CN113916820B - Method for rapidly determining total carotenoid content in bacterial liquid - Google Patents
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- 235000021466 carotenoid Nutrition 0.000 title claims abstract description 103
- 150000001747 carotenoids Chemical class 0.000 title claims abstract description 103
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- 238000000034 method Methods 0.000 title claims abstract description 41
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- 241001052560 Thallis Species 0.000 abstract description 2
- 238000012423 maintenance Methods 0.000 abstract description 2
- 241000797752 Erythrobacter pelagi Species 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 10
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Abstract
The invention relates to the technical field of biology, in particular to a method for rapidly determining total carotenoid content in bacterial liquid, which is characterized in that the optical density value of bacterial liquid of bacteria and the concentration of the bacterial liquid have positive linear relation, and the concentration of bacterial liquid of bacteria and the carotenoid content of the bacterial liquid of bacteria also have positive linear relation. By constructing a standard curve of the concentration value of the bacterial liquid and the total carotenoid content in the bacterial liquid, a linear regression equation between the bacterial liquid concentration value and the total carotenoid content can be established, and then the carotenoid content in the bacterial liquid at any concentration can be calculated by utilizing the equation. Compared with the traditional method, the method has the advantages of fewer steps, no need of using organic solvents, no need of drying thalli, no need of treating bacteria and capability of maintaining the biological activity of carotenoid. Therefore, the invention has the advantages of rapidness, simplicity, environmental protection, low cost, maintenance of carotenoid bioactivity and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for rapidly determining total carotenoid content in bacterial liquid.
Background
Carotenoids (carotenoids) are a general term for a class of natural pigments that are widely found in nature, and are synthesized mainly by plants, bacteria and fungi. Natural carotenoid has various bioactive characteristics, can be used as a colorant and a food additive, can be used as an antioxidant, and has important biological functions, such as high-efficiency quenching of singlet oxygen; enhancing immune function of the organism; the bacteria participate in various vital activities, and the adaptability of the bacteria to the environment can be improved. At present, carotenoids are widely used in the fields of foods, cosmetics, health products and the like. With the pursuit of healthy life, natural carotenoids have a huge market demand, and the global market of 2026 is expected to reach 69 billions of dollars. Therefore, the development of products rich in natural carotenoids is significant.
Bacteria are an important source of natural carotenoids for humans, and the use of cultured bacteria to obtain natural carotenoids is one of the major current approaches. The method for measuring the total carotenoid content in the bacterial liquid by the traditional method comprises the following steps of: 1. sample preparation: the bacterial liquid is centrifuged at 5000g for 10 minutes to obtain bacterial cells. 2. Wall breaking treatment: breaking wall by grinding, ultrasonic crushing, high-speed bead grinding, repeated freezing and thawing, etc. 3. Extracting with an organic solvent: extracting carotenoid in thallus with organic solvent such as methanol and acetone at room temperature in shaking table under dark condition until thallus becomes white. 4. Supernatant acquisition: centrifuging at 5000g for 10 min to obtain supernatant containing carotenoid, and oven drying and weighing thallus precipitate. 5. Total carotenoid content determination: the absorbance of the supernatant was measured at 480nm wavelength by an ultraviolet spectrophotometer. Finally, the total carotenoid content produced by the bacteria was calculated according to the following formula:
total carotenoid content (TCC, μg/g) =a×d×v/(e×w)
Wherein A is absorbance at 480nm of total carotenoid content, D is dilution ratio, V is volume (mL) of extract, E is extinction coefficient of 0.16 of total carotenoid, and W is dry weight (g) of bacterial cells.
The disadvantage of this method is that: 1. the steps are complicated, the time consumption is long, and the whole process needs 12 hours. 2. Not environment-friendly, and needs a large amount of organic solvents such as methanol, acetone and the like. 3. The biological activity of carotenoid is destroyed, natural carotenoid has biological activity characteristic, and has strong sensitivity to light, heat, etc. 4. The cost is high, for example, the organic solvent required by measuring the carotenoid content in 1L of bacterial liquid is 3-5 Yuanrenzhi, and the acetone is 10-15 Yuanzhi.
