阅读说明：本技术 一种原花青素-多糖新型天然固体组合抑菌剂及制备方法和应用 (Novel natural solid combined bacteriostatic agent containing procyanidine and polysaccharide as well as preparation method and application of bacteriostatic agent ) 是由 王静祎 汪超 吴茜 孙智达 谢笔钧 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种原花青素-多糖新型天然固体组合抑菌剂及制备方法和应用,组合抑菌剂由一定比例的原花青素和多糖组成,步骤是：A、按比例取原花青素和多糖；B、将原花青素和多糖置于混合容器中,混合均匀,得混合物；C、向混合物加入蒸馏水并均匀混合,再避光静置,获得复合物；D、将获得的复合物进行低温减压干燥,获得新型天然固体组合抑菌剂。同时,还涉及了天然固体组合抑菌剂在食品保鲜及保健食品中的应用。多糖的加入逆转了原花青素在低浓度下促进致病菌生长的不利效应,改善了原花青素的抑菌特性,制备方法简便、状态稳定,集合了原花青素和多糖的优点,使该组合抑菌剂可代替抗生素、防腐剂被应用于食品保鲜和保健食品中。(The invention discloses a proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent, a preparation method and application thereof, wherein the combined bacteriostatic agent consists of proanthocyanidin and polysaccharide in a certain proportion, and the steps are as follows: A. taking procyanidine and polysaccharide according to a proportion; B. placing procyanidine and polysaccharide in a mixing container, and mixing uniformly to obtain a mixture; C. adding distilled water into the mixture, uniformly mixing, and standing in a dark place to obtain a compound; D. and drying the obtained compound at low temperature under reduced pressure to obtain the novel natural solid combined bacteriostatic agent. Meanwhile, the application of the natural solid combined bacteriostatic agent in food preservation and health-care food is also related. The addition of the polysaccharide reverses the adverse effect of procyanidine for promoting the growth of pathogenic bacteria at low concentration, improves the bacteriostatic property of procyanidine, has simple and convenient preparation method and stable state, and integrates the advantages of procyanidine and polysaccharide, so that the combined bacteriostatic agent can replace antibiotics and preservatives to be applied to food preservation and health-care food.)

1. A proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent is characterized in that: the compound proanthocyanidin polysaccharide is compounded by proanthocyanidin and polysaccharide, and consists of the following raw materials in percentage by mass:

raw materials%

Procyanidin 15-85%

15-85% of polysaccharide;

a preparation method of a novel natural solid combined bacteriostatic agent comprises the following steps:

A. taking procyanidine and polysaccharide according to a certain proportion;

B. placing procyanidine and polysaccharide in a mixing container, and mixing uniformly to obtain a mixture;

C. slowly adding distilled water 1-10 times of the total mass of the mixture, uniformly mixing, and standing for 28-32min at 4 ℃ in a dark place to obtain a compound;

D. and (C) drying the compound obtained in the step (C) at low temperature and reduced pressure, wherein the temperature is controlled to be-10 to-50 ℃, and the pressure is controlled to be 0.1-5 pa, so as to obtain the novel natural solid combined bacteriostatic agent.

2. The novel natural solid proanthocyanidin-polysaccharide conjugate as set forth in claim 1, wherein the natural solid proanthocyanidin-polysaccharide conjugate as set forth is prepared by mixing:

raw materials%

Procyanidin 40-80%

20-60% of polysaccharide.

3. The novel natural solid proanthocyanidin-polysaccharide conjugate as set forth in claim 1, wherein the natural solid proanthocyanidin-polysaccharide conjugate as set forth is prepared by mixing:

raw materials%

Procyanidin 65-75%

25 to 35 percent of polysaccharide.

4. The novel natural solid proanthocyanidin-polysaccharide conjugate as set forth in claim 1, wherein the natural solid proanthocyanidin-polysaccharide conjugate as set forth is prepared by mixing:

raw materials%

Procyanidin 75%

25% of polysaccharide.

5. The novel natural solid proanthocyanidin-polysaccharide conjugate as set forth in claim 1, wherein the natural solid proanthocyanidin-polysaccharide conjugate as set forth is prepared by mixing: the procyanidin is extracted from Nelumbonaceae plant: lotus seed pot or lotus leaf or lotus seed shell or lotus root skin or lotus root node, grape or green snake fruit or lychee, procyanidin monomer compound catechin or epicatechin gallate or epigallocatechin gallate, and the mass ratio of procyanidin to polysaccharide is 1: 6-3: 1.

6. The novel natural solid proanthocyanidin-polysaccharide conjugate as set forth in claim 1, wherein the natural solid proanthocyanidin-polysaccharide conjugate as set forth is prepared by mixing: the polysaccharide comprises carboxymethyl pachyman: degree of substitution of carboxymethyl 0.20 to 0.95, or carboxymethyl chitosan: a degree of substitution of carboxymethyl 0.50 to 1.90, or pectin: the degree of esterification is 50-95%.

7. The use of the novel natural solid proanthocyanidin-polysaccharide conjugate as bacteriostatic agent for preserving food as claimed in claim 1.

8. The use of the novel natural solid combination bacteriostatic agent of procyanidin-polysaccharides as claimed in claim 1 in health food.

The technical field is as follows:

the invention belongs to the technical field of food preservation and functional food, and particularly relates to a novel natural solid combined bacteriostatic agent with synergistic effect, a preparation method of a solid combined bacteriostatic agent containing procyanidine and polysaccharide, and application of the solid combined bacteriostatic agent containing procyanidine and polysaccharide in food preservation and health-care food.

Background art:

food-borne diseases are a public health problem of great concern, and data statistics in recent years show that about one fourth of the world population faces the risk of infection with food-borne pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella, shigella and the like. The pathogenic bacteria are spread by means of polluting water, food and the like, grow and propagate during the growth, transportation and storage of the food, finally enter human bodies and cause diseases such as abdominal pain, diarrhea, enteritis and the like, thus seriously harming the health of consumers and bringing negative effects to the quality and shelf life of the food.

