CN100376684C

CN100376684C - Method for preparing bacteriostatic peptide from low-grade tobacco leaf protein

Info

Publication number: CN100376684C
Application number: CNB2006100346931A
Authority: CN
Inventors: 赵谋明; 潘伟才; 崔春; 饶国华; 周瑞
Original assignee: South China University of Technology SCUT; China National Tobacco Corp Guangdong Branch
Current assignee: South China University of Technology SCUT; China National Tobacco Corp Guangdong Branch
Priority date: 2006-03-28
Filing date: 2006-03-28
Publication date: 2008-03-26
Anticipated expiration: 2026-03-28
Also published as: CN1827773A

Abstract

本发明涉及用低次烟叶蛋白制备抑菌肽的方法，包括：利用碱溶酸沉法提取低次烟叶中的蛋白质制备烟叶蛋白浆；采用复合蛋白酶对烟叶蛋白浆进行限制性酶解后，灭酶，制备烟叶蛋白酶解液；采用超滤膜对烟叶蛋白酶解液进行分级分离获得烟叶蛋白抑菌肽混合液；将烟叶蛋白抑菌肽混合液冷冻干燥或喷雾干燥；得到的烟叶蛋白抑菌肽混合物能有效抑制革兰氏阴性菌的生长繁殖，最低抑菌浓度为0.05%，可以作为食品添加剂广泛应用于食品及相关领域中；本发明通过碱溶酸沉法可显著提高烟叶蛋白的提取率；通过生物酶解可有效控制蛋白的降解程度；通过超滤膜分离酶解液富集生物活性肽，简化分离操作工艺，提高生物活性肽的有效浓度。The invention relates to a method for preparing bacteriostatic peptides from low-grade tobacco leaf protein, comprising: extracting protein in low-grade tobacco leaves by alkali-dissolving acid precipitation method to prepare tobacco leaf protein slurry; Enzyme to prepare enzymatic hydrolyzate of tobacco leaf protein; use ultrafiltration membrane to fractionate and separate the enzymatic hydrolyzate of tobacco leaf protein to obtain mixture of tobacco leaf protein bacteriostatic peptide; freeze-dry or spray-dry the mixture of tobacco leaf protein bacteriostatic peptide; obtain tobacco leaf protein bacteriostatic peptide The mixture can effectively inhibit the growth and reproduction of Gram-negative bacteria, the minimum inhibitory concentration is 0.05%, and can be widely used in food and related fields as a food additive; the invention can significantly improve the extraction rate of tobacco leaf protein by alkali-soluble acid precipitation method ; The degree of protein degradation can be effectively controlled through biological enzymatic hydrolysis; the bioactive peptide is enriched through the ultrafiltration membrane separation enzymatic solution, the separation operation process is simplified, and the effective concentration of the bioactive peptide is increased.

Description

The method for preparing bacteriostatic peptide with discarded tobacco leaf protein

Technical field

The present invention relates to the method for preparing bacteriostatic peptide with discarded tobacco leaf protein.

Background technology

China is tobacco planting and big producing country, and its cultivated area and output all occupy first place in the world.In the tobacco planting and the course of processing, produced tobacco wastes such as a large amount of discarded tobacco leafs, offal, offal and cigarette seed, account for 25% of tobacco leaf ultimate production.To the comprehensive utilization of discarded tobacco leaf resource, be subjected to domestic and international investigator's attention always.A lot of research reports are arranged: from discarded tobacco leaf, extract similar soy proteinaceous tobacco leaf protein matter, as food proteins source and high-quality feed additive.From the extracting method of tobacco leaf protein, the tobacco leaf protein that early stage research worker is extracted often has color and taste, and complex manufacturing, and investment is big, yields poorly, and cost is difficult to be accepted.Many afterwards researchers are purified to the tobacco leaf protein in the tobacco leaf, have obtained highly purified tobacco leaf protein, and wherein particularly Kung and Tso, Lowe have done very big contribution to the progressively simplification of FI albumen method of purification in the tobacco leaf.But make a general survey of the extracting method that domestic and international researcher institute research summary goes out, hypervelocity or the freezing centrifugation step of high speed all need be arranged, and process need be controlled at about 0～4 ℃ mostly; Also there are methods such as the isoelectrofocusing of employing, gel electrophoresis, ultrafiltration to extract tobacco leaf protein.Though these methods can obtain purified tobacco leaf protein, cost is too high, and the output that the unit time extracts is lower, is difficult to adapt to large-scale industrial production.The research work of China's development and use tobacco leaf protein is started late, and only the extracting method to tobacco leaf protein has carried out some preliminary discussions, but the technical barrier of mass-producing extraction tobacco leaf protein is still waiting to solve.