In view of the important role of bacteria in obtaining natural carotenoids in humans, the development of a method for measuring the yield of carotenoids produced by bacteria, which is rapid, simple, environment-friendly and low-cost, has a broad application prospect. However, so far no report has been made on the rapid detection of bacterial total carotenoid content by constructing a linear regression equation using bacterial broth concentration and total carotenoid content. The invention can rapidly detect the total carotenoid content in the bacterial liquid by utilizing a linear regression equation constructed by the bacterial liquid concentration and the total carotenoid content, and can solve the problems existing in the traditional method. Therefore, the invention has the advantages of rapidness, simplicity, environmental protection, low cost, capability of maintaining the bioactivity of the carotenoid and the like.
Disclosure of Invention
The invention aims to overcome the defects of complicated steps, long time consumption, environmental protection, high cost, destroyed biological activity of carotenoid and the like in the traditional method for measuring the total carotenoid content of bacterial liquid, and simultaneously provides a quick, simple, environment-friendly measuring method which has low cost and can keep the biological activity of carotenoid.
The principle of the invention is that the concentration of bacterial liquid of bacteria at a certain wavelength has positive linear relation with the optical density, and the concentration of bacterial liquid of bacteria can be rapidly determined by utilizing a spectrophotometer. We find that: the bacterial liquid concentration and the carotenoid content have positive linear relation, so that the linear regression equation between the bacterial liquid concentration and the total carotenoid content in the bacterial liquid is established by constructing a standard curve of the bacterial liquid concentration and the total carotenoid content in the bacterial liquid, and then the carotenoid content in the bacterial liquid at any concentration can be calculated by utilizing the equation. The method comprises the following steps: (1) obtaining the concentration of carotenoid producing bacteria liquid; (2) Establishing a linear regression equation for measuring the total carotenoid content and bacterial liquid concentration of bacterial liquid; (3) And obtaining the total carotenoid content of the bacterial liquid according to a linear regression equation. Compared with the traditional method, the method has the advantages of fewer steps, no need of using organic solvents, no need of drying thalli, no need of treating bacteria and capability of maintaining the biological activity of carotenoid. Therefore, the invention has the advantages of rapidness, simplicity, environmental protection, low cost, maintenance of carotenoid bioactivity and the like.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for rapidly determining the total carotenoid content in bacterial liquid comprises the following steps:
A. preparing standard bacterial solutions with different concentrations, measuring the optical density value of the standard bacterial solution as X, drawing a standard curve by taking the concentration of the standard bacterial solution as Y, and establishing a linear regression equation for rapidly measuring the concentration of the bacterial solution: y=ax+b.
B. The total carotenoid content of the standard bacterial liquid is measured as y by adopting a traditional method, the concentration of the standard bacterial liquid is used as x, a standard curve is drawn, and a linear regression equation for rapidly measuring the total carotenoid content of the bacterial liquid is established: y=ax+b.
C. And D, obtaining an optical density value of the unknown bacterial liquid, substituting the optical density value into the linear regression equation established in the step A to obtain the concentration of the unknown bacterial liquid, and substituting the linear regression equation established in the step B to obtain the total carotenoid content of the unknown bacterial liquid.
Preferably, the bacterium is a bacterium capable of producing carotenoids.
Preferably, the optical density value is obtained using an ultraviolet spectrophotometer.
Preferably, in the step a, the concentration of the bacterial liquid is obtained by a plate colony counting method, and the standard bacterial liquid is prepared.
Preferably, the method further comprises the following steps:
D. and (3) checking: and C, obtaining the total carotenoid content of the unknown bacterial liquid by adopting a traditional method, wherein compared with the total carotenoid content of the unknown bacterial liquid obtained in the step C, no significant difference exists.
The application of the method for rapidly determining the total carotenoid content in the bacterial liquid is characterized in that the total carotenoid content of any bacterial liquid is rapidly obtained.
After establishing a double linear regression equation and checking, for the same bacterial liquid, the optical density value of the bacterial liquid can be directly obtained, and the total carotenoid content can be quickly obtained by substituting the optical density value into the double linear regression equation, so that convenience is provided for related experiments and researches of bacterial carotenoid production.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
(1) Quick and simple. The traditional method for measuring the total carotenoid content in bacteria requires the steps of centrifugation, wall breaking, extraction, drying and the like, and the whole process is completed for 12 hours, which is tedious and time-consuming; the invention can obtain the result by only two steps: (1) obtaining the concentration of the bacterial liquid. (2) Obtaining the total carotenoid content of the bacterial liquid according to the equation; the whole process can be completed in a few minutes. Therefore, the invention is quick and simple.
