that can promote the growth of Paracoccus and improve the yield of astaxanthin produced by Paracoccus. SUMMARY

[0005] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a culture medium for improving the yield of astaxanthin produced by Paracoccus.

[0006] Another purpose of the present application is to provide the use of the culture medium for improving the yield of astaxanthin produced by Paracoccus.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A culture medium for improving the yield of astaxanthin produced by Paracoccus, including the following components in terms of concentration: yeast extract 4-20 g / L; beef extract 8-20 g / L; bacteriological peptone 4-20 g / L; glucose 10-30 g / L; KH2PO4 0.1-0.2 g / L; Na2HPO4·12H2O 0.1-0.5 g / L; MgSO4·7H2O 0.1-1 g / L; FeSO4·7H2O 0.1-1 g / L; CaCl2·2H2O 0.02-0.1 g / L; pH 7.2±0.2.

[0009] The culture medium for improving astaxanthin production of Paracoccus comprises the following components in the following concentrations: yeast extract 4-20 g / L; beef extract 8-20 g / L; bacteriological peptone 4-20 g / L; glucose 10-30 g / L; KH2PO4 0.188 g / L; Na2HPO4·12H2O 0.475 g / L; MgSO4·7H2O 0.7 g / L; FeSO4·7H2O 0.3 g / L; CaCl2·2H2O 0.05 g / L; and pH 7.2±0.2.

[0010] The Paracoccus comprises at least one of Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis, and preferably Paracoccus marcusii CGMCC 1.8602.

[0011] The total nitrogen source (the sum of yeast extract, beef extract and bacteriological peptone) in the culture medium has a concentration of 16-40 g / L, preferably 16-30 g / L, and more preferably 20 g / L.

[0012] The concentration of yeast extract in the culture medium is preferably 4-8 g / L, and more preferably 4 g / L.

[0013] The concentration of beef extract in the culture medium is preferably 8-16 g / L, and more preferably 8 g / L.

[0014] The concentration of bacteriological peptone in the culture medium is preferably 8-16 g / L, and more preferably 8 g / L.

[0015] The concentration of glucose in the culture medium is preferably 30 g / L.

[0016] The culture medium for improving astaxanthin production of Paracoccus is used for culturing Paracoccus and / or producing astaxanthin.

[0017] The culture medium can promote the proliferation of Paracoccus, and can be used for culturing Paracoccus, and can also promote the accumulation of astaxanthin by Paracoccus, and can be used for producing astaxanthin.

[0018] The Paracoccus includes at least one of Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis; preferably Paracoccus marcusii CGMCC 1.8602.

[0019] A culture method for improving astaxanthin production of Paracoccus, comprising the following steps:

[0020] S1, inoculating the activated Paracoccus into a seed culture medium and culturing to the logarithmic growth phase to obtain a Paracoccus seed solution;

[0021] S2, inoculating the Paracoccus seed solution into the culture medium for improving astaxanthin production of Paracoccus described above, and continuing to culture to promote the proliferation of Paracoccus and the accumulation of astaxanthin, thereby improving the content and / or yield of astaxanthin.

[0022] The Paracoccus described in step S1 includes at least one of Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis; preferably Paracoccus marcusii CGMCC 1.8602.

[0023] The activation condition described in step S1 is 25±1℃ for 1-3 days of activation culture; preferably 25℃ for 2 days of activation culture.

[0024] The seed culture medium described in step S1 is LB culture medium, and its formula is as follows: tryptone 10 g / L, yeast powder 5 g / L, glucose 1-5 g / L, NaCl 5 g / L, pH 7.2.

[0025] The culture temperature described in steps S1 and S2 is preferably 25±1℃.

[0026] The culture time described in step S1 is 1-3 days; preferably 2 days.

[0027] The culture described in step S1 is carried out on a constant temperature shaker, and the rotation speed is 100-160 rpm (preferably 160 rpm).

[0028] The inoculation amount of the Paracoccus seed solution described in step S2 is calculated according to 5-10% of the volume percentage in the culture system; preferably calculated according to 10% of the volume percentage in the culture system.