Summary of the invention

The objective of the invention is to defective, a kind of method for preparing bacteriostatic peptide with discarded tobacco leaf protein is provided at the prior art existence.The present invention is by high efficiency extraction discarded tobacco leaf protein (tobacco leaf protein extraction rate reached to 86.71%, exceed more than one times than the extraction yield in the bibliographical information), the enzymolysis process, gradocol membrane of effectively controlling tobacco leaf protein separate enzymolysis solution enriched biological bioactive peptide, have that technological operation is easy, production cost is low, without any pollute, gained bacteriostatic activity peptide biological activity is stable, minimum inhibitory concentration is 0.05%, advantages such as security height, can be widely used in the field of food, for the deep processing and utilization that hangs down this tobacco leaf provides a new approach.

Of the present inventionly prepare the method for bacteriostatic peptide, comprise the steps: with discarded tobacco leaf protein

(1) protein that extracts in the discarded tobacco leaf with the alkali extraction and acid precipitation method prepares the tobacco leaf protein slurry;

(2) after the employing compound protease carries out limited enzymatic hydrolysis to the tobacco leaf protein slurry, the enzyme that goes out, preparation tobacco leaf protein enzymolysis solution;

(3) adopting ultra-filtration membrane that the tobacco leaf protein enzymolysis solution is carried out fractional separation acquisition molecular weight is the tobacco leaf protein bacteriostatic peptide mixed solution of 1000Da-10000Da;

(4) with mixed solution lyophilize of tobacco leaf protein bacteriostatic peptide or spraying drying.

Preferred version is as follows:

In the step (1), it is last that discarded tobacco leaf is ground into 60-100 purpose tobacco leaf powder, and the tobacco leaf powder is mixed with 1: 10～15 with water, 40～60 ℃ of defibrinations, adjust pH value most 7.5～8.5 with sodium hydroxide, stir after 1 hour with the filtration of 200 order filter clothes, filtrate is that alkali is carried tobacco leaf protein solution; Filter residue adopts above-mentioned technology molten 1-2 time of alkali again, and filtering and impurity removing merges alkali and carries tobacco leaf protein solution; Adjust pH value to 2.5～3.5 that alkali is carried tobacco leaf protein solution with hydrochloric acid, behind 4-10 ℃ of sour down heavy 6-10hr, protein precipitation by centrifugation promptly gets the tobacco leaf protein slurry.

Centrifugal condition in the described protein precipitation by centrifugation is 4800rpm, 4-10 ℃, and 20-30min.

In the step (2), the tobacco leaf protein slurry is dissolved in the water, make protein concentration reach 4-89%, mix the back and the pH value of solution value is adjusted to 6.0-7.5 with 0.1M hydrochloric acid or 0.1M sodium hydroxide, temperature remains on 40-50 ℃, adds protease 3 000-4500U/g albumen, and degree of hydrolysis is controlled at 3-7%, the enzyme that goes out obtains the tobacco leaf protein enzymolysis solution.

The tobacco leaf protein enzymolysis solution that step (2) obtains must see through liquid after removing macromolecular substance by the ultra-filtration membrane of molecular weight 10000Da; The ultra-filtration membrane that with molecular weight cut-off is 1000Da carries out second ultrafiltration and rejects small-molecular peptides, amino acid and other impurity components seeing through liquid, promptly getting molecular weight is the tobacco leaf protein bacteriostatic peptide mixed solution of 1000Da-10000Da, and its lyophilize or spraying drying are preserved.

Enzyme described in the step (2) adopts Protamex ^TMOr papoid.

In the step (2), after enzyme digestion reaction finishes,, enzymolysis product is cooled to 25 ℃ at 100 ℃ of heating 10min enzyme that goes out.

The present invention compared with prior art, have following advantage: the albumen that the alkali extraction and acid precipitation method is extracted in the discarded tobacco leaf can significantly improve the discarded tobacco leaf protein extraction yield, compare with the organic solvent extraction method of being mentioned in the bibliographical information and the extraction yield of water extract method, protein extracting ratio is doubled many; By the palliating degradation degree of control discarded tobacco leaf protein, obtain having the biologically active peptides of obvious anti-microbial activity; By the gradocol membrane isolation technique, make its effective bacteriostatic peptide be able to enrichment, minimum inhibitory concentration is 0.05%.Technological operation of the present invention is easy, production cost is low, without any pollute, gained bacteriostatic activity peptide biological activity is stable, safe, can be widely used in the field of food.