(2) Environmental protection. The traditional method needs a large amount of organic solvents such as methanol, acetone and the like, and is not environment-friendly; the present invention does not require the use of organic solvents. Therefore, the invention is environment-friendly.
(3) The cost is low. The traditional method needs a large amount of organic solvents such as methanol, acetone and the like, and adopts electric drying or nitrogen drying, which takes a certain cost. The invention does not need to use organic solvent and does not need to be dried. Therefore, the cost is low.
(4) The bacterial activity and the biological activity of carotenoid are maintained. The traditional method requires wall breaking and extraction treatment of bacteria, and the bacteria die and easily deactivate carotenoid degradation. The invention can maintain bacterial activity and carotenoid bioactivity without treating bacteria.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
It is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments in the present invention are within the protection scope of the present invention.
In order to avoid redundancy of application text, the present invention is described by taking Erythrobacter pelagi bacteria of the genus gibberellic, family gibberellic as an example. It is a bacterium capable of producing carotenoids isolated from the intestinal tract of Chlamys nobilis "Nana golden scallop". The bacteria are orange, aerobic, rod-shaped gram-negative bacteria. Based on 16S rRNA and phylogenetic analysis, the sequence similarity of the strain and the 16S rRNA gene of Erythrobacter pelagi UST081027-248 is 99.2%.
Those skilled in the art know that similar experimental results are obtained using other bacteria capable of producing carotenoids.
Example 1: at 25 ℃, the invention is adopted to measure the total carotenoid content of E.pelargi bacterial liquid
Time: 2021, 5, 6, location: university of Shanzhi.
The operation steps are as follows:
quick determination of E.pelagi bacterial liquid concentration: bacterial liquid concentration (in/mL) was obtained by plate colony counting. Serial dilutions of bacterial solutions were made into 6 bacterial solutions of different concentrations. The optical density values measured at the wavelength of ultraviolet spectrophotometer λ=600 nm were 0.037, 0.057, 0.102, 0.262, 0.399, 0.619. Their corresponding plate colonies were counted (. Times.10) 6 and/mL) results were 21, 40, 45, 170, 290, 550. Drawing a standard curve by taking the optical density value of the bacterial liquid as X and the counting result of the plate colony as Y, and establishing a linear regression equation: y= 889.25X-32.754 (R 2 ＝0.9808)。
Checking a standard curve linear regression equation: the bacterial concentrations (. Times.10) obtained by substituting the measured optical density values of the bacterial liquids of 0.117, 0.138 and 0.257 into the equation 6 and/mL) was 71, 90, 196. Bacterial concentration (10) obtained from actual plate colony count 6 Per mL) 40,50. 140 through T test analysis of SPSS 19.0 statistical software, no significant difference (p) between the linear regression equation of bacterial fluids and the plate colony count was determined>0.05)。
2. Establishing a linear regression equation of the concentration of the bacterial liquid and the total carotenoid content of the bacterial liquid:
8 concentration gradient dilutions are carried out on E.pelagi bacterial liquid according to a certain proportion, 500 mu L of diluted bacterial liquid is accurately removed, and the optical density values of the E.pelagi bacterial liquid are measured to be 0.47, 0.598, 0.833, 1.062, 1.292, 1.309, 1.360 and 1.440 by an ultraviolet spectrophotometer under the wavelength of lambda=600 nm. Obtaining the bacterial liquid concentration (x 10) according to the linear regression method of the bacterial liquid optical density value and the bacterial liquid concentration 6 The number of the components/mL) is: 385. 499, 708, 912, 1116, 1131, 1177, 1248.
