Culture medium for increasing yield of astaxanthin in paracoccus and application of culture medium

Abstract

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

Description

Technical Field

[0001] The invention relates to the field of producing astaxanthin by fermentation, and in particular to a culture medium for improving the astaxanthin yield of Paracoccus and application thereof. Background Art

[0002] Astaxanthin is chemically named 3,3'-dihydroxy-β,β'-carotene-4,4'-diketone and its chemical formula is C 40 H 52 O4, usually found in various marine bacteria, microalgae and crustacean shells and salmon and trout meat. Natural astaxanthin is composed of a long conjugated double bond carbon chain and a six-membered ring of α-hydroxyketone at both ends. Its unique structure makes it have stronger antioxidant activity than vitamin E and β-carotene. It can enhance the body's immunity, inhibit oxidative damage to cells, and protect against ultraviolet radiation. Due to its strong antioxidant properties and coloring in muscles and egg yolks, astaxanthin is increasingly used in the fields of medicine, food, feed, health products and cosmetics. It is currently one of the carotenoids with the highest economic value and application value.

[0003] Commercially available astaxanthin is mainly derived from Haematococcus pluvialis, Xanthophyllomyces dendrorhous (the asexual form of Phaffia rhodozyma) and chemical synthesis, and has the following limitations: the astaxanthin content in Haematococcus pluvialis is 1.5% to 4.0% dry weight, and the configuration is L-astaxanthin, but its light culture cycle is long and the growth conditions are harsh, making astaxanthin expensive; the astaxanthin in marine red yeast is mostly D-astaxanthin, which has 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 mixed configuration astaxanthin, which has low antioxidant capacity and poor safety.

[0004] Paracoccus belongs to the Proteobacteria, α-Proteobacteria, Rhodobacterales, Rhodobacteraceae, Paracoccus, and is a non-pathogenic Gram-negative bacterium. Some species of this genus can produce carotenoids, such as astaxanthin, β-carotene, canthaxanthin and zeaxanthin, and their nutrition mode is chemoheterotrophic, aerobic and does not require light. As one of the sources of natural astaxanthin, Paracoccus has the following advantages: strong stress resistance, easy cultivation, large-scale high-density fermentation culture using a variety of carbon and nitrogen sources, and a fermentation production cycle of 3 to 10 days; thin cell wall, easy to break, easy to extract pigments; the produced astaxanthin is left-handed, with strong antioxidant activity and high safety. Therefore, the production of astaxanthin by fermentation of Paracoccus can greatly reduce the production cost of natural astaxanthin, which is of great significance to the future commercial application of natural astaxanthin. However, there are few studies on the production of astaxanthin by fermentation of Paracoccus, and the astaxanthin content and yield of wild-type Paracoccus are low, which is difficult to meet the requirements of commercial production. At present, the patent (EP2548968B1, Method of producing astaxanthin by fermentation) of ANCI, a subsidiary of ENEOS, discloses a culture medium for producing carotenoids Paracoccus carotinifaciens, but it is easy to produce precipitation, 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 production of astaxanthin by Paracoccus. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a culture medium for increasing the astaxanthin production of Paracoccus.

[0006] Another object of the present invention is to provide application of the culture medium for improving the astaxanthin production of Paracoccus.

[0007] The purpose of the present invention is achieved through the following technical solutions:

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

[0009] The culture medium for improving the astaxanthin production of Paracoccus preferably includes the following components, calculated by 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 0.188 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.

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

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

[0012] The concentration of the 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 the astaxanthin production of Paracoccus is used in culturing Paracoccus and / or producing astaxanthin.

[0017] The culture medium can promote the proliferation of Paracoccus, can be used to cultivate Paracoccus, and can also promote the accumulation of astaxanthin by Paracoccus, and is used to produce astaxanthin.

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

[0019] A method for increasing the production of astaxanthin in Paracoccus sp. comprises the following steps:

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

[0021] S2. Inoculate the Paracoccus seed liquid into the above-mentioned culture medium for improving the astaxanthin production of Paracoccus, and continue culturing to promote the proliferation of Paracoccus and the accumulation of astaxanthin, thereby increasing the astaxanthin content and / or production.

[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 conditions in step S1 are: activation culture at 25±1°C for 1 to 3 days; preferably: activation culture at 25°C for 2 days.

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

[0025] The temperature of the culture described in steps S1 and S2 is preferably 25±1°C.