Embodiment

Embodiment 1

The first step: it is last that discarded tobacco leaf is ground into 100 purpose tobacco leaf powders, mixes with 1: 10 with water, and 40 ℃ of defibrinations are adjusted pH value most 7.5 with sodium hydroxide, stirs after 1 hour with the filtration of 200 order filter clothes, and filtrate is that alkali is carried tobacco leaf protein solution.Filter residue adopts above-mentioned technology molten 2 times of alkali again, and filtering and impurity removing merges alkali and carries tobacco leaf protein solution; Adjust the pH value to 2.5 that alkali is carried tobacco leaf protein solution with hydrochloric acid, behind 4 ℃ of sour down heavy 6hr, 4800rpm, 4 ℃, centrifugal 20min, protein precipitation gets the tobacco leaf protein slurry, and its rate of recovery reaches 86.71%.

Second step: the discarded tobacco leaf protein slurry is dissolved in the water, makes protein concentration reach 4%, mix the back and with 0.1M hydrochloric acid the pH value of solution value is adjusted to 6.0, temperature remains on 40 ℃, adds compound protease (Protamex ^TM) 4500U/g albumen, degree of hydrolysis is controlled at 39%, obtains enzymolysis tobacco leaf protein liquid, 100 ℃ of heating 10min enzymes that go out;

The 3rd goes on foot: must see through liquid after the control of tobacco leaf protein enzymolysis solution is removed macromolecular substance by the ultra-filtration membrane of molecular weight 10000Da; The employing molecular weight cut-off is that the ultra-filtration membrane of 1000Da carries out second ultrafiltration rejecting small-molecular peptides, amino acid and other impurity components to seeing through liquid, promptly getting molecular weight is the tobacco leaf protein bacteriostatic peptide mixed solution of 1000Da-10000Da, its lyophilize is preserved, and its minimum inhibitory concentration is 0.09%.

Embodiment 2

The first step: it is last that discarded tobacco leaf is ground into 80 purpose tobacco leaf powders, mixes with 1: 12 with water, and 50 ℃ of defibrinations are adjusted pH value most 8 with sodium hydroxide, stirs after 1 hour with the filtration of 200 order filter clothes, and filtrate is that alkali is carried tobacco leaf protein solution.Filter residue adopts above-mentioned technology molten 2 times of alkali again, and filtering and impurity removing merges alkali and carries tobacco leaf protein solution; Adjust the pH value to 3.0 that alkali is carried tobacco leaf protein solution with hydrochloric acid, behind 4 ℃ of sour down heavy 6hr, 4800rpm, 4 ℃, centrifugal 20min gets the tobacco leaf protein slurry with protein precipitation, and its rate of recovery reaches 84.24%.

Second step: the discarded tobacco leaf protein slurry is dissolved in the water, make protein concentration reach 4-8%, mix the back and the pH value of solution value is adjusted to 7.0 with 0.1M sodium hydroxide, temperature remains on 45 ℃, add papoid 3000U/g albumen, degree of hydrolysis is controlled at 5%, obtains enzymolysis tobacco leaf protein liquid, 100 ℃ of heating 10min enzymes that go out;

The 3rd goes on foot: must see through liquid after the control of tobacco leaf protein enzymolysis solution temperature is removed macromolecular substance by the ultra-filtration membrane of molecular weight 10000Da; The employing molecular weight cut-off is that the ultra-filtration membrane of 1000Da carries out second ultrafiltration rejecting small-molecular peptides, amino acid and other impurity components to seeing through liquid, promptly getting molecular weight is the tobacco leaf protein bacteriostatic peptide mixed solution of 1000Da-10000Da, its lyophilize is preserved, and its minimum inhibitory concentration is 0.07%.

Embodiment 3

The first step: it is last that discarded tobacco leaf is ground into 60 purpose tobacco leaf powders, mixes with 1: 15 with water, and 50 ℃ of defibrinations are adjusted pH value most 8.5 with sodium hydroxide, stirs after 1 hour with the filtration of 200 order filter clothes, and filtrate is that alkali is carried tobacco leaf protein solution.Filter residue adopts above-mentioned technology molten 1 time of alkali again, and filtering and impurity removing merges alkali and carries tobacco leaf protein solution; Adjust the pH value to 3.0 that alkali is carried tobacco leaf protein solution with hydrochloric acid, behind 4 ℃ of sour down heavy 10hr, 4800rpm, 4 ℃, centrifugal 20min, protein precipitation promptly gets the tobacco leaf protein slurry, and its rate of recovery reaches 83.53%.