At present, the use of antibiotics and chemically synthesized antiseptics for killing or inhibiting the growth and reproduction of pathogenic bacteria is still considered to be an effective and preferred method for controlling the pollution of food-borne pathogenic bacteria and prolonging the shelf life of food. However, it is worth noting that the antibiotics added into the feed are finally enriched along with the ingestion of meat, eggs, milk and other livestock and poultry and processed products thereof into human bodies, and not only do the antibiotics harm human health (such as anaphylactic reaction and the like), but also the long-term high antibiotic residue can cause the organism to generate bacterial resistance, thereby posing a great threat to public health. On the other hand, the use of chemically synthesized preservatives in food production and packaging may increase the risk of the body to develop diseases such as epilepsy, paralysis, mental retardation, hypertension and cancer. Therefore, the search for natural, highly effective and safe antibacterial agents to prevent food contamination caused by pathogenic bacteria and the consequent overdose and illegal addition of antibiotics and antiseptics is one of the important problems to be solved in the field of food safety.

With the maturation of natural product exploration and development and the continuous improvement of life quality of people, consumers are more willing to accept healthy, safe and green natural preservatives. Therefore, active ingredients in fruits, vegetables and herbs, which are safe, cheap, have little side effects, are bacteriostatic, slightly or non-toxic, and are not easy to have drug resistance, have attracted attention of many researchers. Procyanidins is a secondary metabolite widely distributed in plants and fruits and vegetables, and is a major flavonoid compound ingested by the human body through daily diet. The basic structural unit is formed by connecting (+) -catechin or (-) -epicatechin with each other by C-C bonds, procyanidin can be divided into oligomers (polymerization degree is 1-4) and polymers (polymerization degree is more than 5) according to the number (polymerization degree) of catechin or epicatechin, and the oligomers can be divided into A-type procyanidin (connected by C4-C6 or C4-C8 bonds) and B-type procyanidin (connected by C2-O-C7 or C2-O-C5 bonds) according to the connection mode among the units. Most fruit and vegetable plants in nature such as receptaculum nelumbinis, green snake fruits and the like are rich in B-type procyanidine, and a small part of foods such as litchis, cranberries, cinnamon and the like mainly contain A-type procyanidine. In recent years, due to the nature of plant polyphenols and the broad spectrum of bacteriostatic activity, many studies have been focused on the bacteriostatic activity of plant polyphenols such as procyanidins, and some progress has been made. The experimental results of the previous laboratory show that the procyanidin from lotus seedpod has excellent inhibition effect on the growth of various enterotoxigenic escherichia coli (K88ac, F18ac and K99), and the MIC values are different from 0.8-1.5 mg/mL; has extremely low MIC values (0.020-0.047mg/mL) for four strains of staphylococcus aureus, such as ATCC25923, ATCC27217 and the like. However, a great deal of research shows that the polyphenols can promote the growth of pathogenic bacteria at a certain concentration. Catechin as one of the important monomeric compounds of procyanidine can promote the growth of Escherichia coli in a low concentration range of 0.05-1.6mg/mL, and the previous researches in our laboratory also show that the procyanidine of lotus has a growth promoting effect on enterotoxigenic Escherichia coli K88ac, F18ac and K99 when the procyanidine of lotus is less than 0.8mg/mL, so that the reason for the adverse phenomenon is not exactly concluded, and the solution is not reported.

In conclusion, polyphenols represented by procyanidins have great potential to be applied to the fields of food safety and food preservation as natural and efficient bacteriostatic agents. However, the prior art has the problem that the adverse phenomenon that the low-concentration procyanidin promotes the growth of pathogenic bacteria greatly limits the application of polyphenols represented by procyanidin in the aspect of resisting harmful microorganisms. In recent years, the synergistic effect of polyphenol and another common natural source active substance, namely polysaccharide, is gradually emphasized, and researches show that the non-covalent interaction (mainly comprising hydrophobic, hydrogen bond, electrostatic and molecular field interaction and the like) between polyphenol and certain polysaccharide has important significance in improving the biological activity of the compound. Research shows that effective interaction can change the physical, chemical and structural characteristics of polyphenol and polysaccharide, so as to have positive influence on the biological activity of the compound.

Therefore, on the basis that the synergistic effect which is beneficial to improving the biological activity is generated between polyphenol and polysaccharide discovered in the early stage of a laboratory, on the premise that the polyphenol and the polysaccharide generate non-covalent interaction, a plurality of natural source procyanidins and polysaccharides with biological activities such as bacteriostasis and the like are screened out through a large number of researches and experiments, and the novel natural solid combined bacteriostatic agent of procyanidins and polysaccharide with the synergistic effect is provided. The novel combined bacteriostatic agent has excellent bacteriostatic activity and application expansibility, and simultaneously utilizes the non-covalent interaction between polysaccharide and procyanidine to successfully solve the negative effect that procyanidine at low concentration promotes the growth of pathogenic bacteria, which has not been reported yet before, so that a theoretical basis and a scientific basis are provided for developing procyanidine-polysaccharide compounds into natural and efficient antibacterial agents and replacing antibiotics and preservatives. In addition, the procyanidine and the polysaccharide have proved to have excellent organism health improving capability in vivo, can play roles in multiple aspects such as oxidation resistance, inflammation resistance, intestinal flora regulation and the like, and further enhances the actual application range and effect of the combined bacteriostatic agent.

