阅读说明：本技术 一种利用肌酐处理特氏杜氏藻生产类胡萝卜素的方法 (Method for producing carotenoid by treating Dunaliella tertiolecta with creatinine ) 是由 姜建国 吴芳纯 梁明华 李瑜 何羽婧 陈浩宏 梁志聪 于 2020-12-15 设计创作，主要内容包括：本发明公开了一种利用肌酐处理特氏杜氏藻生产类胡萝卜素的方法,属于食品科学技术领域。该方法为：将特氏杜氏藻细胞接种至培养基中,培养,待OD_(630)达到0.7～1.1(对数生长期),加入肌酐至终浓度为400～800ppm,于6000Lux光照条件下继续培养20～28天,提取色素,获得目标产物。本发明方法成本低廉、简单易行,加入肌酐之后可有效提高特氏杜氏藻中多种类胡萝卜素的含量,而且特氏杜氏藻培养简便,不易被污染。本发明是一种可行且经济的提高特氏杜氏藻中类胡萝卜素含量的有效方法,具有广泛的应用前景。(The invention discloses a method for producing carotenoid by treating Dunaliella tertiolecta with creatinine, belonging to the technical field of food science. The method comprises the following steps: inoculating Dunaliella tertiolecta cells into culture medium, culturing until OD is reached 630 And (3) reaching 0.7-1.1 (logarithmic growth phase), adding creatinine till the final concentration is 400-800 ppm, continuously culturing for 20-28 days under the condition of 6000Lux illumination, and extracting the pigment to obtain the target product. The method has low cost, is simple and feasible, can effectively improve the content of multiple carotenoids in the Dunaliella tertiolecta after adding the creatinine, is simple and convenient to culture, and is not easy to pollute. The invention is a feasible and economic method for improving carotenoid in Dunaliella tertiolectaThe effective method of the content has wide application prospect.)

1. A method for producing carotenoid by treating Dunaliella tertiolecta with creatinine is characterized in that:

inoculating Dunaliella tertiolecta cells into culture medium, culturing until OD is reached₆₃₀And when the concentration reaches 0.7-1.1, adding creatinine to the final concentration of 400-800 ppm, continuously culturing for 20-28 days under the condition of 6000Lux illumination, and extracting the pigment to obtain the target product.

2. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the addition amount of the creatinine is calculated according to the final concentration of the creatinine of 600 ppm.

3. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the Dunaliella tertiolecta is Dunaliella tertiolecta FACHB-821.

4. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the inoculation amount of the Dunaliella tertiolecta cells is 5 percent by volume.

5. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the conditions for continuous culture are as follows: the light-dark period is 14h/10h, the temperature is 28-33 ℃, and shake culture is carried out at the rotating speed of 50 r/min.

6. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the specific operation of extracting the pigment is as follows: centrifuging the culture solution at 8000rpm for 1min, discarding supernatant, adding acetone at volume ratio of 10:1, vortexing for 2min, leaching for 20min, centrifuging at 10000rpm for 10min, transferring supernatant into new centrifuge tube, adding 60% KOH at volume ratio of 100:1, standing for layering, and filtering with 0.45 μm filter membrane.

7. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the culture medium comprises the following components: NaNO₃0.420g/L，NaCl87.69g/L，NaH₂PO₄·2H₂O0.156g/L，NaHCO₃0.840g/L，KCl0.074g/L，MgSO₄·7H₂O1.230g/L，CaCl₂·2H₂O0.044g/L, 0.5mL/L of 0.1% Fe-EDTA solution, and 1mL/L of A5 microelement solution;

the A5 microelement solution comprises the following components: h₃BO₃ 2.86g/L，MnCl₂·4H₂O 1.81g/L，ZnSO₄·7H₂O 0.22g/L，CuSO₄·5H₂O 0.08g/L，(NH₄)₆Mo₇O₂₄·4H₂O 0.04g/L；

The Fe-EDTA solution comprises the following components: na (Na)₂EDTA 0.189g/L，FeCl₃·6H₂O 0.244g/L。

8. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the culture medium is sterilized before use and cooled to room temperature for later use.