[0029] The concentration of the seed solution of the Paracoccus sp. in step S2 is 1 x 10 6 ~ 2 x 10 7 cells / mL; preferably 5 x 10 6 ~ 2 x 10 7 cells / mL; more preferably 1 x 10 7 cells / mL.

[0030] The culture time in step S2 is 7 ~ 14 days; preferably 7 ~ 12 days; more preferably 7 ~ 10 days.

[0031] The present application has the following advantages and effects relative to the prior art:

[0032] The present application optimizes the nitrogen source composition of the Paracoccus sp. culture medium, and finally selects a mixture of yeast extract, beef extract and bacteriological peptone as the nitrogen source for promoting the accumulation of astaxanthin by Paracoccus sp. Under the optimal mixed nitrogen source condition (orthogonal test group 1), the astaxanthin content and yield of Paracoccus sp. are significantly improved compared to the single nitrogen source condition. Under the optimal nitrogen source combination condition of the present application, the highest astaxanthin content is 0.70 ± 0.04 mg / g, which is increased by 59.09%, 48.94% and 70.73% respectively compared to the optimal concentration condition when yeast extract, beef extract and bacteriological peptone are used as single nitrogen source. The highest astaxanthin yield is 3.74 ± 0.17 mg / L, which is increased by 38.01%, 55.83% and 69.23% respectively compared to the optimal concentration condition when yeast extract, beef extract and bacteriological peptone are used as single nitrogen source. It is shown that the combination of yeast extract, beef extract and bacteriological peptone in the mixed nitrogen source of the present application can synergistically promote the biomass and astaxanthin accumulation of Paracoccus sp., and at the same time solve the problem of precipitation after sterilization of the culture medium. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a graph of the biomass yield, astaxanthin content and yield of Paracoccus sp. under different yeast extract concentrations in Example 2.

[0034] Figure 2 Figure 2 is a graph of the biomass yield, astaxanthin content and yield of Paracoccus sp. under different beef extract concentrations in Example 3.

[0035] Figure 3 Figure 3 is a graph of the biomass yield, astaxanthin content and yield of Paracoccus sp. under different bacteriological peptone concentrations in Example 4.

[0036] Figure 4 Figure 4 is a graph of the biomass yield, astaxanthin content and yield of Paracoccus sp. in orthogonal test groups 1-9 in Example 5.

[0037] Figure 5Figure for biomass yield, astaxanthin content and yield of the Paracoccus sp. of the 10th-18th groups of the orthogonal experiment groups in Example 5.

[0038] Figure 6 Figure for biomass yield, astaxanthin content and yield of the Paracoccus sp. of the 19th-27th groups of the orthogonal experiment groups in Example 5.

[0039] Figure 7 Figure for appearance comparison of the Paracoccus sp. culture medium prepared according to the present application and the Paracoccus sp. culture medium prepared according to other patent formula; wherein, A is the Paracoccus sp. culture medium prepared according to other patent formula (precipitate is generated after sterilization); B is the Paracoccus sp. culture medium prepared according to the present application. DETAILED DESCRIPTION

[0040] The present application will be further described in conjunction with the following examples, but the embodiments of the present application are not limited thereto. Unless otherwise specified, the reagents, methods and devices used in the present application are the conventional reagents, methods and devices in the technical field. The test methods in the following examples, unless otherwise specified, are usually carried out according to the conventional experimental conditions or according to the experimental conditions suggested by the manufacturers. Unless otherwise specified, the reagents and raw materials used in the present application are commercially available.

[0041] The detection methods adopted in the embodiments of the present application can refer to the following descriptions:

[0042] (I) Determination of biomass yield

[0043] The dry weight method is used to determine the biomass yield. The empty tube weight is recorded as W1. After the cultivation is completed, the culture solution is placed in the tube and centrifuged at 8000 r / min for 10 min. The sludge is repeatedly washed and centrifuged twice with distilled water. The total weight W2 of the sludge after freeze-drying is determined and recorded. Three parallel samples are set for each sample, and the average value is taken and the standard deviation is calculated. The biomass yield is calculated according to the following formula:

[0044]

[0045] In the formula, W1 is the empty tube weight, unit: g; W2 is the total weight of the dried centrifuge tube and Paracoccus sp. biomass, unit: g; V is the sample volume, unit: L.