Disclosure of Invention

The invention aims to provide a novel proanthocyanidin-polysaccharide natural solid combined bacteriostatic agent with a synergistic effect, wherein the proanthocyanidin and the polysaccharide have a remarkable synergistic bacteriostatic effect through non-covalent interaction, so that the combined bacteriostatic agent has better bacteriostatic activity compared with the combined bacteriostatic agent which is singly used with the natural functional factors. In addition, the polysaccharide is utilized for the first time to reverse the adverse effect of procyanidine for promoting the growth of pathogenic bacteria under low concentration, the availability and the application range of procyanidine and polysaccharide as effective antibacterial components are widened, and the possibility that the procyanidine-polysaccharide composition is applied to the field of food instead of antibiotics and preservatives is increased.

The invention also aims to provide a preparation method of the novel natural solid combined bacteriostatic agent of procyanidin-polysaccharide with synergistic effect, which is easy to implement and simple and convenient to operate, can overcome the defects of the prior art, adopts procyanidin with better bacteriostatic activity and polysaccharide (including carboxymethyl pachyman or carboxymethyl chitosan or pectin) which is another natural functional factor with bacteriostatic effect to carry out non-covalent interaction through special blending to generate the synergistic effect beneficial to the bacteriostatic activity, not only improves the bacteriostatic ability of procyanidin, but also successfully solves the important problem that low-concentration procyanidin promotes the growth of pathogenic bacteria for the first time, so that the compound has better bacteriostatic property.

The invention also aims to provide the application of the novel natural solid combined bacteriostatic agent of procyanidine and polysaccharide with synergistic effect in food preservation and health-care food, wherein procyanidine and certain polysaccharides (including carboxymethyl pachyman, carboxymethyl chitosan and pectin) are used as functional active factors of natural sources, and the bacteriostatic agent has good adaptability in different food media, such as liquid, solid and powdered foods. In addition, the combined bacteriostatic agent not only has excellent growth inhibition effect on pathogenic bacteria in vitro, but also has better biological activities of intestinal oxidation resistance, anti-inflammation, intestinal flora regulation and the like in vivo after being taken. Thereby playing multiple effects of keeping food fresh, improving food safety, improving body health and the like.

In order to achieve the purpose, the invention adopts the following technical measures:

a proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent with synergistic effect is characterized by being prepared by compounding proanthocyanidin and polysaccharide double components and consisting of the following raw materials in percentage by mass:

the mass percentage of the raw materials is%

Procyanidin 15-85%

Polysaccharide 15-85%

A proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent with synergistic effect comprises the following raw materials (in a better range) by mass ratio:

the mass percentage of the raw materials is%

Procyanidin 40-80%

20-60% of polysaccharide;

a proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent with synergistic effect comprises the following raw materials (in a best range) by mass ratio:

the mass percentage of the raw materials is%

Procyanidin 65-75%

25-35% of polysaccharide;

a proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent with synergistic effect comprises the following raw materials by mass ratio (specific value):

the mass percentage of the raw materials is%

Procyanidin 75%

25% of polysaccharide.

The preparation of the novel natural solid combined bacteriostatic agent containing procyanidin and polysaccharide utilizes non-covalent interaction caused by special blending between procyanidin and polysaccharide, including hydrogen bond interaction, electrostatic interaction, hydrophobic interaction and the like, so as to generate a synergistic bacteriostatic effect. Therefore, when the two have the most significant intermolecular non-covalent interaction, namely the procyanidin accounts for 75% by mass, and the polysaccharide accounts for 25% by mass, the combined bacteriostatic agent has stronger bacteriostatic effect in vitro than the combined bacteriostatic agent in other mixing ratios. From the perspective of the chemical structure of the sample, in order to enable stronger intermolecular non-covalent interaction to be generated between the two, thereby improving the bacteriostatic activity of the composition, experiments and researches find that the galloyl is the most key chemical group in the procyanidin, and the interaction strength between the galloyl and the polysaccharide and the subsequent biological activity improvement effect can be obviously improved.

Therefore, for the combined bacteriostatic agent of the present invention, the source of the procyanidin may be a plant extract, or may be a monomeric compound of procyanidin. The procyanidine is extracted from plants of Nelumbonaceae (such as receptaculum Nelumbinis, folium Nelumbinis, testa Nelumbinis, or nodus Nelumbinis Rhizomatis) or fructus Vitis Viniferae, Zaocys, or fructus litchi; the procyanidin monomeric compound comprises catechin (C) or Epicatechin (EC) or epicatechin gallate (ECG) or Epigallocatechin (EGC) or epigallocatechin gallate (EGCG), and can also interact with polysaccharide (the mass ratio of procyanidin to polysaccharide is 1: 6-3: 1) to form a composition with stronger bacteriostatic activity, so the procyanidin monomeric compound also belongs to the content range of the invention. The polysaccharides comprise carboxymethyl pachyman (the degree of carboxymethyl substitution is 0.20-0.95) or carboxymethyl chitosan (the degree of carboxymethyl substitution is 0.50-1.90) or pectin (the degree of esterification is 50% -95%), the polysaccharides from natural sources have the common characteristic of certain negative charge and hydrophobicity, so that hydrogen bond interaction, electrostatic interaction and hydrophobic interaction can be generated between procyanidine and polysaccharides, wherein the anions and the hydrophobicity of the carboxymethyl pachyman are derived from the carboxymethyl with a certain degree of substitution (0.20-0.95), the anions and the hydrophobicity of the carboxymethyl chitosan are also derived from the carboxymethyl with a certain degree of substitution (0.50-1.90), and the pectin is derived from a certain degree of esterification (50% -90%).

