CN114931599B - A composition for inhibiting Staphylococcus aureus biofilm and its preparation method - Google Patents

Abstract

The invention discloses a composition for inhibiting staphylococcus aureus biofilm and a preparation method thereof, and relates to the technical field of medicine and food safety, wherein the composition for inhibiting staphylococcus aureus biofilm comprises the following components in percentage by weight: 60-75% of tea saponin, 10-20% of honeysuckle extract, 7-15% of echinacea extract, 5-10% of lemon extract and 3-5% of punicalagin; the preparation method comprises the following steps: s1: preparing tea-oil camellia saponin; s2: preparing honeysuckle extract; s3: preparing Echinacea purpurea extract; s4: preparing lemon extract; s5: preparing punicalagin; s6: preparing a composition; the composition is high-efficiency, low-toxicity and not easy to generate drug resistance, and the composition is a plant extract, can effectively inhibit biofilm formation of staphylococcus aureus, thereby preventing and treating infection and harm of staphylococcus aureus.

Description

A composition for inhibiting Staphylococcus aureus biofilm and its preparation method

technical field

The invention relates to the technology in the fields of medicine and food safety, in particular to a composition for inhibiting Staphylococcus aureus biofilm and a preparation method thereof.

Background technique

Staphylococcus aureus is the most common Gram-positive pathogenic bacteria in food, which can seriously endanger public health and human health; biofilm formation is the key to the persistence of Staphylococcus aureus under adverse environmental conditions Factors; Bacteria can usually resist the host's immune response after forming a biofilm. Compared with planktonic bacteria, they are more resistant to antibiotics and bacteriostatic agents; looking for natural, efficient and less resistant bacteriostatic substances , has broad application prospects in the development of anti-staphylococcus aureus drugs; therefore, in view of this situation, there is an urgent need to develop a composition and a preparation method for inhibiting staphylococcus aureus biofilm to meet actual needs.

Contents of the invention

In view of this, the present invention aims at the deficiency in the prior art, and its main purpose is to provide a composition for inhibiting Staphylococcus aureus biofilm by using the composition for inhibiting Staphylococcus aureus biofilm provided by the application and The preparation method provides a high-efficiency, low-toxicity, and non-drug-resistant composition, all of which are plant extracts, which can effectively inhibit the formation of biofilms of staphylococcus aureus, thereby preventing the infection and harm of staphylococcus aureus.

To achieve the above object, the present invention adopts the following technical solutions:

A kind of preparation method of the composition that suppresses Staphylococcus aureus biofilm, it comprises the following steps:

S1: Preparation of camellia oleifera saponin: crush the camellia oleifera cake after oil extraction, pass through a 50-100 mesh sieve, extract with the extractant according to the ratio of solid to liquid at 1:10, concentrate and dry the extract, dissolve it in water and carry out Filtration; eluting the filtrate with a macroporous adsorption resin, the eluent is 30%-95% ethanol, gradient elution, collecting the eluted liquid respectively, drying and concentrating to obtain camellia saponin;

S2: Preparation of honeysuckle extract: crush honeysuckle through a 50-100 mesh sieve to obtain honeysuckle powder, add 9 times the amount of water to the honeysuckle powder, mix well, extract in water at 70-80°C for 120 minutes, filter the extract, Concentrate the filtrate to obtain a concentrated solution; add 80%-85% ethanol to extract the concentrated solution for 6 days, and concentrate the extracted liquid to obtain a slurry; use supercritical _CO2 fluid extraction to extract the slurry to obtain honeysuckle extract thing;

S3: Preparation of Echinacea extract: crush Echinacea through a 50-100 mesh sieve to obtain Echinacea powder, add 20%-50% ethanol to the Echinacea powder, heat and reflux three times, filter and combine the filtrates to obtain ethanol extract and filter residue Two parts: add 8 times the amount of 1% lysozyme aqueous solution to the filter residue to extract again, filter, combine the lysozyme extract with the previous ethanol extract to obtain the Echinacea purpurea extract, and concentrate the extract to obtain the Echinacea purpurea extract;

S4: Preparation of lemon extract: crush lemon fruit through a 20-50 mesh sieve, add 3-3.5 times the weight of lemon fruit to reflux extraction for 3-4 times at 50-60°C to obtain an extract, and combine The extract is concentrated on a rotary evaporator to recover ethanol to obtain lemon extract;

S5: Preparation of punicalagin: crush the pomegranate peel through a 20-50 mesh sieve, add water to the crushed pomegranate peel for flash extraction, remove polysaccharides in the supernatant: concentrate the supernatant extract, add 3.5 times Ethanol with a volume of 70%-90%, standing at low temperature for 10 hours to precipitate, centrifuging, filtering, collecting the supernatant, and concentrating the supernatant to obtain punicalagin concentrate;