9. The method for producing carotenoids by creatinine treatment of Dunaliella tertiolecta according to claim 8, wherein:

the sterilization treatment condition is that the high temperature sterilization is carried out for 20min at the temperature of 121 ℃ under the atmospheric pressure of 102.9 kPa.

10. The method for producing carotenoids by treating Dunaliella tertiolecta with creatinine according to claim 1, wherein:

the culture is to OD₆₃₀1.0 is reached;

the time for continuing the culture is 24 days.

Technical Field

The invention belongs to the technical field of food science, and particularly relates to a method for producing carotenoid by treating Dunaliella tertiolecta with creatinine.

Background

Carotenoids are a generic term for an important class of natural pigments. Carotenoids are ubiquitous in animals, higher plants, fungi, algae and bacteria. Carotenoids have been of great interest and research because of their remarkable physiological activities. Research shows that the carotenoid (such as carotene) can protect eyesight, improve nyctalopia and maintain skin health, and has obvious antioxidation and atherosclerosis prevention effects. Nowadays, carotenoids are widely used in the industries of food, feed, cosmetics, pharmaceutical products, and the like.

The human body cannot synthesize the carotenoid by itself and must take in the carotenoid through the outside; however, carotenoids are low in many plants and are difficult to synthesize chemically, mainly by biosynthetic means. At present, there are mainly 3 methods for producing carotenoids: chemical synthesis, natural extraction and microbial synthesis. The carotenoid obtained by chemical synthesis has poor activity and can only be used as pigment generally, and although the carotenoid obtained by natural extraction has multiple functional activities, the extraction rate and the yield are low, and the environment is polluted. The microbial synthesis method can be used for producing the carotenoid in large quantity, and has low raw material cost and little environmental pollution. Microalgae are considered an ideal feedstock for the production of carotenoids and biodiesel. The special Dunaliella (Dunaliella tertiolecta) is common halophytic green algae, has the advantages of fast growth and reproduction, easy large-scale culture, environmental protection, no toxicity and no harm. Furthermore, chemical interference with Dunaliella tertiolecta accumulates carotenoids, such as beta-carotene, in large amounts. Therefore, a substance capable of stimulating Dunaliella tertiolecta to accumulate carotenoids in a large amount is sought, which is beneficial to meeting the demand of the market for carotenoids.

Disclosure of Invention

In order to improve the yield of carotenoid in Dunaliella tertiolecta, the invention aims to provide a method for producing carotenoid by treating Dunaliella tertiolecta with creatinine.

The purpose of the invention is realized by the following technical scheme:

a method for producing carotenoid by treating Dunaliella tertiolecta with creatinine comprises inoculating Dunaliella tertiolecta cell into culture medium, culturing until OD is reached₆₃₀And (3) reaching 0.7-1.1 (logarithmic growth phase), adding creatinine till the final concentration is 400-800 ppm, continuously culturing for 20-28 days under the condition of 6000Lux illumination, and extracting the pigment to obtain the target product.

The Dunaliella tertiolecta strain is preferably Dunaliella tertiolecta FACHB-821.

The culture medium comprises the following components: NaNO₃0.420g/L，NaCl87.69g/L，NaH₂PO₄·2H₂O0.156g/L，NaHCO₃0.840g/L，KCl0.074g/L，MgSO₄·7H₂O1.230g/L，CaCl₂·2H₂O0.044g/L, 0.5mL/L of 0.1% Fe-EDTA solution, and 1mL/L of A5 microelement solution;

the A5 microelement solution comprises the following components: h₃BO₃ 2.86g/L，MnCl₂·4H₂O 1.81g/L，ZnSO₄·7H₂O 0.22g/L，CuSO₄·5H₂O 0.08g/L，(NH₄)₆Mo₇O₂₄·4H₂O 0.04g/L；

The Fe-EDTA solution comprises the following components: na (Na)₂EDTA 0.189g/L，FeCl₃·6H₂O is 0.244 g/L. The concentration of Fe ions in the solution was 0.1 mg/mL.