[0026] The culture time in step S1 is 1 to 3 days, preferably 2 days.

[0027] The culture in step S1 is carried out on a constant temperature shaker at a rotation speed of 100 to 160 rpm (preferably 160 rpm).

[0028] 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; preferably, it is calculated based on 10% of its volume percentage in the culture system.

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

[0030] The culture time in step S2 is 7 to 14 days, preferably 7 to 12 days, and more preferably 7 to 10 days.

[0031] Compared with the prior art, the present invention has the following advantages and effects:

[0032] The present invention optimizes the nitrogen source composition of the Paracoccus culture medium, and finally selects a mixture of yeast extract, beef extract and bacteriological peptone as a nitrogen source to promote the accumulation of astaxanthin by Paracoccus. The astaxanthin content and yield of the Paracoccus obtained under the optimal mixed nitrogen source condition (orthogonal test group 1) are significantly improved compared with the single nitrogen source condition. Under the optimal nitrogen source combination conditions of the present invention, the maximum astaxanthin content is 0.70±0.04mg / g, which is 59.09%, 48.94% and 70.73% higher than the optimal concentration conditions when yeast extract, beef extract and bacteriological peptone are used as a single nitrogen source, respectively; the maximum astaxanthin yield is 3.74±0.17mg / L, which is 38.01%, 55.83% and 69.23% higher than the optimal concentration conditions when yeast extract, beef extract and bacteriological peptone are used as a single nitrogen source, respectively. This indicates that the combination of yeast extract, beef extract and bacteriological peptone in the mixed nitrogen source of the present invention can synergistically promote the accumulation of Paracoccus biomass and astaxanthin, while solving the problem of precipitation after sterilization of the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0037] Figure 5This is a graph showing the biomass yield, astaxanthin content and yield of Paracoccus in groups 10-18 of the orthogonal experimental group in Example 5.

[0038] Figure 6 This is a graph showing the biomass yield, astaxanthin content and yield of Paracoccus in groups 19-27 of the orthogonal experimental group in Example 5.

[0039] Figure 7 This is a comparison diagram of the appearance of the Paracoccus culture medium prepared by the present invention and the Paracoccus culture medium prepared by other patented formulas in Comparative Example 1; wherein A is the Paracoccus culture medium prepared by other patented formulas (precipitate is produced after sterilization); and B is the Paracoccus culture medium prepared by the present invention. DETAILED DESCRIPTION

[0040] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0041] The detection method adopted in the embodiment of the present invention can refer to the following description:

[0042] (I) Determination of biomass yield

[0043] The biomass yield was determined by the dry weight method. The weight of the empty tube was recorded as W1. After the culture was completed, the culture solution was placed in the tube and centrifuged at 8000r / min for 10min. The bacterial sludge was repeatedly washed with distilled water and centrifuged twice. After the bacterial sludge was freeze-dried, the total weight W2 was measured and recorded. Three parallels were set for each sample, the average value was taken and the standard deviation was calculated, and the biomass yield was calculated according to the following formula:

[0044]

[0045] Where: W1 is the weight of the empty tube, in g; W2 is the total weight of the centrifuge tube and Paracoccus biomass after drying, in g; V is the sample volume, in L.

[0046] (II) Determination of astaxanthin content

[0047] Astaxanthin was determined by high performance liquid chromatography. The specific steps are as follows:

[0048] (1) Preparation of standard curve: Accurately weigh 10 mg of astaxanthin standard and dissolve it in 10 mL of constant volume (acetone, containing 0.1% (w / v) 2,6-di-tert-butyl-p-cresol (BHT)) to prepare a standard solution. Then dilute it with constant volume to different concentration gradients, filter it with a 0.22 μm organic filter membrane into a liquid phase vial and test it on the machine. The HPLC system uses Waters dual 1525 pumps, Waters 2996 diode array (PDA) detector, and YMC carotenoid column C30 column (4.6×150 mm, 3 μm). The mobile phases are methanol (A), methyl tert-butyl ether (MTBE) (B) and 1% (v / v) phosphoric acid solution (C). 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. A standard curve of astaxanthin concentration (mg / L, y) versus peak area (x) was drawn.