S6: Preparation of the composition: the tea saponin obtained in S1 above, the honeysuckle extract obtained in S2, the echinacea purpurea extract obtained in S3, the lemon extract obtained in S4, and the punicalagin obtained in S5 are in proportion mixing, adding a preservative to the mixture, and adjusting the pH to 7 to obtain a composition; the composition includes the following components by weight percentage: 60% to 75% of camellia saponin, 10% to 20% of honeysuckle extract, echinacea purpurea 7%-15% of the extract, 5%-10% of the lemon extract and 3%-5% of punicalagin; the honeysuckle extract contains 50%-90% of chlorogenic acid; the polysaccharide content in the echinacea purpurea extract is ≥ 21.50%, polyphenol content ≥ 8.70%, cichoric acid content ≥ 5.00%; total flavonoid content in the lemon extract ≥ 80%.

As a preferred solution: before the eluent is used for elution in the S1, distilled water with 5 times the amount of the resin and an ethanol solution with a concentration of 10% are used for elution to remove impurities in sequence.

As a preferred solution: the extractant in S1 is acetone with a concentration of 70%, the extraction temperature is 65° C., and the extraction time is 2 hours.

As a preferred solution: the S2 is leached twice in 70-80°C water, the supercritical _CO2 fluid extraction conditions: the pressure is 20-22mpa, the fluid ratio is 5-6%, and the temperature is 45-50°C , the time is 120 minutes.

As a preferred solution: in the S3, 20%-50% ethanol is added to the Echinacea purpurea powder, and then Ca(OH) ₂ is used to adjust the pH value to 10.

As a preferred solution: the condition for re-extracting the filter residue in S3 with 8 times the amount of 1% lysozyme aqueous solution is: soak the filter residue in 38° C. lysozyme aqueous solution for 10 hours.

As a preferred solution: the flash extraction conditions in S5 are: the ratio of pomegranate peel powder to water is 1:3; the extraction speed is 6000 rpm; the extraction time is 100 s.

As a preferred solution: when mixing the components in S6, the components include: 60%-75% camellia saponin, 10%-20% honeysuckle extract, 7%-15% echinacea extract, lemon Extract 5%-10% and punicalagin 3%-5%.

Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above-mentioned technical scheme that by adopting the composition and preparation method thereof for inhibiting Staphylococcus aureus biofilm provided by the application, providing high-efficiency, The composition is low-toxic and not easy to produce drug resistance. The composition is all plant extracts, which can effectively inhibit the biofilm formation of Staphylococcus aureus, thereby preventing the infection and harm of Staphylococcus aureus; during the preparation process of camellia saponin Among them, the camellia saponin extracted by the preparation conditions of the present application has high purity and effectively removes other impurities; the preparation of honeysuckle extract: the combination of alcohol precipitation and supercritical _CO2 fluid extraction has high yield and high purity, and can Fully extract the active ingredients of honeysuckle; Echinacea purpurea extract is obtained by extracting Echinacea purpurea medicinal materials with alkaline ethanol solution and lysozyme, which is beneficial to increase the content of polyphenols and cichoric acid in the extract, and greatly improves the efficacy of the obtained extract The preparation of lemon extract and punicalagin in this application simplifies the extraction process; in this composition, camellia saponin is the main component, and camellia saponin is extracted from camellia oleifera cake, a by-product of camellia oleifera processing, that is Camellia oleifera seed is extracted from the residue of camellia oleifera cake after oil extraction, which is a by-product utilization with low cost and sufficient raw materials; at the same time, camellia saponin has a significant effect on inhibiting the formation of Staphylococcus aureus biofilm.

Detailed ways

Example 1

A composition for inhibiting Staphylococcus aureus biofilm, comprising the following components by weight percentage: 60% camellia saponin, 15% honeysuckle extract, 15% echinacea extract, 5% lemon extract and 5% punicalagin %.

The honeysuckle extract contains 50%-90% of chlorogenic acid; the polysaccharide content in the Echinacea purpurea extract ≥ 21.50%, the polyphenol content ≥ 8.70%, and the cichoric acid content ≥ 5.00%; the total flavonoid content in the lemon extract ≥80%.

A kind of preparation method of the composition that suppresses Staphylococcus aureus biofilm, it comprises the following steps:

S1: Preparation of camellia oleifera saponin: crush the camellia oleifera cake after oil extraction, pass through a 50-mesh sieve, extract with an extractant at a ratio of solid to liquid of 1:10, concentrate and dry the extract, dissolve it in water and filter; Use macroporous adsorption resin to elute the filtrate, the eluent is 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol and 95% ethanol, gradient elution, collect the eluted liquid respectively, dry and concentrate Camellia oleifera saponin is obtained; the extractant is acetone with a concentration of 70%, the extraction temperature is 65°C, and the extraction time is 2h; before using the eluent for elution, successively use distilled water of 5 times the amount of resin, and a concentration of 10% ethanol solution Elution is performed to remove impurities.