Determining the total carotenoid content (mug/mL) of the bacterial liquids with different concentration gradients in the step 1 according to the traditional method (as described in the background art) to be 0.338, 0.430, 0.719, 0.828, 1.039, 1.040, 1.116 and 1.163, taking the total carotenoid content (mug/mL) as y, taking the bacterial liquid concentration obtained in the step 1 as x, and drawing a standard curve to establish a linear regression equation for determining the total carotenoid content (mug/mL) of the E.pelargi bacterial liquid: y=0.0009 x-0.0063 (R 2 ＝0.9887)。
3. And (3) effect inspection: randomly taking quantitative bacterial solutions with different concentrations, measuring optical density values of 0.469, 0.983, 1.185 and 1.316 by an ultraviolet spectrophotometer at the wavelength of lambda=600 nm, and substituting the optical density values into the equation of the step 1 to obtain the bacterial solution concentration (multiplied by 10) 6 The number of the components/mL) is: 384. 841, 1021, 1137. Substituting the obtained concentration into the equation of the step 2 to obtain the total carotenoid content (mug/mL) of the bacterial liquid under the concentration of 0.339, 0.751, 0.913 and 1.017. Whereas the total carotenoid content (μg/mL) as determined by conventional methods (as described in the background) was 0.344, 0.856, 1.256, 1.016.SPSS 19.0 statistical software T test analysis linear regression equation shows that there is no significant difference between total carotenoid content calculated from the conventional method and total carotenoid content (P>0.05)。
4. Application: taking 3 groups of E.pelagi bacteria bacterial liquids, and measuring optical density values of 0.586 and 0 under the wavelength of lambda=600 nm of an ultraviolet spectrophotometer087, 1.103, substituting the bacterial concentration (. Times.10) obtained in the equation of step 1 6 And 3/mL) were 488, 45, 948, and the total carotenoid content (μg/mL) of the bacterial liquid at the concentration was 0.433, 0.034, 0.847, which was obtained by substituting the total carotenoid content into the equation of step 2. Then culturing the 3 groups of E.pelagi bacteria bacterial liquid at 25 ℃ for overnight, and substituting the bacterial concentration (x 10) obtained in the equation of the step 1 into the optical density values of 0.608, 0.102 and 1.125 measured at the wavelength of lambda=600 nm of an ultraviolet spectrophotometer 6 And (mu/mL) were 508, 58 and 968, and the total carotenoid content (mu g/mL) of the bacterial liquid at the concentration was 0.451, 0.046 and 0.865, which were obtained by substituting the values into the equation of step 2. The method has the advantages that the bacterial activity is maintained, and the bacterial concentration and carotenoid content are increased to a certain extent after one-night culture.
Example 2: at 30 ℃, the invention is adopted to measure the total carotenoid content of E.pelargi bacterial liquid
Time: 2021, 5 months, 10 days, location: university of Shanzhi
The operation steps are as follows:
quick determination of E.pelagi bacterial liquid concentration: since the same bacteria are used, the linear relation of the positive correlation between the optical density value of the bacterial liquid and the concentration of the bacterial liquid in the embodiment 1 is directly used, and the linear regression equation between the two is obtained: y= 889.25X-32.754 (R 2 ＝0.9808)。
2. Establishing a linear regression equation of the concentration of the bacterial liquid and the total carotenoid content of the bacterial liquid:
and 6 concentration gradient dilutions are carried out on the E.pelagi bacterial liquid according to a certain proportion, 500 mu L of diluted bacterial liquid is accurately removed, and the optical density values of the E.pelagi bacterial liquid are measured to be 0.837, 0.880, 1.229, 1.418, 1.470 and 1.506 by an ultraviolet spectrophotometer under the wavelength of lambda=600 nm. Obtaining the bacterial liquid concentration (x 10) according to the linear regression method of the bacterial liquid optical density value and the bacterial liquid concentration 6 and/mL) 712, 750, 1060, 1228, 1274, 1306.