[0046] (II) Determination of astaxanthin content

[0047] The high performance liquid chromatography method is used to determine astaxanthin. The specific steps are as follows:

[0048] (1) Standard curve preparation: 10 mg of astaxanthin standard was accurately weighed and dissolved in 10 mL of a constant volume solvent (acetone containing 0.1% (w / v) 2,6-di-tert-butyl-p-cresol (BHT)) to prepare a standard solution. The standard solution was diluted to different concentration gradients with the constant volume solvent, and filtered with a 0.22 μm organic filter membrane into a liquid phase vial for testing. The HPLC system used a Waters dual 1525 pump, a Waters 2996 diode array (PDA) detector, and a YMC carotenoid column C30 column (4.6 x 150 mm, 3 μm). The mobile phases were methanol (A), methyl tert-butyl ether (MTBE) (B), and 1% (v / v) phosphoric acid solution (C), respectively. The gradient elution program was as follows: 0-15 min, 81%→66% A, 15%→30% B, 4% C; 15-23 min, 66%→16% A, 30%→80% B, 4% C; 23-27 min, 16%→16% A, 80%→80% B, 4% C. The flow rate was 0.8 mL / min, the injection volume was 20 μL, the detection wavelength was 474 nm, and the column temperature was 25°C. The standard curve of astaxanthin concentration (mg / L, y) versus peak area (x) was plotted.

[0049] (2) Determination of astaxanthin content in the Propionibacterium: 10 mg of freeze-dried bacterial powder was accurately weighed and placed in a cryogenic tube containing ceramic beads, and an extraction solvent (acetone containing 0.1% (w / v) 2,6-di-tert-butyl-p-cresol (BHT)) was added. After cell disruption in a grinder, the extract was collected by centrifugation, and repeated extraction was performed until the bacterial body became colorless. The extracts were combined and nitrogen-dried, and then dissolved in 1 mL of acetone. A 0.22 μm nylon needle filter was used to filter and transfer the solution to a brown sample vial for high-performance liquid chromatography analysis, as described in step (1) above. Qualitative and quantitative analysis of astaxanthin in the sample was performed according to the retention time of astaxanthin and the standard curve.

[0050] (III) Calculation of astaxanthin yield

[0051] Astaxanthin yield (mg / L) = biomass yield (g / L) x astaxanthin content (mg / g).

[0052] In the examples of the present application, Origin 9.0 and SPSS software were used to process the data, and single-factor variance analysis and paired data t-test were used for significance test in statistical analysis. Different letters indicate significant differences between groups (P < 0.05).

[0053] Example 1 Effect of different yeast extract concentrations on biomass yield, astaxanthin content, and yield of Propionibacterium

[0054] 1.1 Strain activation and seed liquid preparation

[0055] A loop of Paracoccus marcusii CGMCC 1.8602 (purchased from China General Microbiological Culture Collection Center) was picked from the LB slope and streaked on the LB plate medium, and then incubated at 25°C for 2 days in an incubator. A single colony was inoculated into 20 mL of LB liquid medium, and then incubated at 25°C and 160 r / min for 2 days in a constant-temperature shaker to obtain a seed liquid. The LB medium contains the following components: 10 g / L of tryptone, 5 g / L of yeast powder, 1-5 g / L of glucose, 5 g / L of NaCl, and pH 7.2.

[0056] 1.2 Preparation of Paracoccus marcusii culture medium with different concentrations of yeast extract

[0057] The Paracoccus marcusii culture medium was prepared according to the formula in Table 1, and 4, 8, 12, 16, and 20 g / L of yeast extract (yeast extract powder, purchased from Guangdong Huanke Microbial Co., Ltd., item number 050090) were added as a nitrogen source. The components in Table 1 were divided into five groups, sterilized (121°C, 15 min), and then mixed, and the pH was controlled at about 7.2.