S2: Preparation of honeysuckle extract: crush honeysuckle through a 50-mesh sieve to obtain honeysuckle powder, add 9 times the amount of water to the honeysuckle powder, mix well and extract in water at 70°C for 120 minutes, filter the extract, and concentrate the filtrate Obtain a concentrated solution; add 85% ethanol to extract the concentrated solution for 6 days, concentrate the extracted liquid to obtain a slurry; use a supercritical _CO2 fluid extraction process to extract the slurry to obtain a honeysuckle extract; immerse in 70 ° C water Two times, the supercritical CO2 fluid extraction conditions: the pressure is 20mpa, the fluid ratio is 5%, the temperature is 45°C, and the time is 120 minutes.

Honeysuckle: The combination of alcohol precipitation and supercritical CO ₂ fluid extraction has high yield and high purity, and can fully extract the active ingredients of honeysuckle.

S3: Preparation of Echinacea extract: crush Echinacea through a 50-mesh sieve to obtain Echinacea powder, add 50% ethanol to the Echinacea powder, adjust the pH value to 10 with Ca(OH) ₂ , heat and reflux 3 times, filter and combine the filtrates , to obtain ethanol extract and filter residue two parts; filter residue plus 8 times the amount of 1% aqueous solution of lysozyme to extract again, the extraction conditions are: filter residue soaked in 38 ℃ aqueous solution of lysozyme for 10 hours; filter, the lysozyme extract and The previous ethanol extracts were combined to obtain the Echinacea purpurea extract, and the extract was concentrated to obtain the Echinacea purpurea extract;

Echinacea contains polysaccharides, polyphenols (including derivatives of cichoric acid and caffeic acid), flavonoids, essential oils, polyynes, alkylamines and alkaloids; it has the effects of improving immunity and antibacterial and anti-inflammatory effects; polysaccharides can stimulate the body's Cells to fight against infection; the extraction methods of Echinacea purpurea extracts currently on the market are mainly simple water immersion and alcohol extraction, resulting in low extraction efficiency, and the disadvantage of high content of certain active ingredients and low content of other active ingredients. Thereby affecting its curative effect; Echinacea purpurea extract is to adopt alkaline ethanol solution and lysozyme to extract Echinacea purpurea medicinal material and obtain, is conducive to improving the content of polyphenols and cichoric acid in the extract, greatly improving the efficacy of the obtained extract; The main components of plant cell walls are cellulose and pectin, and lysozyme can destroy its structure. Adding an appropriate amount of lysozyme to the filter residue after ethanol extraction can destroy the cell walls of Echinacea purpurea cells, making the active ingredients polysaccharides and polyphenols and cichoric acid are further released, improving the extraction rate of Echinacea purpurea.

S4: Preparation of lemon extract: crush the lemon fruit through a 50-mesh sieve, add 3 times the weight of the lemon fruit at 50°C and add ethanol for reflux extraction for 3 times to obtain the extract, combine the extract and put it on a rotary evaporator Concentrate and recover ethanol to obtain lemon extract;

S5: Preparation of punicalagin: crush the pomegranate peel through a 50-mesh sieve, add water to the crushed pomegranate peel for flash extraction, and remove polysaccharides in the supernatant: concentrate the supernatant extract, add 3.5 times the volume of 70 % ethanol, set aside at low temperature for 10 hours to precipitate, centrifuge, filter, collect the supernatant, and concentrate the supernatant to obtain the punicalagin concentrate; the flash extraction condition is: the ratio of pomegranate peel powder to water is 1:3 ; The extraction speed is 6000rpm; the extraction time is 100s.

S6: Preparation of composition: 60% of camellia saponin obtained in S1 above, 15% of honeysuckle extract obtained in S2, 15% of Echinacea purpurea extract obtained in S3, 5% of lemon extract obtained in S4 and S5 Mix the punicalagin 5% obtained in the method according to the ratio, add a preservative to the mixed solution, and adjust the pH to 7 to obtain the composition.

Example 2

A composition for inhibiting Staphylococcus aureus biofilm, comprising the following components by weight percentage: 65% camellia saponin, 17% honeysuckle extract, 10% echinacea extract, 5% lemon extract and punicalagin 3 %.

The honeysuckle extract contains 50%-90% of chlorogenic acid; the polysaccharide content in the Echinacea purpurea extract ≥ 21.50%, the polyphenol content ≥ 8.70%, and the cichoric acid content ≥ 5.00%; the total flavonoid content in the lemon extract ≥80%.