The culture medium is sterilized before use and cooled to room temperature for later use.

The conditions of the sterilization treatment are preferably sterilization at a high temperature of 121 ℃ for 20min under an atmospheric pressure of 102.9 kPa.

The inoculation amount of the Dunaliella tertiolecta cells is 5 percent by volume.

The culture is preferably carried out to OD₆₃₀Up to 1.0.

The addition amount of creatinine is preferably 600ppm in terms of final concentration of creatinine.

The conditions for continuing the culture are preferably: the light-dark period is 14h/10h, the temperature is 28-33 ℃, and shake culture is carried out at the rotating speed of 50 r/min.

The period of time for continuing the culture is preferably 24 days.

The specific operation of extracting the pigment is preferably as follows: centrifuging the culture solution at 8000rpm for 1min, discarding supernatant, adding acetone at volume ratio of 10:1, vortexing for 2min, leaching for 20min, centrifuging at 10000rpm for 10min, transferring supernatant into new centrifuge tube, adding 60% KOH (removing chlorophyll) at volume ratio of 100:1, standing for layering, and filtering with 0.45 μm filter membrane. The pigment sample can be stored in a refrigerator at-20 deg.C.

Separating and detecting carotenoid in acetone phase by using High Performance Liquid Chromatography (HPLC), wherein a chromatographic column is Welch Ultimate XB-C18; the mobile phases used were: phase A: 97% methanol + 3% ultrapure water, phase B was 100% t-butyl methyl ether, the flow rate used was 1.0mL/min, and the column temperature used was 25 ℃.

The conversion formula of the peak area/content of the lutein is as follows: 63.124 x-0.4755;

the conversion formula of the peak area/content of the zeaxanthin is as follows: 47.805 x-8.4906;

the conversion formula of the peak area/content of the alpha-carotene is as follows: 95.696 x-12.952;

the conversion formula of the peak area/content of the beta-carotene is as follows: y 34.791x + 59.973.

The conversion formula is obtained by calibrating a standard product.

The invention has the following effects: the total carotenoid yield can be obviously improved by using creatinine to interfere Dunaliella tertiolecta. When the amount of creatinine added is 600ppm, the total yield and single cell yield of lutein, alpha-carotene and beta-carotene in Dunaliella tertiolecta reach high values. In the invention, the consumption of creatinine is less, the culture of the Dunaliella tertiolecta is simple and convenient, and the effect of interfering the accumulation of carotenoid in the Dunaliella tertiolecta is obvious.

Compared with the prior art, the invention has the advantages and effects that:

(1) after the creatinine is added, the total biomass of the Dunaliella tertiolecta is increased, the Dunaliella tertiolecta has concentration dependence, the total yield and the single cell yield of the contents of lutein, alpha-carotene and beta-carotene in the body of the Dunaliella tertiolecta are obviously increased, the content of zeaxanthin is reduced, and the total carotenoid content is obviously improved.

(2) The Dunaliella tertiolecta used in the invention is an autotrophic eukaryote, has simple culture conditions, fast growth and reproduction, is not easy to be polluted, and is nontoxic and harmless.

(3) The method has the advantages of low creatinine consumption, low cost and good carotenoid accumulation effect on Dunaliella tertiolecta stimulation.

Drawings

FIG. 1 is a liquid phase chromatogram of the change in carotenoid composition of Dunaliella tertiolecta 4 days after addition of creatinine.

FIG. 2 is a liquid phase chromatogram of changes in the carotenoid composition of Dunaliella tertiolecta 8 days after addition of creatinine.

FIG. 3 is a liquid phase chromatogram of changes in carotenoid composition of Dunaliella tertiolecta after 12 days of creatinine.