[0049] (2) Determination of astaxanthin content in Paracoccus: Accurately weigh 10 mg of freeze-dried bacterial powder and place it in a cryovial containing ceramic beads. Add an extractant (acetone containing 0.1% (w / v) 2,6-di-tert-butyl-p-cresol (BHT)). After cell disruption in a grinder, collect the extract by centrifugation. Repeat the extraction operation until the bacteria become colorless. Combine the extracts and blow dry with nitrogen. Make the volume to 1 mL with acetone. Filter with a 0.22 μm nylon needle filter and transfer to a brown injection bottle for high performance liquid chromatography analysis. The method is the same as in step (1). Qualitative and quantitative analysis of astaxanthin in the sample is performed based on the astaxanthin retention time and the standard curve.

[0050] (III) Calculation of astaxanthin production

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

[0052] In the examples of the present invention, Origin 9.0 and SPSS software were used to process the data, and the statistical analysis was performed using one-way analysis of variance and paired data t-test for significance test. Different letters represent significant differences between groups (P<0.05).

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

[0054] 1.1 Activation of bacterial strains and preparation of seed solution

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

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

[0057] The Paracoccus culture medium was prepared according to the formula in Table 1, and yeast extract (yeast extract powder, purchased from Guangdong Huankai Microbiology Co., Ltd., product number 050090) with a final concentration of 4, 8, 12, 16, and 20 g / L was added as a nitrogen source, respectively. The components in Table 1 were divided into 5 groups, and the groups were sterilized (121° C., 15 min) and then mixed, and the pH was controlled at about 7.2.

[0058] Table 1 Paracoccus culture medium formula

[0059]

[0060] 1.3 Fermentation culture of Paracoccus

[0061] The seed solution (1×10 7 cells / mL) was inoculated into the prepared Paracoccus culture medium (shake flask volume of 250mL) at 10% (v / v), and each gradient was replicated three times. The culture conditions were temperature 25°C, rotation speed 160r / min, and the bacterial liquid was collected after 7 days of culture, and the bacterial mud was washed and collected, and the bacterial mud was vacuum freeze-dried to obtain bacterial powder, and the biomass yield was determined. The bacterial powder was stored in a -20°C refrigerator for subsequent analysis to detect astaxanthin content and calculate the yield.

[0062] 1.4 Capacity Analysis Results

[0063] Effects of adding different concentrations of yeast extract to the culture medium on biomass yield, astaxanthin content and yield of Paracoccus 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. The astaxanthin content and yield peaked at 8g / L, which were 0.44±0.02mg / g and 2.71±0.21mg / L, respectively, which were significantly higher than those under other concentration conditions (P<0.05). The biomass yield reached a peak at 12g / L (6.33±0.35g / L), but there was no significant difference with that under 8g / L (6.12±0.19mg / 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 different concentrations of beef extract Paracoccus culture medium

[0068] According to the formula in Table 1, Paracoccus culture medium was prepared, and beef extract (beef extract, purchased from Guangdong Huankai Microbiology Co., Ltd., product number 050060) with a final concentration of 8, 12, 16, and 20 g / L was added respectively, and the culture medium was sterilized in groups (121°C, 15 min) and then mixed, and the pH was maintained at about 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 of beef extract concentration, the biomass yield, astaxanthin content and yield of Paracoccus showed a trend of first increasing and then decreasing. Among them, the biomass yield reached a peak value (6.31±0.09g / L) under the condition of 16g / L, which was significantly higher than that of the other groups (P<0.05). The astaxanthin content was 0.47±0.05mg / g and 0.41±0.02mg / g under the conditions of 12g / L and 16g / L, respectively, and the astaxanthin yield 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] The method is the same as 1.1.

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

[0077] The Paracoccus culture medium was prepared according to the formula in Table 1, and bacteriological peptone (purchased from Guangdong Huankai Microbiology Co., Ltd., product number 050170) with a final concentration of 4, 8, 12, 16, and 20 g / L was added respectively, sterilized in groups (121° C., 15 min) and then mixed, and the pH was maintained at about 7.2.

[0078] 3.3 Fermentation culture of Paracoccus

[0079] The method is the same as 1.3.

[0080] 3.4 Capacity analysis results

[0081] Effects of adding different concentrations of bacteriological peptone to the culture medium on biomass yield, astaxanthin content and yield of Paracoccus Figure 3 As shown. The results showed that with the increase of bacteriological peptone concentration, the biomass yield, astaxanthin content and yield of Paracoccus first increased and then decreased. The biomass yield, astaxanthin content and yield reached their peak values ​​at 16g / L, which were 5.42±0.24g / L, 0.41±0.04mg / g and 2.21±0.12g / L, respectively. Under the conditions of 12-20g / L, there were no significant differences among the indicators; under the condition of 4g / L, the indicators were significantly lower than those of the other groups.