A kind of preparation method of the composition that suppresses Staphylococcus aureus biofilm, it comprises the following steps:

S1: Preparation of camellia oleifera saponin: crush the camellia oleifera cake after oil extraction, pass through a 50-mesh sieve, extract with an extractant at a ratio of solid to liquid of 1:10, concentrate and dry the extract, dissolve it in water and filter; Use macroporous adsorption resin to elute the filtrate, the eluent is 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol and 95% ethanol, gradient elution, collect the eluted liquid respectively, dry and concentrate Camellia oleifera saponin is obtained; the extractant is acetone with a concentration of 70%, the extraction temperature is 65°C, and the extraction time is 2h; before using the eluent for elution, successively use distilled water of 5 times the amount of resin, and a concentration of 10% ethanol solution Elution is performed to remove impurities.

S2: Preparation of honeysuckle extract: crush honeysuckle through a 50-mesh sieve to obtain honeysuckle powder, add 9 times the amount of water to the honeysuckle powder, mix well and extract in water at 70°C for 120 minutes, filter the extract, and concentrate the filtrate Obtain a concentrated solution; add 85% ethanol to extract the concentrated solution for 6 days, concentrate the extracted liquid to obtain a slurry; use a supercritical _CO2 fluid extraction process to extract the slurry to obtain a honeysuckle extract; immerse in 70 ° C water Two times, the supercritical CO2 fluid extraction conditions: the pressure is 20mpa, the fluid ratio is 5%, the temperature is 45°C, and the time is 120 minutes.

S3: Preparation of Echinacea extract: crush Echinacea through a 50-mesh sieve to obtain Echinacea powder, add 50% ethanol to the Echinacea powder, adjust the pH value to 10 with Ca(OH) ₂ , heat and reflux 3 times, filter and combine the filtrates , to obtain ethanol extract and filter residue two parts; filter residue plus 8 times the amount of 1% aqueous solution of lysozyme to extract again, the extraction conditions are: filter residue soaked in 38 ℃ aqueous solution of lysozyme for 10 hours; filter, the lysozyme extract and The previous ethanol extracts were combined to obtain the Echinacea purpurea extract, and the extract was concentrated to obtain the Echinacea purpurea extract;

S4: Preparation of lemon extract: crush the lemon fruit through a 50-mesh sieve, add 3 times the weight of the lemon fruit at 50°C and add ethanol for reflux extraction for 3 times to obtain the extract, combine the extract and put it on a rotary evaporator Concentrate and recover ethanol to obtain lemon extract;

S5: Preparation of punicalagin: crush the pomegranate peel through a 50-mesh sieve, add water to the crushed pomegranate peel for flash extraction, and remove polysaccharides in the supernatant: concentrate the supernatant extract, add 3.5 times the volume of 70 % ethanol, set aside at low temperature for 10 hours to precipitate, centrifuge, filter, collect the supernatant, and concentrate the supernatant to obtain the punicalagin concentrate; the flash extraction condition is: the ratio of pomegranate peel powder to water is 1:3 ; The extraction speed is 6000rpm; the extraction time is 100s.

S6: Preparation of composition: 65% of camellia saponin obtained in S1 above, 17% of honeysuckle extract obtained in S2, 10% of Echinacea purpurea extract obtained in S3, 5% of lemon extract obtained in S4 and S5 The 3% punicalagin obtained in the method was mixed according to the proportion, and a preservative was added to the mixed liquid, and the pH was adjusted to 7 to obtain the composition.

Example 3

A composition for inhibiting Staphylococcus aureus biofilm, comprising the following components by weight percentage: Camellia saponin 70%, honeysuckle extract 17%, Echinacea purpurea extract 5%, lemon extract 5% and punicalagin 3% %

The honeysuckle extract contains 50%-90% of chlorogenic acid; the polysaccharide content in the Echinacea purpurea extract ≥ 21.50%, the polyphenol content ≥ 8.70%, and the cichoric acid content ≥ 5.00%; the total flavonoid content in the lemon extract ≥80%.

A kind of preparation method of the composition that suppresses Staphylococcus aureus biofilm, it comprises the following steps:

S1: Preparation of camellia oleifera saponin: crush the camellia oleifera cake after oil extraction, pass through a 50-mesh sieve, extract with an extractant at a ratio of solid to liquid of 1:10, concentrate and dry the extract, dissolve it in water and filter; Use macroporous adsorption resin to elute the filtrate, the eluent is 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol and 95% ethanol, gradient elution, collect the eluted liquid respectively, dry and concentrate Camellia oleifera saponin is obtained; the extractant is acetone with a concentration of 70%, the extraction temperature is 65°C, and the extraction time is 2h; before using the eluent for elution, successively use distilled water of 5 times the amount of resin, and a concentration of 10% ethanol solution Elution is performed to remove impurities.