[0058] Table 1 Formula of Paracoccus marcusii culture medium

[0059]

[0060] 1.3 Fermentation culture of Paracoccus marcusii

[0061] The seed liquid (1×10 7 cells / mL) grown to the logarithmic growth phase was inoculated into the prepared Paracoccus marcusii culture medium (250 mL in a shake flask) at 10% (v / v), and three parallel samples were prepared for each gradient. The culture conditions were 25°C and 160 r / min, and the bacterial liquid was collected after 7 days of culture. The bacterial slurry was washed and collected, and the bacterial slurry was vacuum freeze-dried to obtain bacterial powder. The biomass yield was determined. The bacterial powder was stored in a refrigerator at -20°C for subsequent analysis of astaxanthin content and calculation of yield.

[0062] 1.4 Results of energy production analysis

[0063] The effects of different concentrations of yeast extract added to the culture medium on the biomass yield, astaxanthin content, and yield of Paracoccus marcusii are shown in Table 2. Figure 1The results showed that with the increase of yeast extract concentration, the biomass yield, astaxanthin content and yield of Paracoccus first increased and then decreased. Among them, the astaxanthin content and yield reached peak values ​​at 8 g / L, which were 0.44±0.02 mg / g and 2.71±0.21 mg / L, respectively, which were significantly higher than those under other concentration conditions (P<0.05). The biomass yield reached its peak at 12 g / L (6.33±0.35 g / L), but there was no significant difference from the 8 g / L condition (6.12±0.19 mg / L). The biomass yield of both groups was significantly higher than that of the other groups (P<0.05).

[0064] Example 2 Effects of different beef extract concentrations on biomass yield, astaxanthin content and yield of Paracoccus

[0065] 2.1 Activation of bacterial strains and preparation of seed solution

[0066] The method is the same as 1.1.

[0067] 2.2 Preparation of Paracoccus beef extract culture medium with different concentrations

[0068] Paracoccus culture medium was prepared according to the formula in Table 1, and beef extract (beef extract, purchased from Guangdong Huankai Microbiology Co., Ltd., product number 050060) was added at final concentrations of 8, 12, 16, and 20 g / L, respectively. The culture medium was sterilized in groups (121°C, 15 min) and then mixed, and the pH was maintained at around 7.2.

[0069] 2.3 Fermentation culture of Paracoccus

[0070] The method is the same as 1.3.

[0071] 2.4 Capacity Analysis Results

[0072] Effects of adding different concentrations of beef extract to the culture medium on the biomass yield, astaxanthin content and yield of Paracoccus Figure 2 As shown. The results showed that with the increase in beef extract concentration, the biomass yield, astaxanthin content, and production of Paracoccus showed a trend of first increasing and then decreasing. Among them, the biomass yield reached a peak (6.31±0.09g / L) at 16g / L, which was significantly higher than that of the other groups (P<0.05). The astaxanthin content at 12g / L and 16g / L was 0.47±0.05mg / g and 0.41±0.02mg / g, respectively, and the astaxanthin production was 2.40±0.22mg / L and 2.61±0.07mg / L, respectively, which were significantly higher than those of the other concentration conditions (P<0.05).

[0073] Example 3 Effects of different bacteriological peptone concentrations on biomass yield, astaxanthin content and yield of Paracoccus

[0074] 3.1 Activation of bacterial strains and preparation of seed solution

[0075] Method same as 1.1.

[0076] 3.2 Preparation of Paracoccus culture medium with different concentrations of bacteriological peptone

[0077] Paracoccus culture medium was prepared according to the formula in Table 1, and bacteriological peptone (purchased from Guangdong Huan Kai Microbial Co., Ltd., item number 050170) was added at a final concentration of 4, 8, 12, 16, and 20 g / L, respectively. After sterilization (121°C, 15 min) and mixing, the pH was maintained at about 7.2.

[0078] 3.3 Fermentation culture of Paracoccus

[0079] Method same as 1.3.

[0080] 3.4 Results of energy production analysis

[0081] The effects of adding different concentrations of bacteriological peptone to the culture medium on the biomass yield, astaxanthin content, and yield of Paracoccus are shown in Table 3. Figure 3 The results showed that with the increase of the concentration of bacteriological peptone, the biomass yield, astaxanthin content, and yield of Paracoccus showed a trend of first increasing and then decreasing. The biomass yield, astaxanthin content, and yield reached a peak at 16 g / L, which were 5.42 ± 0.24 g / L, 0.41 ± 0.04 mg / g, and 2.21 ± 0.12 g / L, respectively. At 12-20 g / L, there was no significant difference between the indicators; at 4 g / L, each index was significantly lower than the rest.

