Summary of the invention
The present invention proposes a kind of special extract and extracting method of oil palm leaves total protein matter, effectively can remove the thick substances in supernatant liquor, improve total protein extraction productive rate and purity.
Technical scheme of the present invention is achieved in that
A kind of special extract of oil palm leaves total protein matter, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprise the component of following mass concentration: complexing agent 35-40g/L, sodium tetraborate 15-20g/L, reductive agent 20-40g/L, sucrose 200-400g/L; The special extract pH value of described oil palm gross protein is 4-7.
Further, described complexing agent is ENTA or EDTA-2Na.
Further, described reductive agent is the mixture of vitamins C and beta-mercaptoethanol.
Further, ascorbic mass concentration is 5-10g/L, and the mass concentration of beta-mercaptoethanol is 15-28g/L.
Further, the special extract pH value of described oil palm leaves total protein matter is 4-5.
Further, described pH value regulates reagent used to be hydrochloric acid.
A kind of extracting method of oil palm leaves total protein matter, comprise the following steps: oil palm blade is pulverized last, add described oil palm leaves total protein matter special extract, room temperature whirlpool concussion 4-6min, remove supernatant liquor after centrifugal, then carry out the extraction of gross protein according to BPP method.
Further, with leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 4-6 (V/W) times amount.
Further, in described centrifugal process, temperature is 3-5 DEG C, centrifugal force 10000-17000RCF, and centrifugation time is 8-12min.
Beneficial effect of the present invention:
Oil palm special extract of the present invention, join after oil palm blade grinds in powder, effectively can remove the dope in supernatant liquor, greatly reduce the difficulty of experimental implementation, and leaching process is not by the impact of temperature, summer or winter all can carry out experimental implementation, easy and simple to handle, for oil palm leaf protein Quality Research provides the foundation.Extract the protein soln color clarification obtained, not or only have minute quantity thick substances, purity does not improve greatly.Carry out detection by quantitative through Bradford method to the protein soln extracted, the total protein of the oil palm blade adopting BPP method to extract, gained extracting solution of protein concentration is generally 5-7 μ g μ L
-1, and adopting extracting method of the present invention, gained total protein concentration generally can reach 8 μ g μ L
-1above, most concentration is at 10 μ g μ L
-1left and right.Meanwhile, after testing, carry out the extraction of oil palm leaves total protein by adding oil palm special extract of the present invention, gross protein extraction yield improves greatly.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
A special extract for oil palm leaves total protein matter, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 35g/L, sodium tetraborate 15g/L, vitamins C 5g/L, beta-mercaptoethanol 15g/L, sucrose 200g/L; Add concentrated hydrochloric acid and regulate pH value to 4.
An extracting method for the special extract that oil palm leaves total protein matter is extracted, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 4 (V/W) times amount, and room temperature whirlpool concussion 4min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.Concrete operation step is as follows:
(1) get oil palm blade 2g, liquid nitrogen grinding powdered, proceed to 50mL centrifuge tube;
(2) add 8mL oil palm leaves total protein matter special extract, room temperature whirlpool concussion 4min, removes supernatant after centrifugal 8min, repeats this step 1 time under temperature 3 DEG C, centrifugal force 10000RCF condition;
(3) 10mL precooling extracting solution, 1mL 10%SDS is added, room temperature whirlpool concussion 5min;
(4) equal-volume Tris balance phenol (pH7.8) is added, room temperature whirlpool concussion 10min;
(5) 4 DEG C, 17000RCF, centrifugal 15min, upper-layer phenol goes to 10mL centrifuge tube mutually;
(6) equal-volume precooling extracting solution is added, room temperature whirlpool concussion 10min;
(7) 4 DEG C, 17000RCF, centrifugal 15min, upper-layer phenol goes to new pipe mutually;
(8) add the pre-cold methanol of 5 times of volumes containing supersaturated ammonium sulfate, mixing, puts-20 DEG C of more than precipitating proteins 6h or spends the night.
(9) supernatant is removed, resuspended 2 times of protein precipitation precooling methanol solution after centrifugal (4 DEG C, 17000RCF, 10min);
(10) remove supernatant after centrifugal (4 DEG C, 17000RCF, 10min), with precooling acetone soln resuspended for 2 times;
(11) centrifugal (4 DEG C, 17000RCF, 10min), precipitate air-dry after add appropriate lysate, more than room-temperature dissolution 2h.