Second step: the discarded tobacco leaf protein slurry is dissolved in the water, make protein concentration reach 8%, mix the back and the pH value of solution value is adjusted to 8.5 with sodium hydroxide, temperature remains on 50 ℃, add papoid 4000U/g albumen, degree of hydrolysis is controlled at 7%, obtains enzymolysis tobacco leaf protein liquid, 100 ℃ of heating 10min enzymes that go out;

The 3rd goes on foot: must see through liquid after the control of tobacco leaf protein enzymolysis solution temperature is removed macromolecular substance by the ultra-filtration membrane of molecular weight 10000Da; The employing molecular weight cut-off is that the ultra-filtration membrane of 1000Da carries out second ultrafiltration rejecting small-molecular peptides, amino acid and other impurity components to seeing through liquid, promptly getting molecular weight is the tobacco leaf protein bacteriostatic peptide mixed solution of 1000Da-10000Da, its lyophilize is preserved, and its minimum inhibitory concentration is 0.05%.

Claims

1. a method for preparing bacteriostatic peptide with low-grade tobacco leaf protein, is characterized in that comprising the steps:

(1) Utilize the alkali-dissolving and acid-precipitating method to extract the protein in low-order tobacco leaves to prepare tobacco leaf protein slurry;

(2) After restrictive enzymolysis of the tobacco leaf protein slurry by using a compound protease, the enzyme is inactivated to prepare a tobacco leaf protein enzymatic hydrolysis solution;

(3) using an ultrafiltration membrane to fractionate the tobacco leaf protein enzymatic hydrolyzate to obtain a tobacco leaf protein bacteriostatic peptide mixture with a molecular weight of 1000Da-10000Da;

(4) Freeze-drying or spray-drying the tobacco leaf protein antimicrobial peptide mixture.

2. The method according to claim 1, characterized in that in step (1), after the low-grade tobacco leaves are crushed into 60-100 mesh tobacco leaf powder, the tobacco leaf powder and water are mixed at 1: 10-15, 40-60 Refining at ℃, adjusting the pH value to 7.5-8.5 with sodium hydroxide, stirring for 1 hour and then filtering with a 200-mesh filter cloth, the filtrate is the alkali-extracted tobacco leaf protein solution; Mix, combine the alkali-extracted tobacco leaf protein solution; adjust the pH value of the alkali-extracted tobacco leaf protein solution to 2.5-3.5 with hydrochloric acid, and after acid precipitation at 4-10°C for 6-10 hours, centrifuge to precipitate the protein to obtain tobacco leaf protein slurry.

3. The method according to claim 1, characterized in that in step (2), the tobacco leaf protein slurry is dissolved in water so that the protein concentration reaches 4-8%, and after mixing evenly, use 0.1M hydrochloric acid or 0.1M sodium hydroxide The pH value of the solution is adjusted to 6.0-7.5, the temperature is kept at 40-50° C., 3000-4500 U/g protein is added with protease, the degree of hydrolysis is controlled at 3-7%, and the enzyme is inactivated to obtain a tobacco leaf proteolysis solution.

4. the method according to claim 1 is characterized in that the tobacco leaf proteolysis solution obtained by step (2) is passed through the ultrafiltration membrane of molecular weight 10000Da to remove macromolecular substances and obtains permeate; The permeated liquid is subjected to secondary ultrafiltration by the membrane to remove small molecule peptides, amino acids and other impurity components, and the tobacco leaf protein bacteriostatic peptide mixture with a molecular weight of 1000Da-10000Da is obtained, which is freeze-dried or spray-dried for storage.

5. The method according to claim 2, characterized in that the centrifugation conditions in the centrifugal protein precipitation are 4800rpm, 4-10°C, 20-30min.

6. The method according to claim 1 or 3, characterized in that the enzyme in step (2) is Protamex ^™ .

7. The method according to claim 1 or 3, characterized in that in step (2), after the enzymolysis reaction is completed, the enzyme is extinguished by heating at 100° C. for 10 minutes, and the enzymolysis product is cooled to 25° C.