S2: Preparation of honeysuckle extract: crush honeysuckle through a 50-mesh sieve to obtain honeysuckle powder, add 9 times the amount of water to the honeysuckle powder, mix well and extract in water at 70°C for 120 minutes, filter the extract, and concentrate the filtrate Obtain a concentrated solution; add 85% ethanol to extract the concentrated solution for 6 days, concentrate the extracted liquid to obtain a slurry; use a supercritical _CO2 fluid extraction process to extract the slurry to obtain a honeysuckle extract; immerse in 70 ° C water Mention twice, the supercritical CO ₂ fluid extraction conditions: the pressure is 20mpa, the fluid ratio is 5%, the temperature is 45°C, and the time is 120 minutes.

S3: Preparation of Echinacea extract: crush Echinacea through a 50-mesh sieve to obtain Echinacea powder, add 20% ethanol to the Echinacea powder, adjust the pH value to 10 with Ca(OH) ₂ , heat and reflux three times, filter and combine the filtrates , to obtain ethanol extract and filter residue two parts; filter residue plus 8 times the amount of 1% aqueous solution of lysozyme to extract again, the extraction conditions are: filter residue soaked in 38 ℃ aqueous solution of lysozyme for 10 hours; filter, the lysozyme extract and The previous ethanol extracts were combined to obtain the Echinacea purpurea extract, and the extract was concentrated to obtain the Echinacea purpurea extract;

S4: Preparation of lemon extract: crush the lemon fruit through a 50-mesh sieve, add 3 times the weight of the lemon fruit at 50°C and add ethanol for reflux extraction for 3 times to obtain the extract, combine the extract and put it on a rotary evaporator Concentrate and recover ethanol to obtain lemon extract;

S5: Preparation of punicalagin: crush the pomegranate peel through a 50-mesh sieve, add water to the crushed pomegranate peel for flash extraction, and remove polysaccharides in the supernatant: concentrate the supernatant extract, add 3.5 times the volume of 70 % ethanol, set aside at low temperature for 10 hours to precipitate, centrifuge, filter, collect the supernatant, and concentrate the supernatant to obtain the punicalagin concentrate; the flash extraction condition is: the ratio of pomegranate peel powder to water is 1:3 ; The extraction speed is 6000rpm; the extraction time is 100s.

S6: Preparation of composition: 70% of Camellia saponin obtained in S1 above, 17% of honeysuckle extract obtained in S2, 5% of Echinacea purpurea extract obtained in S3, 5% of lemon extract obtained in S4 and S5 The 3% punicalagin obtained in the method was mixed according to the proportion, and a preservative was added to the mixed liquid, and the pH was adjusted to 7 to obtain the composition.

Example 4

A composition for inhibiting Staphylococcus aureus biofilm, comprising the following components by weight percentage: 75% camellia saponin, 10% honeysuckle extract, 7% echinacea extract, 5% lemon extract and punicalagin 3 %.

The honeysuckle extract contains 50%-90% of chlorogenic acid; the polysaccharide content in the Echinacea purpurea extract ≥ 21.50%, the polyphenol content ≥ 8.70%, and the cichoric acid content ≥ 5.00%; the total flavonoid content in the lemon extract ≥80%.

A kind of preparation method of the composition that suppresses Staphylococcus aureus biofilm, it comprises the following steps:

S1: Preparation of camellia oleifera saponin: crush the camellia oleifera cake after oil extraction, pass through a 50-mesh sieve, extract with an extractant at a ratio of solid to liquid of 1:10, concentrate and dry the extract, dissolve it in water and filter; Use macroporous adsorption resin to elute the filtrate, the eluent is 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol and 95% ethanol, gradient elution, collect the eluted liquid respectively, dry and concentrate Camellia oleifera saponin is obtained; the extractant is acetone with a concentration of 70%, the extraction temperature is 65°C, and the extraction time is 2h; before using the eluent for elution, successively use distilled water of 5 times the amount of resin, and a concentration of 10% ethanol solution Elution is performed to remove impurities.

S2: Preparation of honeysuckle extract: crush honeysuckle through a 50-mesh sieve to obtain honeysuckle powder, add 9 times the amount of water to the honeysuckle powder, mix well and extract in water at 70°C for 120 minutes, filter the extract, and concentrate the filtrate Obtain a concentrated solution; add 85% ethanol to extract the concentrated solution for 6 days, concentrate the extracted liquid to obtain a slurry; use a supercritical _CO2 fluid extraction process to extract the slurry to obtain a honeysuckle extract; immerse in 70 ° C water Mention twice, the supercritical CO ₂ fluid extraction conditions: the pressure is 20mpa, the fluid ratio is 5%, the temperature is 45°C, and the time is 120 minutes.