(12) centrifugal (4 DEG C, 17000RCF, 30min), draws supernatant for subsequent use to 1.5mL centrifuge tube.
Embodiment 2
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA-2Na 37g/L, sodium tetraborate 18g/L, vitamins C 8g/L, beta-mercaptoethanol 22g/L, sucrose 300g/L; Add acetic acid and regulate pH value to 5.5.
An extracting method for the special extract that oil palm leaves total protein matter is extracted, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 5 (V/W) times amount, and room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, the add-on of step (2) oil palm leaves total protein matter special extract is 10mL, room temperature whirlpool concussion 5min; Under temperature 4 DEG C, centrifugal force 13000RCF condition, remove supernatant liquor after centrifugal 10min, all the other operations are identical with embodiment 1.
Embodiment 3
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 40g/L, sodium tetraborate 20g/L, vitamins C 10g/L, beta-mercaptoethanol 28g/L, sucrose 400g/L; Add hydrochloric acid and regulate pH value to 7.
An extracting method for oil palm leaves total protein matter, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 8 (V/W) times amount, and room temperature whirlpool concussion 6min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, step (2) adds 16mL oil palm leaves total protein matter special extract, room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal 12min, repeats this step 1 time under temperature 5 DEG C, centrifugal force 17000RCF condition; All the other operations are identical with embodiment 1.
Embodiment 4
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 35g/L, sodium tetraborate 18g/L, vitamins C 5g/L, beta-mercaptoethanol 28g/L, sucrose 300g/L; Add hydrochloric acid and regulate pH value to 5.
An extracting method for oil palm leaves total protein matter, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 6 (V/W) times amount, and room temperature whirlpool concussion 6min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, step (2) adds 12mL oil palm leaves total protein matter special extract, room temperature whirlpool concussion 6min, removes supernatant liquor after centrifugal 12min, repeats this step 1 time under temperature 5 DEG C, centrifugal force 10000RCF condition; All the other operations are identical with embodiment 1.
Embodiment 5
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 37g/L, sodium tetraborate 19g/L, vitamins C 10g/L, beta-mercaptoethanol 22g/L, sucrose 200g/L; Add hydrochloric acid and regulate pH value to 4.5.
An extracting method for oil palm leaves total protein matter, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 5 (V/W) times amount, and room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, step (2) adds 10mL oil palm leaves total protein matter special extract, and room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal 10min under temperature 3 DEG C, centrifugal force 17000RCF condition; All the other operations are identical with embodiment 1.
In above embodiment, if thick substances is more in supernatant liquor, step (2) can be repeated 1-2 time.The special extract of oil palm leaves total protein of the present invention and extracting method effect expedition
Get 2g oil palm blade respectively, respectively according to extracting method and the conventional BPP method extraction leaves total protein matter of the present invention's three embodiments, each process repeats 10 operations.Obtaining extracting solution of protein to extraction adopts Bradford method to carry out quantitative assay, and calculate its mean value, result is as shown in table 1.
Table 1 extracting method extraction effect of the present invention compares
As shown in Table 1, the method that three embodiments of the present invention provide, the supernatant liquid measure that proteins extraction obtains, protein concn are all significantly increased.The ultimate capacity of 2g oil palm leaves total protein matter is all at more than 3mg; And adopting BPP method, the protein ultimate capacity of gained is at about 2mg.
Adopt BPP method, the supernatant fluid color extracting gained is faint yellow, and obvious thickness, foreign matter content is high, brings very large impact to follow-up research.And the treatment process described in the present invention's three embodiments, do not extract the supernatant liquor that obtains not or only have the thick substances of minute quantity, color is clarified, and foreign matter content reduces greatly, facilitates the carrying out of subsequent experimental research.In addition, extracting method provided by the present invention, extract operation not by the restriction of envrionment temperature, temperature high (summer), temperature low (winter) all can carry out the extraction of oil palm leaves total protein matter, greatly improve the efficiency that oil palm leaves total protein extracts, the correlative study also for carrying out oil palm leaf protein under Various Seasonal and weather condition creates condition.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.