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

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

[0084] The method is the same as 1.1.

[0085] 4.2 Preparation of different nitrogen source combination culture media for Paracoccus

[0086] Using L 27 (3 13 ) Orthogonal test table Design a three-factor three-level orthogonal test (Table 2), select the level of each factor based on the test results of Examples 1, 2, and 3: the level of yeast extract (X factor) is selected between 4g / L and 12g / L, and the level of beef extract (Y factor) and bacteriological peptone (Z factor) is selected in the growth section 8g / L to 16g / L on the left side of the peak. The step length is 4g / L. Prepare according to the formula in Table 1, sterilize in groups (121°C, 15min) and then mix, and keep the pH at about 7.2.

[0087] Table 2 L 27(3 13 ) Orthogonal Experimental Design

[0088]

[0089]

[0090] 4.3 Fermentation culture of Paracoccus

[0091] The method is the same as 1.3.

[0092] 4.4 Capacity analysis results

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

[0094] The results of variance analysis of biomass yield are shown in Table 5. Yeast extract and beef extract have extremely significant effects 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 can be obtained as X1Y1Z1. The primary and secondary degrees of influence 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. Yeast extract, beef extract and bacteriological peptone have extremely significant effects on the astaxanthin content of Paracoccus (P < 0.01). There is an interaction between yeast extract and bacteriological peptone and has a significant effect on astaxanthin production (P < 0.05). 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 can be obtained as X1Y1Z1. The primary and secondary degrees of influence of each factor on astaxanthin content are Z>X>Y>(Y×Z)>(X×Z)>(X×Y).

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

[0097] Therefore, the optimal combination of nitrogen sources that is beneficial to improving the biomass, astaxanthin content and yield of Paracoccus is 4g / L yeast extract, 8g / L beef extract and 8g / L bacteriological peptone. Under this condition, Paracoccus was cultured with a biomass yield of 5.35±0.33g / L, an astaxanthin content of 0.70±0.04mg / g and a yield of 3.74±0.17mg / 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 Analysis of variance of biomass yield

[0104]

[0105]

[0106] Table 6 Analysis of variance of astaxanthin content

[0107] factor Degrees of Freedom Sum of squared deviations from the mean Mean Square F-number P-value Significance X 2 0.14 0.07 32.27 0.00 ** Y 2 0.05 0.03 12.76 0.00 ** <![CDATA[(X×Y)1]]> 2 0.01 0.00 1.37 0.31 <![CDATA[(X×Y)2]]> 2 0.01 0.00 1.51 0.28 Z 2 0.19 0.09 44.35 0.00 ** <![CDATA[(X×Z)1]]> 2 0.02 0.01 3.98 0.06 * <![CDATA[(X×Z)2]]> 2 0.01 0.01 2.99 0.11 <![CDATA[(Y×Z)1]]> 2 0.02 0.01 3.74 0.07 <![CDATA[(Y×Z)2]]> 2 0.00 0.00 0.42 0.67 error 8 0.02 0.00 total 26 0.45

[0108] Table 7 Analysis of variance of astaxanthin production

[0109] factor Degrees of Freedom Sum of squared deviations from the mean Mean Square F-number P-value Significance X 2 7.45 3.72 26.76 0.00 ** Y 2 2.99 1.50 10.75 0.01 ** <![CDATA[(X×Y)1]]> 2 0.60 0.30 2.16 0.18 <![CDATA[(X×Y)2]]> 2 0.18 0.09 0.63 0.56 Z 2 4.85 2.43 17.44 0.00 ** <![CDATA[(X×Z)1]]> 2 1.44 0.72 5.18 0.04 * <![CDATA[(X×Z)2]]> 2 0.39 0.19 1.40 0.30 <![CDATA[(Y×Z)1]]> 2 0.19 0.10 0.68 0.53 <![CDATA[(Y×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 invention and other Paracoccus culture mediums

[0111] According to the invention patent (application number EP11756284.3, publication number EP2548968B1, title "Method of producing astaxanthin by fermentation"), the culture medium was prepared according to the formula in Table 8, the pH was adjusted to about 7.2, and sterilized at 121°C for 15 minutes. Figure 7 As shown in A: A large amount of precipitation appears in the culture medium, mainly because the corn syrup will produce large flocculent precipitation after sterilization, and iron salts, calcium salts, etc. will react with phosphate to form slightly soluble / insoluble phosphates. The precipitation in the culture medium is not conducive to the growth of Paracoccus. According to the optimal combination of nitrogen sources in Example 4 of the present invention and the formula in Table 1, the culture medium prepared is as follows Figure 7As shown in B: It can be seen that the culture medium of the present invention does not have precipitation and can promote the growth of Paracoccus.