The preparation method of the procyanidin refers to the prior art (patent grant number: CN 02115423.6), and is slightly modified according to different raw materials: processing fresh receptaculum Nelumbinis or grape skin or exocarpium litchi or green snake fruit into small pieces, adding 70% (V/V) ethanol or pure water at a feed-liquid ratio of 1:10(m/V), and leaching at 60-80 deg.C for 5 hr. And then, recovering ethanol from the filtrate by a rotary evaporator (water temperature is 45 ℃), passing the concentrated solution through AB-8 macroporous resin, removing impurities by 10 times of volume of distilled water, eluting by 70% (v/v) ethanol, passing the eluent through the rotary evaporator again, recovering ethanol from the eluent, placing the concentrated solution in a dark environment at 4 ℃ for precipitating overnight, carrying out suction filtration on the supernatant, extracting by ethyl acetate with a certain volume, carrying out rotary evaporation on the organic phase by the rotary evaporator (water temperature is 40 ℃) until the organic phase is dry, dissolving by a small amount of distilled water, and finally carrying out low-temperature reduced-pressure drying (-10 to-50 ℃ and 0.1-5 pa) to obtain a yellow or brownish red powdered procyanidin product.

The preparation method of the carboxymethyl pachyman or carboxymethyl chitosan is slightly modified based on the prior art (patent No. CN 105348407A). 3.0g of alkali-soluble pachymaran or chitosan was accurately weighed, dispersed in a mixed solution of 40mL of NaOH (20%) and 100mL of isopropanol, and stirred in an ice bath state until completely dissolved. A mixed solution of 100mL of isopropyl alcohol (containing 24.0g of monochloroacetic acid) and 40mL of 20% sodium hydroxide (NaOH) was slowly added dropwise thereto, and the mixture was stirred at room temperature (20-25 ℃ C., the same applies hereinafter) for about 3 hours. Subsequently, after the reaction system was warmed to 50 ℃ to 60 ℃ and stirred for 30min, a mixed solution of 100mL of isopropyl alcohol (containing 24.0g of monochloroacetic acid) and 40mL of 20% sodium hydroxide (NaOH) was again slowly dropped, and stirring was continued for 1h while maintaining the temperature range of 50 ℃ to 60 ℃. Finally, the reaction was rapidly cooled to room temperature by an ice bath and the derivatization was terminated by adjusting the pH of the reaction to 7.0 with 10% (v/v) glacial acetic acid solution and recovering the isopropanol by rotary evaporation. Then, the carboxymethyl pachyman aqueous solution is transferred into a cellulose dialysis bag (the molecular weight cut-off is 3500g/mol) and dialyzed by distilled water for 6 days, the solution is concentrated by a rotary evaporator, then protein is removed by a DEAE cellulose chromatographic column, and finally the solution is dried under reduced pressure at low temperature (-10 to-50 ℃ and 0.1pa to 5pa) to obtain white foam carboxymethyl pachyman or white powdery carboxymethyl chitosan.

The preparation method of the pectin is slightly modified based on the prior art (patent No. CN 105237653A), and since the pectin is widely distributed in peels of oranges, lemons, pomelos, mangoes and the like, the extraction of the mango peel pectin is taken as an example: peeling fresh mango, steaming mango peel at 95 deg.C for 1min for inactivating enzyme, drying at 50 deg.C to constant weight, pulverizing, and sieving with 100 mesh sieve. Mixing the sieved mango peel powder with a citric acid aqueous solution with the pH of 2.5 according to the feed-liquid ratio of 1:10g/mL, and readjusting the pH to 2.5. After a water bath at 80 ℃ for 2.5h, the extract solution was immediately cooled in an ice bath and centrifuged at 8000r/min for 15min at 4 ℃. Precipitating with 95% ethanol at a feed-liquid ratio of 1:4g/mL overnight, filtering, adding 50mL of acetone, washing for 5min, filtering, washing again under the same conditions, drying in a 50 ℃ oven to constant weight, dissolving the dried sample with 50mL of distilled water, dialyzing for 72h by using a 3500Da dialysis bag, concentrating by using a rotary evaporator, removing protein by using a DEAE cellulose chromatographic column, and finally drying at low temperature under reduced pressure (-10 to-50 ℃, 0.1-5 pa) to obtain the sample pectin.

It should be noted that the refined fractions of carboxymethyl polysaccharide and pectin obtained by different chromatography resins can also interact with procyanidins or monomeric compounds in a non-covalent manner, so as to improve the bacteriostatic activity.

A preparation method of a novel natural solid combined bacteriostatic agent of procyanidine-polysaccharide with synergistic effect comprises the following steps:

A. taking procyanidine and polysaccharide according to a certain proportion;

B. placing procyanidine and polysaccharide in a mixing container, and mixing uniformly to obtain a mixture;

C. slowly adding distilled water 1-10 times of the total mass of the mixture, uniformly mixing, and standing for 28-32min at 4 ℃ in a dark place to obtain a compound;

D. and D, drying the compound obtained in the step C at low temperature and reduced pressure, wherein the temperature is controlled to be-10 to-50 ℃, and the pressure is controlled to be 0.1pa to 5pa, so as to obtain the novel natural solid combined bacteriostatic agent.

In the technical measure steps, the most important is that the procyanidine and the polysaccharide are uniformly mixed and then are kept stand for 28-32min in a dark place at 4 ℃. In the process, polysaccharide and procyanidin can fully carry out non-covalent interaction to form a novel compound. Meanwhile, compared with the prior art, the technical measures of the steps mainly solve the growth promoting effect of the procyanidine on the pathogenic bacteria under low concentration, and simultaneously generate a synergistic effect under the influence of non-covalent interaction between the procyanidine and the pathogenic bacteria, so that the combined bacteriostatic agent has more excellent bacteriostatic activity and bacteriostatic property compared with the raw materials when the combined bacteriostatic agent is used alone.