The total carotenoid content (μg/mL) in the bacterial liquids of different concentration gradients in step 1 was determined as 0.681, 0.730, 1.019, 1.077, 1.279, 1.324 by conventional methods (as described in the background), and was taken as y, which was used in step 1The concentration of the obtained bacterial liquid is used as x, a standard curve is drawn, and a linear regression equation for determining the total carotenoid content (mug/mL) of the E.pelargi bacterial liquid can be established: y=0.001x+0.007 (R 2 ＝0.9712)。
3. And (3) effect inspection: randomly taking quantitative bacterial solutions with different concentrations, measuring optical density values of 0.887, 1.263 and 1.444 by an ultraviolet spectrophotometer at the wavelength of lambda=600 nm, and substituting the optical density values into the equation of the step 1 to obtain the bacterial solution concentration (multiplied by 10) 6 and/mL) 756, 1090, 1251. Substituting the obtained concentration into the equation of the step 2 to obtain the total carotenoid content (mug/mL) of the bacterial liquid under the concentration of 0.763, 1.097 and 1.258. Whereas the total carotenoid content (μg/mL) was 0.825, 0.928, 1.251 as determined by conventional methods (as described in the background). SPSS 19.0 statistical software T test analysis linear regression equation shows that there is no significant difference between total carotenoid content calculated from the conventional method and total carotenoid content (P>0.05)。
4. Application: taking 3 groups of E.pelagi bacteria bacterial liquids, and substituting the bacterial concentrations (multiplied by 10) obtained in the equation of the step 1 into the optical density values of 0.632, 1.398 and 1.123 measured at the wavelength of lambda=600 nm of an ultraviolet spectrophotometer 6 Each mL) was 529, 1210 and 966, and the total carotenoid content (μg/mL) of the bacterial liquid at this concentration was 0.536, 1.217 and 0.973, which were obtained by substituting them into the equation of step 2. Then culturing the 3 groups of E.pelagi bacteria bacterial liquid at 30 deg.C overnight, measuring optical density value of 0.662, 1.458, 1.189 at ultraviolet spectrophotometer lambda=600 nm wavelength, substituting into the bacterial concentration (x 10) obtained in the equation of step 1 6 The total carotenoid content (mug/mL) of the bacterial liquid at the concentration is 0.563, 1.271 and 1.032, which are obtained by substituting 556, 1264 and 1025 into the equation of the step 2. The method has the advantages that the bacterial activity is maintained, and the bacterial concentration and carotenoid content are increased to a certain extent after one-night culture.
In the 2 embodiments, the total carotenoid content of the bacterial liquids with different concentrations at the same temperature can be directly calculated and obtained by using a linear regression equation. Compared with the traditional method, the method has the advantages of rapidness, simplicity, convenience, environmental protection, low cost, capability of keeping the biological activity of the carotenoid and the like.
In addition, the calculation of the total carotenoid content of other bacterial liquids by a linear regression equation is also the idea of the invention.
The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent changes may be made in the claims herein without departing from the scope of the invention.
Claims (5)
1. The method for rapidly determining the total carotenoid content in the bacterial liquid is characterized by comprising the following steps of:
A. preparing standard bacterial solutions with different concentrations, measuring the optical density value of the standard bacterial solution as X, drawing a standard curve by taking the concentration of the standard bacterial solution as Y, and establishing a linear regression equation for rapidly measuring the concentration of the bacterial solution: y=ax+b;
B. the total carotenoid content of the standard bacterial liquid is measured as y by adopting a traditional method, the concentration of the standard bacterial liquid is used as x, a standard curve is drawn, and a linear regression equation for rapidly measuring the total carotenoid content of the bacterial liquid is established: y=ax+b;
C. obtaining an optical density value of the unknown bacterial liquid, substituting the optical density value into a linear regression equation established in the step A to obtain the concentration of the unknown bacterial liquid, and substituting the linear regression equation established in the step B to obtain the total carotenoid content of the unknown bacterial liquid;
the bacteria areErythrobacter pelagiBacteria.
2. The method for rapidly determining total carotenoid content in bacterial fluids according to claim 1, wherein the optical density value is obtained using an ultraviolet spectrophotometer.
3. The method of claim 1, wherein in step a, the concentration of the bacterial fluid is obtained by plate colony counting and configured as the standard bacterial fluid.
4. The method for rapidly determining total carotenoid content in bacterial fluids according to claim 1, further comprising the steps of:
D. and (3) checking: and C, obtaining the total carotenoid content of the unknown bacterial liquid by adopting a traditional method, wherein compared with the total carotenoid content of the unknown bacterial liquid obtained in the step C, no significant difference exists.
5. Use of the method for rapid determination of total carotenoid content in bacterial liquids according to claim 1, characterized in that rapid acquisition is achievedErythrobacter pelagiTotal carotenoid content of the bacterial liquid.
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