[0112] Table 8 Paracoccus culture medium formula

[0113]

[0114]

[0115] Comparative Example 2 Effects of various nitrogen sources on biomass yield, astaxanthin content and yield of Paracoccus

[0116] 1. Activation of bacterial strains and preparation of seed solution

[0117] The method is the same as 1.1.

[0118] 2. Preparation of culture medium

[0119] The experiment was divided into 4 groups, and 4 kinds of Paracoccus culture media were prepared according to the formulas in Table 1 and Table 9, in which the single nitrogen source group (experimental group 1) used corn syrup (product of Aladdin Biochemical Technology Co., Ltd., item number C116026-100g) as the nitrogen source, and the mixed nitrogen source groups (experimental groups 2-4) used yeast extract as the fixed nitrogen source (the concentration ratio of yeast extract to other nitrogen sources was controlled to be 1:2), respectively compounded with soy peptone (Guangdong Huankai Microbiology Co., Ltd., item number 050220), bacteriological peptone and beef extract. They were sterilized (121°C, 15min) according to the grouping method in Table 1 and then mixed, and the pH was kept at about 7.2.

[0120] Table 9 Nitrogen source composition

[0121]

[0122] 3. Fermentation culture of Paracoccus

[0123] The method is the same as 1.3.

[0124] 4. Capacity analysis results

[0125] As shown in Table 10, the culture medium with corn syrup as nitrogen source has the least effect on the production performance of Paracoccus, and all indicators are significantly lower than those of the other groups. The biomass yield (4.89 ± 0.03 g / L) of the beef extract compound group, the astaxanthin content (0.68 ± 0.03 mg / g) and the yield (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 with 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 shows 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 implementation modes of the present invention, but the implementation modes 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A culture medium for improving the astaxanthin production of Paracoccus, 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; FeSO4·7H2O0.1-1 g / L; CaCl2·2H2O 0.02-0.1 g / L; pH 7.2±0.

2.

2. The culture medium for improving the astaxanthin production of Paracoccus astaxanthin according to claim 1, 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 0.188 g / L; Na2HPO4·12H2O 0.475 g / L; MgSO4·7H2O 0.7 g / L; FeSO4·7H2O0.3 g / L; CaCl2·2H2O 0.05 g / L; pH 7.2±0.

2.

3. The culture medium for improving the astaxanthin production of Paracoccus according to claim 1 or 2, characterized in that: The paracoccus includes at least one of Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis.

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

5. The culture medium for improving the production of astaxanthin in Paracoccus as claimed in claim 4, characterized in that: 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.

6. Use of the culture medium for improving the astaxanthin production of Paracoccus as claimed in any one of claims 1 to 5 in culturing Paracoccus and / or producing astaxanthin.

7. The use according to claim 6, characterized in that: The paracoccus includes at least one of Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis.

8. A method for increasing the production of astaxanthin in Paracoccus sp., characterized in that: The steps include: S1, inoculating the activated Paracoccus into a seed culture medium, and culturing to a logarithmic growth phase to obtain a Paracoccus seed solution; S2. Inoculate the Paracoccus seed liquid into the culture medium for improving the astaxanthin production of Paracoccus according to any one of claims 1 to 5, and continue culturing to promote the proliferation of Paracoccus and the accumulation of astaxanthin, thereby increasing the astaxanthin content and / or production.

9. The method for cultivating Paracoccus astaxanthin to improve its yield according to claim 8, characterized in that: The Paracoccus described in step S1 includes at least one of Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis.

10. The method for cultivating Paracoccus astaxanthin to improve its yield according to claim 8, characterized in that: 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 culture described in steps S1 and S2 is 25 ± 1°C; The inoculation amount of the Paracoccus seed solution in step S2 is calculated based on the volume percentage of 5-10% in the culture system; The culture time described in step S2 is 7 to 14 days.

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