Therefore, the novel natural solid combined bacteriostatic agent of procyanidine and polysaccharide has the advantages of high efficiency, no public nuisance, no drug resistance, no toxicity and the like, can replace antibiotics and preservatives to be applied in the field of food, improves the food safety by exerting bacteriostatic activity, and simultaneously forms a novel composition by combining two natural active substances with different bioactivity biases and utilizing the non-covalent interaction between the two natural active substances, so that the novel natural solid combined bacteriostatic agent can exert multiple effects of excellent intestinal oxidation resistance, anti-inflammation, immunity improvement, intestinal flora regulation and the like in vivo. Provides scientific basis for deeply analyzing the application of the compound use of natural active substances in the aspect of food safety.

The application of the proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent in health-care food comprises the following steps:

A. procyanidine is extracted from plants of Nelumbonaceae (Lotus seed pod or lotus leaf or lotus seed shell or lotus root skin or lotus node) or grape skin or litchi shell or green snake fruit (patent authorization number: CN 02115423.6); carboxymethyl pachymaran or carboxymethyl chitosan is slightly modified to be subjected to carboxymethylation derivatization on the basis of the prior art (patent No. CN105348407A) so as to improve the water solubility and the biological activity of the carboxymethyl pachymaran or carboxymethyl chitosan, and has no toxic or side effect; pectin is slightly modified based on the prior art (patent No. CN 105237653A), and is extracted from pericarp of citrus, lemon, pomelo, and mango (conventional technology).

B. The procyanidin and polysaccharide are used as main medicinal active ingredients in health food, and the procyanidin-polysaccharide composition and solid or liquid excipient or adjuvant (such as starch, dextrin, glycerol, xylitol and the like) can be prepared into any one of pharmaceutically acceptable dosage forms of health food, food additive and dietary supplement, such as capsules, microcapsules, powder, tablets, oral liquid and the like by using modern pharmaceutical means. Adding a thickening agent beta-cyclodextrin into the procyanidin-polysaccharide composition, adding 5-10% of water for wetting, finally adding a disintegrating agent carboxymethyl starch sodium, granulating according to a conventional process, and encapsulating to obtain the health food containing the procyanidin-polysaccharide composition, wherein the health food can regulate intestinal flora and improve the intestinal health level after being eaten. Wherein the excipient or adjuvant is generally 40% -60% and the procyanidin-polysaccharide composition is 30% -50%.

The application of the proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent as a food additive in food preservation comprises the following steps:

A. the natural solid combined bacteriostatic agent is directly added into some foods which are easy to eat or are often eaten in a fresh state, such as beverages, milk, cheese and the like (the adding amount is usually 100-500 mg/kg), and the bacteriostatic agent can exert the food fresh-keeping effect on meat by means of spraying, smearing and the like.

B. The proanthocyanidin-polysaccharide composition has good bacteriostatic effect on perishable food, remarkably improves the safety of food, has the effects of resisting oxidation and inflammation, improving immunity, regulating intestinal flora and the like, and is beneficial to human health.

Preparing the procyanidin-polysaccharide combined bacteriostatic agent into 0.5-2g/L liquid with sterile distilled water, soaking meat in the fresh-keeping liquid at room temperature (20-25 deg.C) for 15-20min, draining water under sterile environment, and vacuum packaging. The results show that the use of the combination bacteriostatic agent can improve the microbial safety of meat during storage.

In the prior art, generally, due to the defect that polyphenols such as procyanidine and the like have obvious low concentration to promote the growth of pathogenic bacteria, and the fact that the actual use concentration of bacteriostatic substances is difficult to determine due to the complexity of environment and food when the bacteriostatic substances are used, the bacteriostatic effect of the phenols such as procyanidine and the like in food preservation is still to be demonstrated.

Therefore, compared with the prior art, the invention has the following advantages and effects:

by utilizing the non-covalent interaction which is beneficial to improving the biological activity and occurs between the procyanidine and the polysaccharide, the procyanidine and the polysaccharide can be used for inhibiting bacteria synergistically, and the unfavorable phenomenon that the procyanidine has low concentration and promotes the growth of pathogenic bacteria is eliminated. The food preservative can effectively replace antibiotics and antiseptics to improve food safety, and has the advantages of safety, no toxicity, no bacterial drug resistance and the like. And the existing results in our laboratory show that the composition can also improve the activity of probiotics in the yoghourt during the storage period, enhance the survivability of the probiotics and improve the quality of the yoghourt. Meanwhile, the research in our laboratory shows that the composition can also exert antioxidant activity during the storage period of meat, inhibit the protein oxidation and lipid oxidation of meat, and improve the quality of meat. In addition, the composition can also exert antioxidant activity after being ingested into a body along with food, and simultaneously improve the structure of intestinal flora and strengthen the health level of intestinal tracts.

Description of the drawings:

FIG. 1 is a graph (12h) showing the concentration-effect curve of procyanidins from Nelumbo nucifera for inhibiting the growth of Escherichia coli, Staphylococcus aureus, Salmonella, and Shigella.

FIG. 2 is a graph of concentration-effect curves of a carboxymethyl pachyman inhibiting the growth of E.coli, Staphylococcus aureus, Salmonella, and Shigella (12 h).

FIG. 3 is a graph of concentration-effect curves of a combined bacteriostatic agent of lotus procyanidins and carboxymethylpachymaran at different ratios for inhibiting the growth of Escherichia coli (12 h).

FIG. 4 is a schematic diagram of Isbologram analyzing the synergistic bacteriostatic effect of lotus procyanidin and carboxymethyl pachyman (12 h).

FIG. 5 is a graph of concentration-effect of the main fraction of procyanidins from Nelumbo nucifera (FIG. 5A) and the main fraction of carboxymethylpachymaran (FIG. 5B) alone on inhibition of E.coli growth (12 h). The procyanidin monomer comprises: catechin C, epicatechin EC, epicatechin gallate ECG, procyanidin dimers B1 and B2; the main fraction of carboxymethyl pachyman comprises CMP_W(CMP was adsorbed on DEAE-FF Sepharose and eluted with distilled waterThe following fractions) and CMP_N(CMP is a fraction adsorbed to DEAE-FF Sepharose and eluted with 0.5mol/L NaCl).