[0082] Example 4 Effects of different combinations of mixed nitrogen sources on the biomass yield, astaxanthin content, and yield of Paracoccus

[0083] 4.1 Activation of strains and preparation of seed solution

[0084] Method same as 1.1.

[0085] 4.2 Preparation of Paracoccus culture medium with different combinations of nitrogen sources

[0086] A three-factor three-level orthogonal test was designed using L 27 (3 13 ) orthogonal test table (Table 2). The levels of each factor were selected based on the test results of Examples 1, 2, and 3: the level of yeast extract (X factor) was selected between 4 g / L and 12 g / L, and the levels of beef extract (Y factor) and bacteriological peptone (Z factor) were selected in the growth segment on the left side of the peak, i.e., 8 g / L-16 g / L. The step size was 4 g / L. The culture medium was prepared according to the formula in Table 1, sterilized (121°C, 15 min) and mixed, and the pH was maintained at about 7.2.

[0087] Table 2 L 27(3 13 )orthogonal experimental design

[0088]

[0089]

[0090] 4.3 Fermentation of Paracoccus

[0091] The method is the same as 1.3.

[0092] 4.4 Results of energy production analysis

[0093] L 27 (3 13 )orthogonal experimental results are shown in Table 3, and the range analysis table is shown in Table 4. The effects of each medium on the biomass of Paracoccus, astaxanthin content and yield are shown in Table 4. Figures 4-6

[0094] The results of variance analysis of biomass yield are shown in Table 5, and yeast extract and beef extract have a very significant effect on the biomass yield of Paracoccus (P<0.01), while the interaction between nitrogen sources has no significant effect on astaxanthin yield (P>0.05). According to the k value in Table 4, the optimal level of the three factors is X1Y1Z1. The primary and secondary degrees of the effects of each factor on biomass yield are X>Y>Z>(X×Y)>(Y×Z)>(X×Z).

[0095] The results of variance analysis of astaxanthin content are shown in Table 6, and yeast extract, beef extract and bacteriological peptone have a very significant effect on the astaxanthin content of Paracoccus (P<0.01), there is an interaction between yeast extract and bacteriological peptone and it has a significant effect on astaxanthin yield (P<0.05), while the interaction between other nitrogen sources has no significant effect on astaxanthin content (P>0.05). According to the k value in Table 4, the optimal level of the three factors is X1Y1Z1. The primary and secondary degrees of the effects of each factor on astaxanthin content are Z>X>Y>(Y×Z)>(X×Z)>(X×Y).

[0096] The results of variance analysis of astaxanthin yield are shown in Table 7, and yeast extract, beef extract and bacteriological peptone have a very significant effect on the astaxanthin yield of Paracoccus (P<0.01), there is an interaction between yeast extract and bacteriological peptone and it has a significant effect on astaxanthin yield (P<0.05), while the interaction between other nitrogen sources has no significant effect on astaxanthin yield (P>0.05). According to the k value in Table 4, the optimal level of the three factors is X1Y1Z1. The primary and secondary degrees of the effects of each factor on astaxanthin yield are X>Z>Y>(X×Z)>(X×Y)>(Y×Z).

[0097] ​Therefore, the optimal combination of nitrogen sources for improving the biomass of Paracoccus, the astaxanthin content and yield is 4 g / L yeast extract, 8 g / L beef extract and 8 g / L bacteriological peptone. Under this condition, the Paracoccus culture has a biomass yield of 5.35±0.33 g / L, an astaxanthin content of 0.70±0.04 mg / g and a yield of 3.74±0.17 mg / L.