S3: Preparation of Echinacea extract: crush Echinacea through a 50-mesh sieve to obtain Echinacea powder, add 20%% ethanol to the Echinacea powder, adjust the pH value to 10 with Ca(OH) ₂ , heat and reflux 3 times, filter and combine Filtrate, to obtain two parts of ethanol extract and filter residue; add 8 times the amount of 1% lysozyme aqueous solution to the filter residue to extract again, the extraction conditions are: soak the filter residue in 38 ℃ lysozyme aqueous solution for 10 hours; filter, and lysozyme extract Combine with the previous ethanol extract to obtain the Echinacea purpurea extract, and concentrate the extract to obtain the Echinacea purpurea extract;

S4: Preparation of lemon extract: crush the lemon fruit through a 50-mesh sieve, add 3 times the weight of the lemon fruit at 50°C and add ethanol for reflux extraction for 3 times to obtain the extract, combine the extract and put it on a rotary evaporator Concentrate and recover ethanol to obtain lemon extract;

S5: Preparation of punicalagin: crush the pomegranate peel through a 50-mesh sieve, add water to the crushed pomegranate peel for flash extraction, and remove polysaccharides in the supernatant: concentrate the supernatant extract, add 3.5 times the volume of 70 % ethanol, set aside at low temperature for 10 hours to precipitate, centrifuge, filter, collect the supernatant, and concentrate the supernatant to obtain the punicalagin concentrate; the flash extraction condition is: the ratio of pomegranate peel powder to water is 1:3 ; The extraction speed is 6000rpm; the extraction time is 100s.

S6: Preparation of composition: 75% of camellia saponin obtained in S1 above, 10% of honeysuckle extract obtained in S2, 7% of Echinacea purpurea extract obtained in S3, 5% of lemon extract obtained in S4 and S5 The 3% punicalagin obtained in the method was mixed according to the proportion, and a preservative was added to the mixed liquid, and the pH was adjusted to 7 to obtain the composition.

Comparative example 1

A composition for inhibiting Staphylococcus aureus biofilm, comprising the following components by weight percentage: 15% of honeysuckle extract, 15% of Echinacea purpurea extract, 5% of lemon extract and 5% of punicalagin.

The honeysuckle extract contains 50%-90% of chlorogenic acid; the polysaccharide content in the Echinacea purpurea extract ≥ 21.50%, the polyphenol content ≥ 8.70%, and the cichoric acid content ≥ 5.00%; the total flavonoid content in the lemon extract ≥80%.

A kind of preparation method of the composition that suppresses Staphylococcus aureus biofilm, it comprises the following steps:

S1: Preparation of honeysuckle extract: crush honeysuckle through a 50-mesh sieve to obtain honeysuckle powder, add 9 times the amount of water to the honeysuckle powder, mix well and extract in water at 70°C for 120 minutes, filter the extract, and concentrate the filtrate Obtain a concentrated solution; add 85% ethanol to extract the concentrated solution for 6 days, concentrate the extracted liquid to obtain a slurry; use a supercritical _CO2 fluid extraction process to extract the slurry to obtain a honeysuckle extract; immerse in 70 ° C water Mention twice, the supercritical CO ₂ fluid extraction conditions: the pressure is 20mpa, the fluid ratio is 5%, the temperature is 45°C, and the time is 120 minutes.

S2: Preparation of Echinacea extract: crush Echinacea through a 50-mesh sieve to obtain Echinacea powder, add 50% ethanol to the Echinacea powder, adjust the pH value to 10 with Ca(OH) ₂ , heat and reflux 3 times, filter and combine the filtrates , to obtain ethanol extract and filter residue two parts; filter residue plus 8 times the amount of 1% aqueous solution of lysozyme to extract again, the extraction conditions are: filter residue soaked in 38 ℃ aqueous solution of lysozyme for 10 hours; filter, the lysozyme extract and The previous ethanol extracts were combined to obtain the Echinacea purpurea extract, and the extract was concentrated to obtain the Echinacea purpurea extract;

S3: Preparation of lemon extract: crush lemon fruit through a 50-mesh sieve, add ethanol 3 times the weight of lemon fruit at 50°C for reflux extraction for 3 times to obtain an extract, combine the extracts and put them on a rotary evaporator Concentrate and recover ethanol to obtain lemon extract;

S4: Preparation of punicalagin: crush the pomegranate peel through a 50-mesh sieve, add water to the crushed pomegranate peel for flash extraction, and remove polysaccharides in the supernatant: concentrate the supernatant extract, add 3.5 times the volume of 70 % ethanol, set aside at low temperature for 10 hours to precipitate, centrifuge, filter, collect the supernatant, and concentrate the supernatant to obtain the punicalagin concentrate; the flash extraction condition is: the ratio of pomegranate peel powder to water is 1:3 ; The extraction speed is 6000rpm; the extraction time is 100s.