FIG. 4 is a liquid phase chromatogram of changes in carotenoid composition of Dunaliella tertiolecta after 16 days of creatinine.

FIG. 5 is a graph of the effect of creatinine on carotenoids in Dunaliella tertiolecta.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Dunaliella tertiolecta FACHB-821 used in the following examples was purchased from the institute for aquatic organisms of the Chinese academy of sciences (Wuhan) and stored in a liquid medium under reduced light; creatinine was purchased from Aladdin, product number C108393, CAS number 60-27-5.

Example 1

(1) Preparation of Dunaliella tertiolecta liquid culture medium

The components are as follows: NaNO₃ 0.420g/L，NaCl 87.69g/L，NaH₂PO₄·2H₂O 0.156g/L，NaHCO₃0.840g/L，KCl 0.074g/L，MgSO₄·7H₂O 1.230g/L，CaCl₂·2H₂0.044g/L of O, 0.5mL/L of 0.1% Fe-EDTA solution and 1mL/L of A5 trace element solution. The Fe-EDTA solution comprises the following components: na (Na)₂EDTA 0.189g/L，FeCl₃·6H₂O is 0.244 g/L. The trace element A5 solution had the following composition: h₃BO₃2.86g/L，MnCl₂·4H₂O 1.81g/L，ZnSO₄·7H₂O 0.22g/L，CuSO₄·5H₂O 0.08g/L，(NH4)₆Mo₇O₂₄·4H₂O is 0.04 g/L. The basic culture solution is subpackaged into 500mL triangular flasks per 200mL bottle, sealed with four layers of gauze, and sterilized at 121 deg.C under 102.9kPa for 20 min. Cooled at room temperature and then used.

(2) Induced culture

Centrifuging Dunaliella tertiolecta FACHB-821 algae liquid at 4000r/min for 3min, removing culture liquid, adding algae cells into a triangular flask filled with fresh sterilized culture liquid according to the inoculation amount of 5% of volume ratio, wrapping the bottleneck with four layers of gauze, and fastening with a rubber band. Placing the flask in a light incubator, and allowing Dunaliella tertiolecta cells to be in logarithmic growth phase (OD)₆₃₀About 1), adding creatinine (0 (control group, T), 400, 600, 800ppm) with different final concentrations into the algae solution, and culturing for 24 days (culture condition of 6000 Lux; the light-dark period is 14h/10 h; the temperature is 28-33 ℃, shaking culture is carried out at the rotating speed of 50 r/min), the culture is carried out for 24 days, samples are taken every 4 days, and the carotenoid content is measured.

(3) Extraction of carotenoids

Centrifuging 20mL of algae solution at 8000rpm for 1min, discarding supernatant, adding 2mL of acetone again, vortexing for 2min, leaching for 20min, centrifuging at 10000rpm for 10min, transferring supernatant to 4mL centrifuge tube, adding 0.2mL of 60% KOH (removing chlorophyll), standing for layering, filtering with 0.45 μm filter membrane, and storing at-20 deg.C in refrigerator.

(4) Determination of carotenoid content

Separating and detecting carotenoid in acetone phase by using High Performance Liquid Chromatography (HPLC), wherein a chromatographic column is Welch Ultimate XB-C18; the mobile phases used were: phase A: 97% methanol + 3% ultrapure water, phase B was 100% t-butyl methyl ether, the flow rate used was 1.0mL/min, and the column temperature used was 25 ℃.

The conversion formula of the peak area/content of the lutein is as follows: 63.124 x-0.4755;

the conversion formula of the peak area/content of the zeaxanthin is as follows: 47.805 x-8.4906;

the conversion formula of the peak area/content of the alpha-carotene is as follows: 95.696 x-12.952;

the conversion formula of the peak area/content of the beta-carotene is as follows: y 34.791x + 59.973.

The conversion formula is obtained by calibrating a standard product.

The results of the stepwise HPLC separation detection of carotenoids are shown in FIGS. 1-4. The change in total carotenoid content is shown in FIG. 5.