[0098] Table 3L 27 (3 13 ) Orthogonal test results

[0099]

[0100]

[0101] Table 4L 27 (3 13 ) Orthogonal test range analysis table

[0102]

[0103] Table 5 Biomass yield variance analysis table

[0104]

[0105]

[0106] Table 6 Astaxanthin content variance analysis table

[0107] Factor Degrees of Freedom Mean Square Deviation Mean Square F Value P Value Significance X 2 0.14 0.07 32.27 0.00 ** Y 2 0.05 0.03 12.76 0.00 ** (X x Y)1 2 0.01 0.00 1.37 0.31 (X x Y)2 2 0.01 0.00 1.51 0.28 Z 2 0.19 0.09 44.35 0.00 ** (X x Z)1 2 0.02 0.01 3.98 0.06 * (X x Z)2 2 0.01 0.01 2.99 0.11 (Y x Z)1 2 0.02 0.01 3.74 0.07 [(Y x Z)2] 2 0.00 0.00 0.42 0.67 Error 8 0.02 0.00 Total 26 0.45

[0108] Table 7 Astaxanthin yield variance analysis table

[0109] Factor Degrees of Freedom Mean Square Deviation Mean Square F Value P Value Significance X 2 7.45 3.72 26.76 0.00 ** Y 2 2.99 1.50 10.75 0.01 ** (X x Y)1 2 0.60 0.30 2.16 0.18 (X x Y)2 2 0.18 0.09 0.63 0.56 Z 2 4.85 2.43 17.44 0.00 ** (X x Z)1 2 1.44 0.72 5.18 0.04 * (X x Z)2 2 0.39 0.19 1.40 0.30 (Y x Z)1 2 0.19 0.10 0.68 0.53 (Y x Z)2 2 0.10 0.05 0.37 0.70 Error 8 1.11 0.14 Total 26 19.30

[0110] Comparative Example 1 Differences between the Paracoccus culture medium of the present application and other Paracoccus culture media

[0111] According to the application patent (application number EP11756284.3, publication number EP2548968B1, name "Method of producing astaxanthin by fermentation"), the medium is prepared according to the formula in Table 8, the pH is adjusted to about 7.2, and the high-temperature high-pressure sterilization is performed at 121°C for 15 min. After sterilization, the medium is as shown in Table 8: Figure 7 A: a large amount of precipitate appears in the medium, mainly due to the formation of flocculent precipitate in large pieces after sterilization of corn syrup, as well as the formation of slightly soluble / insoluble phosphate by iron salt, calcium salt and phosphate. The precipitate in the medium is not conducive to the growth of Paracoccus. According to the optimal combination of nitrogen sources of Example 4 of the present application and the formula in Table 1, the prepared medium is as shown in Table 1: Figure 7B. As can be seen, the culture medium of the present application does not have precipitate and can promote the growth of D. hansenii.

[0112] Table 8. D. hansenii culture medium formula

[0113]

[0114]

[0115] Example 2. Effect of various nitrogen sources on D. hansenii biomass yield, astaxanthin content and yield

[0116] 1. Strain activation and seed liquid preparation

[0117] Method same as 1.1.

[0118] 2. Preparation of culture medium

[0119] The experiment was divided into 4 groups, and 4 D. hansenii culture media were prepared according to the formula in Table 1 and Table 9. In the single nitrogen source group (experimental group 1), corn syrup (product of Aolind Biochemical Technology Co., Ltd., item number C116026-100g) was used as the nitrogen source. In the mixed nitrogen source group (experimental groups 2-4), yeast extract was used as the fixed nitrogen source (the concentration ratio of yeast extract to other nitrogen sources was controlled at 1:2), and was respectively compounded with soybean peptone (Guangdong Huan Kai Microbial Co., Ltd., item number 050220), bacteriological peptone and beef extract. After sterilization (121°C, 15 min) according to the grouping mode in Table 1, they were mixed, and the pH was maintained at about 7.2.

[0120] Table 9. Nitrogen source composition table

[0121]

[0122] 3. Fermentation culture of D. hansenii

[0123] Method same as 1.3.