S5: Preparation of the composition: 15% of the honeysuckle extract obtained in the above S1, 15% of the Echinacea purpurea extract obtained in S2, 5% of the lemon extract obtained in S3 and 5% of punicalagin obtained in S4 according to the proportion Mixing is carried out, a preservative is added to the mixture, and the pH is adjusted to 7 to obtain a composition.

Effect Example 1

Composition is measured to Staphylococcus aureus minimum inhibitory concentration (MIC):

Get equal amounts of the compositions prepared in Example 1 and Example 4 and dissolve them in sterile water respectively. 16mg/mL, 32mg/mL and 64mg/mL Camellia saponin solutions were stored in a 4°C refrigerator for later use; preparation of Staphylococcus aureus strain activation: prepare 1 L of nutrient agar medium (NA), and sterilize at 121°C for 20 minutes Take it out, pour it into a sterilized petri dish, and obtain a solid medium after cooling. Take the strain from the -8°C refrigerator, pick up the bacteria with an inoculation loop, mark the line on the solid medium, and place it in a constant temperature incubator for 18 hours. The temperature is 37°C; the preparation of the bacterial suspension: use an inoculation loop to pick a single colony and inoculate it into the liquid medium, and cultivate it on a constant temperature shaker at 150r/min at 37°C for 14 hours; then inoculate 1% of the inoculum into 10mL of sterile water, Sequentially dilute, by turbidimetric method, the number of colonies is 106cfu/mL; minimum inhibitory concentration (MIC) measurement: adopt two-fold dilution method, get two groups of sterile test tubes, each group is 6 sterile test tubes, carry out numbering, Then add 2mL of culture medium to each test tube, in the first group of sterile test tubes: add 2mL of the composition solution prepared in Example 1 to the first tube, take 2mL after mixing and add it to the second test tube, and dilute in turn, After mixing the last test tube, take 2mL of liquid and discard it; in the second group of sterile test tubes: add 2mL of the composition solution prepared in Example 4 to the first tube, take 2mL and add it to the second test tube after mixing, Dilute sequentially. After the last test tube is mixed, take 2 mL of liquid and discard it; add 100 μL of 106 cfu/mL bacterial suspension to the above test tubes respectively, place in a constant temperature incubator at 37 ° C for 24-48 hours, and observe whether the liquid in the test tube changes. Turbidity, repeat the above experiment 3 times; see Table 1 for the degree of turbidity of the liquid in the test tube;

Table 1: The degree of turbidity of the liquid in the two groups of test tubes:

Analysis of test results:

As can be seen from Table 1, when the concentration of camellia saponin in Example 1 was 32mg/mL, the solution began to be turbid, and the MIC of camellia saponin in Example 1 was 64mg/mL; when the concentration of camellia saponin in Example 4 When it was 16mg/mL, the solution began to be turbid, and the MIC of camellia saponin in Example 4 was 32mg/mL.

Effect Example 2

Composition to staphylococcus aureus quorum sensing inhibition test:

Inoculate Staphylococcus aureus for overnight culture, and adjust the OD value of the culture medium to 0.1; take 200 μL of the bacterial solution and add it to 100 mL of melted LB agar medium (50 °C), and add 200 μL of the signal molecule C6-HSL (final concentration: 5 μM) and Ca Namycin (final concentration 20 μg/mL); evenly poured on 6 plates after mixing; after the plates condensed, place a sterile Oxford cup; The composition prepared in Comparative Example 1 and the control group (sterile water) were added to an Oxford cup, incubated at room temperature for 1 hour, and then cultured overnight at 28°C; quorum sensing was assessed based on the formation of a colorless halo around the well with viable cell growth Inhibition, use a caliper to measure the colony diameter of each treatment, and the unit is mm; use the cross method to measure the diameter vertically each time, and take the average value; calculate the mycelial growth inhibition rate according to the following formula.

Mycelium inhibition rate (%)=(control colony growth diameter-treatment colony growth diameter)/control colony growth diameter*100

Composition is shown in Table 2 to Staphylococcus aureus inhibitory effect:

Table 2: The inhibitory effect of the compositions prepared by Examples 1-4, Comparative Example 1 and the control group on Staphylococcus aureus

The results in Table 2 show that the compositions prepared in Examples 1-4 have higher inhibitory activity to Staphylococcus aureus, and the implementation of Examples 1 to 4 increases the proportion of camellia saponin for the inhibition of Staphylococcus aureus. The effect is better; the composition prepared when Camellia saponin 75%, honeysuckle extract 10%, Echinacea purpurea extract 7%, lemon extract 5% and punicalagin 3% has the best inhibitory effect on Staphylococcus aureus; by It can be seen from Comparative Example 1 that the bacteriostasis effect is poor when no camellia saponin is added, indicating that camellia saponin plays a major role in inhibiting Staphylococcus aureus in the prepared composition.