(5) Conclusion

The creatinine interferes with the Dunaliella tertiolecta, and the carotenoid accumulation amount can be obviously improved. From the phase liquid phase diagram, the addition of creatinine not only increased the total biomass of Dunaliella tertiolecta but also had a concentration dependence, with the carotenoid composition varying at different time periods.

On the fourth day of dosing, the changes in the contents of the four major carotenoids in the Dunaliella tertiolecta cells under the action of different concentrations of creatinine can be seen from FIG. 1. The yield of the lutein in the control group is 1.52 mu g/mL, and the yield of the lutein after the creatinine is added to the concentration of 400 mu g/mL reaches 1.6 mu g/mL, which is improved by 5 percent. The lutein yield of the experimental group with 600 mug/mL reaches 1.78 mug/mL, which is improved by 17 percent compared with the control group. The single cell yield of 400. mu.g/mL and 600. mu.g/mL groups were increased by 6% and 18%, respectively. The total zeaxanthin content in the 400. mu.g/mL group was reduced to 78%, while that in the 600. mu.g/mL group was reduced to 57% and the single cell content was reduced to 57%. The total content of alpha-carotene is increased, and the total content of the group with 600 mug/mL is increased by 35 percent. The total amount of beta-carotene and the content of single cells are obviously improved along with the concentration, and the creatinine concentration reaches the maximum when being 600 mug/mL, and is respectively increased by 33 percent and 36 percent.

On the eighth day after the addition of the drug, the content of four main carotenoids in the Dunaliella tertiolecta cells can be seen from FIG. 2 under the action of creatinine with different concentrations. After the action concentration is 600 mug/mL for eight days, the total content of lutein is improved by 28%, and the content of single cells is improved by 24%. The total zeaxanthin content and the single cell content are still obviously reduced. The alpha-carotene content was less but also increased with increasing drug concentration. The beta-carotene has higher content when the action concentration is 600 mug/mL, the total content is improved by 33 percent, and the content of single cells is improved by 34 percent.

On the twelfth day of the addition of the drug, the content changes of four main carotenoids in the Dunaliella tertiolecta cells under the action of creatinine with different concentrations can be seen from figure 3. The yield of lutein is improved by 14 percent and the yield of single cells is improved by 12 percent by 600 mu g/mL of creatinine; the total content of zeaxanthin and the content of single cells are reduced by about 50%; the total content of alpha-carotene is increased by 7 percent, and the content of single cells is increased by 5 percent; the total amount of beta-carotene is increased by 20 percent, and the content of single cells is increased by 18 percent.

On the sixteenth day after the addition of the drug, the content of four major carotenoids in the Dunaliella tertiolecta cells was changed by the action of creatinine at different concentrations as shown in FIG. 4. By integrating the change trends of the four pigments, the creatinine effect of 600 mug/mL can obviously improve the contents of lutein, alpha-carotene and beta-carotene and simultaneously reduce the content of zeaxanthin. The yield of the 600 mu g/mL creatinine is increased to 3.02 mu g/mL compared with the yield of the lutein of a control group which is 2.32 mu g/mL, the contrast is improved by 30 percent, and the content of single cells is also improved by nearly 30 percent; the total alpha-carotene is increased by 15 percent, and the content of single cells is increased by 16 percent; the total beta-carotene is increased by 40 percent, and the content of single cells is increased by 30 percent; the total content of zeaxanthin is reduced by 50%, and the content of single cells is reduced by 40%.

In a word, creatinine treatment of t's algae can increase the total carotenoid content, increase the contents of lutein, alpha-carotene and beta-carotene, while reducing the zeaxanthin content. The effect of the drug was consistent from day four to day sixteenth from the phasic liquid phase diagram. The concentration with the best effect on increasing the three pigments is 600 mug/mL, and the total yield and the single cell yield of the lutein, the alpha-carotene and the beta-carotene reach higher values.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.