[0124] 4. Energy production analysis results

[0125] As shown in Table 10, the culture medium with corn steep liquor as nitrogen source has the least promoting effect on the production performance of Paracoccus, and each index is significantly lower than that of the other groups. The biomass yield (4.89 ± 0.03 g / L) of the beef extract compound group and the astaxanthin content (0.68 ± 0.03 mg / g) and output (2.89 ± 0.11 mg / L) of the bacteriological peptone compound group are significantly higher than those of the other groups, and each index of the soy peptone compound group is significantly lower than that of the other compound groups. Compared to soy peptone, beef extract and bacteriological peptone are more conducive to the growth and accumulation of astaxanthin of Paracoccus, but are lower than the combination of yeast extract, beef extract and bacteriological peptone in Example 4 of the present invention. It is shown that yeast extract, beef extract and bacteriological peptone can synergistically promote the biomass and astaxanthin accumulation of Paracoccus.

[0126] Table 1 Effects of various nitrogen sources on biomass yield, astaxanthin content and yield of Paracoccus

[0127]

[0128]

[0129] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A culture medium for increasing the astaxanthin production of Paracoccus sp., characterized in that: Calculated by concentration, it includes the following components: yeast extract 4-20 g / L; beef extract 8-20 g / L; bacteriological peptone 4-20 g / L; glucose 10-30 g / L; KH2PO4 0.1-0.2 g / L; Na2HPO4·12H2O 0.1-0.5 g / L; MgSO4·7H2O 0.1-1 g / L; FeSO4·7H2O 0.1-1 g / L; CaCl2·2H2O 0.02-0.1 g / L; pH 7.2±0.2; The Paracoccus is Paracoccus martensii ( Paracoccus marcusii )CGMCC 1.8602.

2. The culture medium for improving astaxanthin production of Paracoccus astaxanthin according to claim 1, wherein Calculated by concentration, it includes the following components: yeast extract 4-20 g / L; beef extract 8-20 g / L; bacteriological peptone 4-20 g / L; glucose 10-30 g / L; KH2PO4 0.188 g / L; Na2HPO4·12H2O 0.475 g / L; MgSO4·7H2O 0.7 g / L; FeSO4·7H2O 0.3 g / L; CaCl2·2H2O 0.05 g / L; pH 7.2±0.

2.

3. The culture medium for improving astaxanthin production of Paracoccus according to claim 1 or 2, characterized in that: The total nitrogen source concentration in the culture medium is 16 to 40 g / L; The concentration of yeast extract in the culture medium is 4 to 8 g / L; The concentration of beef extract in the culture medium is 8 to 16 g / L; The concentration of bacteriological peptone in the culture medium is 8-16 g / L.

4. The culture medium for improving astaxanthin production in Paracoccus sp. according to claim 3, wherein: The concentration of yeast extract in the culture medium is 4 g / L; The concentration of beef extract in the culture medium is 8 g / L; The concentration of bacteriological peptone in the culture medium is 8 g / L; The concentration of glucose in the culture medium is 30 g / L.

5. Use of the culture medium for improving the astaxanthin production of Paracoccus according to any one of claims 1 to 4 in culturing Paracoccus and / or producing astaxanthin, characterized in that: The Paracoccus is Paracoccus martensii ( Paracoccus marcusii )CGMCC 1.8602.

6. A method for increasing the astaxanthin production of Paracoccus sp., characterized in that: The steps include: S1. inoculating the activated Paracoccus into a seed culture medium and culturing the culture medium until the logarithmic growth phase to obtain a Paracoccus seed solution; S2. inoculating the Paracoccus seed liquid into the culture medium for improving the astaxanthin production of Paracoccus according to any one of claims 1 to 4, and continuing to culture to promote the proliferation of Paracoccus and the accumulation of astaxanthin, thereby increasing the astaxanthin content and / or production; The Paracoccus is Paracoccus martensii ( Paracoccus marcusii )CGMCC 1.8602.

7. The method for increasing the astaxanthin production of Paracoccus sp. according to claim 6, wherein: The activation conditions described in step S1 are: activation culture at 25 ± 1°C for 1 to 3 days; The seed culture medium described in step S1 is LB culture medium; The temperature of the incubation described in steps S1 and S2 was 25 ± 1 °C; The inoculation amount of the Paracoccus seed solution in step S2 is calculated based on 5-10% of its volume percentage in the culture system; The culture time described in step S2 is 7 to 14 days.

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