FIG. 6 shows CMP of main fractions of procyanidin (ECG) and carboxymethylpachymaran) with strong antibacterial activity_NThe formed combined bacteriostat has a synergistic bacteriostasis effect schematic diagram (12 h).

Detailed Description

Example 1:

a proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent with a synergistic effect is prepared from the following raw materials in percentage by mass:

raw materials by mass percent

Procyanidin 70%

30% of polysaccharide.

Experimental materials and methods of preparation of the compositions:

procyanidine is extracted from plants of Nelumbonaceae (Nelumbo nucifera Gaertn. or exocarpium litchi or fruit of Serpentis (patent No. CN 02115423.6); carboxymethyl pachymaran or carboxymethyl chitosan is slightly modified to be subjected to carboxymethylation derivatization on the basis of the prior art (patent No. CN105348407A) so as to improve the water solubility and the biological activity of the carboxymethyl pachymaran or carboxymethyl chitosan, and has no toxic or side effect; pectin is extracted from pericarp of citrus, lemon, grapefruit, mango, etc. slightly modified based on the prior art (patent No. CN 105237653A) (conventional technology).

A preparation method of a novel natural solid combined bacteriostatic agent of procyanidine-polysaccharide with synergistic effect comprises the following steps:

A. taking procyanidine and polysaccharide according to a certain proportion;

B. placing procyanidine and polysaccharide in a mixing container, and mixing uniformly to obtain a mixture;

C. slowly adding distilled water 1-10 times of the total mass of the mixture, uniformly mixing, and standing for 28-32min at 4 ℃ in a dark place to obtain a compound;

D. and C, drying the compound obtained in the step C at low temperature and reduced pressure, wherein the temperature is controlled to be-10 or-20 or-25 or-38 or-45 or-50 ℃, and the pressure is controlled to be 0.1pa or 0.5 or 1.1 or 1.6 or 2.4 or 3.5 or 4.5 or 5pa, so as to obtain the novel natural solid combined bacteriostatic agent.

Examples 2 to 6:

a proanthocyanidin-polysaccharide novel natural solid combined bacteriostatic agent with a synergistic effect is composed of the following raw materials in percentage by mass:

raw materials and examples	2	3	4	5	6
						Procyanidins	15	30	50	60	72
Polysaccharides	85	70	50	40	28
						Total of	100	100	100	100	100

The preparation procedure was the same as in example 1:

example 7:

the determination of the concentration-effect curve of the individual action of the procyanidin and the polysaccharide on pathogenic bacteria and the MIC (minimum inhibitory concentration)/MBC (minimum bactericidal concentration) value

1. Materials and methods

1.1 Experimental strains and activation and culture

The four pathogenic bacteria (Escherichia coli, salmonella, staphylococcus aureus and shigella, provided by agricultural institute of Hubei province) frozen stock solution is subjected to streak culture overnight (37 ℃) on an LB agar culture medium plate through an inoculating loop, a single colony is picked and subjected to streak culture again overnight, and the plate after secondary activation is stored in an environment at 4 ℃. The single colonies used during the experiment were all taken from bacterial plates activated every three weeks to ensure the viability of the bacteria.

After activation, a single pathogenic bacterium colony is taken to be placed in a sterile LB (Luria-Bertani) broth culture medium and is placed in a water bath shaker (200rpm/min) to be cultured at constant temperature (37 ℃) until logarithmic phase (6-8 h). Subsequently, the culture was centrifuged (6300g/min, 5min) at 4 ℃ to collect the bacteria, washed twice with sterile physiological saline and resuspended in sterile physiological saline to prepare a bacterial suspension. Subsequently, the OD at 600nm is measured in a 0.5McFarland turbidity tube (concentration 10)⁸Approximate OD value of CFU/mL bacterial suspension) as a reference, and measuring the OD600 of the bacterial suspension and adjusting the concentration by sterile physiological saline to obtain the concentration of 10⁸CFU/mL of initial bacterial suspension of pathogenic bacteria.

1.2 bacteriostatic concentration-effect curves of procyanidins and carboxymethyl pachyman

Method for treating procyanidine of lotus and carboxymethyl pachyman by microporous broth methodThe effect of sugars on the growth curves of the four pathogens was determined. The samples were dissolved in MH (Mueller-Hinton) broth and filtered through a 0.2 μm sterile filter, then 200 μ L of the sample solution was added to a 96-well plate, and the sample solution (200 μ L) was obtained at a concentration ranging from 5 to 0.078125mg/mL by a gradient dilution method. Pathogenic bacteria in the logarithmic growth phase were prepared as described in 1.1, and 10. mu.L of inoculum was added to 96-well plates to give a final bacterial concentration of about 5X 10 per well⁵CFU/mL. Meanwhile, MH broth medium was used as a blank control instead of the sample solution. And finally, placing the 96-well plate in a constant-temperature incubator at 37 ℃ for incubation for 12h, and measuring the OD600 value by a microplate reader every 2h to obtain the pathogenic bacteria growth curve under the influence of different samples. The experiment was independently repeated three times, each time in triplicate. The bacteriostatic rate is calculated by the following formula:

wherein A is₀Represents the OD600 value determined in 12h for the blank control group, and A_SRepresents the OD600 value determined in 12h of the sample group.

1.3 MIC/MBC value of inhibition of lotus procyanidin and carboxymethyl pachyman.

The MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of the different samples against the four pathogenic bacteria were determined by broth dilution. The samples were dissolved in MH broth and filtered through sterile filter membranes, transferred to 96-well plates and diluted in gradient. Subsequently, the bacterial suspension in logarithmic growth phase was added to 96-well plates so that the final bacterial concentration per well was about 5X 10⁵CFU/mL, and the sample concentration is 5-0.078125 mg/mL. Finally, the 96-well plate was incubated at 37 ℃ for 15 h. Where MIC is defined as the lowest concentration that inhibits pathogenic bacterial growth (OD 600 before and after incubation is unchanged), MBC is defined as the lowest concentration at which the solution in the wells is plated after 15h of incubation and incubation continues for 20h, where no colonies grow visible to the naked eye. The experiment was independently repeated three times, with three replicates for each assay.

2. Results and analysis

2.1 MIC/MBC value of inhibition of lotus procyanidin and carboxymethyl pachyman.

TABLE 1 minimum inhibitory and bactericidal concentrations of procyanidins from Nelumbo nucifera and Poria carboxymethylpachymaran against four pathogenic bacteria

^aMIC, MBC unit is mg/mL;^b- -means that MIC and MBC of the sample were not detected

The results in table 1 show that the procyanidin from lotus seeds not only can completely inhibit the growth of pathogenic bacteria, but also has a bactericidal effect. In addition, procyanidins from lotus seedpod have the lowest MIC and MBC for Staphylococcus aureus and the highest MIC and MBC for Shigella. Meanwhile, the MIC and MBC of the carboxymethyl pachyman to the four pathogenic bacteria are not detected in the experimental concentration range, which indicates that the inhibition effect of the carboxymethyl pachyman to the growth of the four pathogenic bacteria is far inferior to that of the lotus procyanidin.

2.2 bacteriostatic concentration-effect curve of flos Nelumbinis procyanidin and carboxymethyl pachyman

After independently acting on the four pathogenic bacteria (12h), the procyanidine from the lotus seeds plays a good role in inhibiting growth and is influenced by the types of the pathogenic bacteria (figure 1). Wherein the IC of Escherichia coli is inhibited₅₀The value was 0.98mg/mL, 1.02mg/mL for Salmonella, 1.82mg/mL for Shigella, and 0.284mg/mL for Staphylococcus aureus. In addition, the procyanidin from lotus seedpod has the adverse effect of promoting proliferation at low concentration on tested pathogenic bacteria: the growth of the escherichia coli is promoted from 0.8mg/mL, and the highest growth promotion rate is 27.5%; the growth of staphylococcus aureus is promoted from 0.26mg/mL, and the highest growth promotion rate is 36.8%; the growth of the Shigella is promoted from 1.47mg/mL, and the highest growth promotion rate is 35.4%; the growth of the salmonella is promoted from 0.59mg/mL, the highest growth promotion rate is 32.7%, which is obviously not beneficial to the application of the bacteriostatic activity of the procyanidine. And for the carboxymethyl pachymaran (figure 2), the carboxymethyl pachymaran has certain inhibition effect on the growth of tested pathogenic bacteria after 12 hours of action. Wherein the shigella is best inhibited from growingThe effect is that the highest inhibition rate can reach 77.54 percent, and IC₅₀A value of 0.82 mg/mL; the highest inhibition rate for Escherichia coli is 54.12%, IC₅₀The value was 2.48 mg/mL; the highest inhibition rate to staphylococcus aureus is 51.23 percent, IC₅₀The value was 4.93 mg/mL; the highest inhibition rate of salmonella is only 28.02%. In addition, the low concentration of carboxymethyl pachyman does not have the phenomenon that the low concentration promotes the growth of pathogenic bacteria.

Example 8:

the determination of the synergistic bacteriostatic effect between the procyanidine and the polysaccharide in the natural solid combined bacteriostatic agent is disclosed.

1. Materials and methods

1.1 Experimental strains and activation and culture

Same as in example 7.

1.2 inhibition effect of lotus procyanidine-carboxymethyl pachyman combination bacteriostat on growth of escherichia coli

The specific experimental method of the synergistic bacteriostatic effect of the carboxymethyl pachyman and the lotus procyanidin (12h) is the same as that of example 7. Wherein, the preparation process of the carboxymethyl pachyman-lotus procyanidin composition is described according to the description, the carboxymethyl pachyman-lotus procyanidin composition is obtained by compounding two samples according to a certain proportion and drying the two samples at low temperature under reduced pressure, then the composition powder is dissolved in MH culture medium according to a certain initial concentration, and the mixture is mixed in equal volume according to a certain proportion after being sterilized by a 0.2 mu m microporous membrane. The experiment was independently repeated three times with three replicates for each assay.

1.3 measurement of synergistic antibacterial effect between procyanidine of lotus and carboxymethyl pachyman

According to the concentration-bacteriostasis rate curve and data when the experiment is carried out for 12h, the synergistic bacteriostasis between the carboxymethyl pachyman and the lotus procyanidin is researched through Isobologram analysis (Isobologram analysis), and the synergistic effect value (gamma) is calculated through a formula:

wherein the IC_50AmixAnd IC_50BmixIs IC of sample A and sample B in the complex₅₀A value; IC (integrated circuit)_50AAnd IC_50BFor IC of sample A and sample B when used alone₅₀The value is obtained. If γ is 1, it means that there is an additive effect between the two; if gamma is<1, the synergistic effect is shown between the two, and the smaller the gamma value is, the stronger the synergistic effect is; if gamma is>1, there is antagonism between the two.

2. Results and analysis of the experiments

2.1 inhibition effect of the lotus procyanidin-carboxymethyl pachyman combination bacteriostatic agent on growth of escherichia coli

The common bacteriostatic effect of flos Nelumbinis procyanidin and carboxymethyl pachyman on Escherichia coli for 12h is shown in FIG. 3 (flos Nelumbinis procyanidin: carboxymethyl pachyman, w/w). For the compositions with different proportions, the bacteriostatic effect of the compounds on the escherichia coli is changed compared with that of the lotus procyanidin alone. For the combined bacteriostatic agents, the introduction of carboxymethyl pachyman decreased the absolute concentration of procyanidins from lotus, resulting in an increase in the MIC of the composition (table 2), i.e. higher doses were required to completely inhibit the growth of e. And the slave IC₅₀The IC is shown when the two are combined at 3:1(w/w)₅₀The value (0.26mg/mL) is far lower than the effect (0.98mg/mL) of the lotus procyanidin when the lotus procyanidin is used alone, and the two show certain synergistic bacteriostasis. While at the remaining proportion the complex inhibits the growth of E.coli₅₀The value is increased compared to when procyanidins from lotus seeds are used alone.

2.2 measurement of synergistic antibacterial Effect between procyanidin of Lotus and carboxymethyl pachyman

And further analyzing and quantifying the synergistic antibacterial effect between the lotus procyanidin and the carboxymethyl pachyman through an isobologram. As can be seen from FIG. 4, the IC's of the compositions in different ratios₅₀All spots are on IC with separate action of procyanidin from flos Nelumbinis and carboxymethyl pachyman₅₀The lower left of the line of addition of values together means that at these formulation ratios, there is synergistic bacteriostatic activity between the two. As can be seen from the calculation results of the synergy value γ (table 2, in which a lower γ value indicates a stronger synergy), the synergy value between both is less than 1, meaning that a synergy exists between them. Especially when the two are compounded at 3:1,the compound takes procyanidin as a main body and is supplemented with the synergistic effect generated by carboxymethyl pachyman, so that the composition has the lowest IC₅₀And MIC values, with the best synergy (lowest gamma values).

TABLE 2 MIC values and synergistic Effect (γ) values for the composition of Nelumbo nucifera procyanidin-carboxymethylpachymaran against E.coli

^aThe compounding ratio is LSOPC: CMP (w/w);^bIC₅₀and^cMIC unit is mg/mL

Example 9:

the application of the natural solid combined bacteriostatic agent in the health food comprises the following steps:

A. procyanidine is extracted from plants of Nelumbonaceae (Nelumbo nucifera Gaertn. or exocarpium litchi or fruit of Serpentis (patent No. CN 02115423.6); carboxymethyl pachymaran or carboxymethyl chitosan is slightly modified to be subjected to carboxymethylation derivatization on the basis of the prior art (patent No. CN105348407A) so as to improve the water solubility and the biological activity of the carboxymethyl pachymaran or carboxymethyl chitosan, and has no toxic or side effect; pectin is extracted from pericarp of citrus, lemon, grapefruit, mango, etc. slightly modified based on the prior art (patent No. CN 105237653A) (conventional method).

B. The procyanidin and polysaccharide are used as main bacteriostatic active ingredients in health food, and the procyanidin-carboxymethyl pachymaran composition and certain solid or liquid excipient or adjuvant (such as starch, dextrin, glycerol, xylitol and the like) can be prepared into any one of pharmaceutically or health food, food additive and dietary supplement acceptable dosage forms of capsules, microcapsules, powder, tablets, oral liquid and the like by using modern pharmaceutical means, wherein the excipient or adjuvant such as starch, dextrin, glycerol and the like usually accounts for 40% or 45% or 50% or 55% or 60%, and the procyanidin-polysaccharide composition accounts for 30% or 35% or 40% or 45% or 50%. The health food has certain application in regulating intestinal flora, inhibiting growth and adhesion of pathogenic bacteria and improving intestinal health level.

Adding a thickening agent beta-cyclodextrin into the procyanidin-polysaccharide composition, adding 5-10% of water for wetting, finally adding a disintegrating agent carboxymethyl starch sodium, granulating according to a conventional process, and encapsulating to obtain the health food containing the procyanidin-polysaccharide composition. The results show that the capsule can effectively regulate intestinal flora and improve the intestinal health level after being eaten.

Example 10:

the application of the natural solid combined bacteriostatic agent as a food additive in food fresh keeping comprises the following steps:

A. procyanidine is extracted from plants of Nelumbonaceae (Nelumbo nucifera Gaertn. or exocarpium litchi or fruit of Serpentis (patent No. CN 02115423.6); carboxymethyl pachymaran or carboxymethyl chitosan is slightly modified to be subjected to carboxymethylation derivatization on the basis of the prior art (patent No. CN105348407A) so as to improve the water solubility and the biological activity of the carboxymethyl pachymaran or carboxymethyl chitosan, and has no toxic or side effect; pectin is extracted from pericarp of citrus, lemon, grapefruit, mango, etc. slightly modified based on the prior art (patent No. CN 105237653A) (conventional method).

B. The procyanidin-polysaccharide combined bacteriostatic agent is directly added into beverages, milk, cheese and the like in an aseptic state, the adding amount is 500mg/kg, and after the materials are uniformly mixed, the materials are stored for later use under the environment of a certain temperature and the like according to the requirements of different kinds of food. For meat and fruits and vegetables, the proanthocyanidin-polysaccharide combined bacteriostatic agent is uniformly sprayed and smeared on the surface of food, and then the food is placed in a fresh-keeping bag for low-temperature refrigeration or frozen storage for later use. Preparing the procyanidin-polysaccharide combined bacteriostatic agent into 0.5-2g/L liquid with sterile distilled water, soaking meat in the fresh-keeping liquid at room temperature (20-25 deg.C) for 15-20min, draining water under sterile environment, and vacuum packaging. The results show that the combined bacteriostatic agent can not only improve the bacteriostatic activity, but also improve the microbial safety of the meat during storage. Meanwhile, the composition can also exert antioxidant activity during the storage period of meat, inhibit protein oxidation and lipid oxidation of meat, and improve the quality of meat.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and it is to be understood that the present invention is not limited to the above description, but is intended to cover all modifications, equivalents, and improvements, which are within the spirit and scope of the present invention.