Effect Example 3

The inhibitory action test of composition to Staphylococcus aureus biofilm formation:

Inoculate a single colony of Staphylococcus aureus into 5mL NB liquid medium, culture at 28°C and 180rpm for 24 hours, adjust the absorbance of the above bacterial solution at 600nm wavelength to 0.06-0.10; take 10μL of the bacterial solution and add it to 5mL NB culture In the base, the culture medium is divided into 6 groups, each group is 10 culture media, the composition prepared in Example 1-4 and Comparative Example 1 is added respectively to 5 groups of culture media among them, and the composition prepared in another group of culture media is added Sterile water was used as a blank control test; 1 mL of the mixed bacterial solution was taken from the culture medium of the 6 groups, and added to the flat-bottomed well plate with a round cover glass at the bottom of the 6 groups, and cultured at 28°C for 24 hours; After culturing, take out the circular coverslips at the bottom of the 6 groups, and wash away the planktonic bacteria with PBS; the capsules on the 6 groups of coverslips are all fixed with 2.5% glutaraldehyde for 12 hours, and then washed with 50% ethanol and 70% ethanol in turn. , 80% ethanol, 90% ethanol and 100% ethanol for gradient dehydration (each concentration eluted for 10min), freeze-dried in a freeze dryer and read the OD570 value on a microplate reader, and each sample was repeated three times to get the average absorption value; this absorption value is directly proportional to the output of film within a certain range, and the sample prepared by using the following formula to calculate embodiment 1-4 and comparative example 1 is to the inhibitory rate of Staphylococcus aureus biofilm formation:

Inhibition rate = (negative control A570 average - sample A570 average) / negative control A570 average × 100%

The inhibitory effect of the composition on Staphylococcus aureus biofilm formation is shown in Table 3;

Table 3: Inhibitory effect of the composition on Staphylococcus aureus biofilm formation

sample Biofilm Formation Ability Biofilm inhibition rate (%) control group stronger 28.2 Example 1 poor 75.8 Example 2 poor 80.5 Example 3 poor 86.7 Example 4 poor 90.2 Comparative example 1 Moderate 60.5

Result analysis:

It can be seen from Table 3 that the inhibitory effect of the control group on the formation of Staphylococcus aureus biofilm is relatively poor, and the resulting composition prepared in Examples 1-4 has a significant inhibitory effect on the formation of Staphylococcus aureus biofilm, and the resulting combination prepared in Comparative Example 1 The substance has a certain inhibitory effect on the formation of Staphylococcus aureus biofilm, and along with the increase of tea oil saponin mass concentration in embodiment 1-4, better for the formation inhibitory effect of Staphylococcus aureus biofilm, presents dose-dependent sex.

In summary; Staphylococcus aureus is the most common Gram-positive pathogenic bacteria in food, which will seriously endanger public health and human health; and the formation of biofilm is the main cause of Staphylococcus aureus under unfavorable environmental conditions. The key factor for persistence; bacteria can usually resist the host’s immune response after forming a biofilm, and they are more resistant to antibiotics and bacteriostats than planktonic bacteria; Drug-resistant antibacterial substances have broad application prospects in the development of anti-Staphylococcus aureus drugs.

Camellia oleifera saponin, a natural product extracted from camellia oleifera cake, is a type of biologically active substance formed by linking oleanane-type pentacyclic triterpenes and oligosaccharides. It consists of saponins (aglycones, usually triterpene ), glycosyl (oligosaccharides) and organic acids; camellia saponin has strong foaming, emulsifying, dispersing, moisturizing effects, as well as antibacterial, anti-inflammatory, analgesic, anti-cancer and other physiological activities.

The biofilm of Staphylococcus aureus is the key factor of its strong pathogenicity and strong drug resistance; the method currently used to prevent and treat the biofilm of Staphylococcus aureus mainly adopts new antibiotics and new anti-biofilm agents (enzyme, gallium-based therapy, etc.), plant extracts, antimicrobial peptides, combined with antibiotics, etc.

The focus of the design of the present invention is to provide a composition with high efficiency, low toxicity, and not easy to produce drug resistance. The composition is all plant extracts, which can effectively inhibit the biofilm formation of Staphylococcus aureus, thereby preventing and treating the infection and disease of Staphylococcus aureus. harm.

The advantages of the present invention are also reflected in the relatively high cost of the plant extracts used to inhibit the biofilm of Staphylococcus aureus, but in this composition, camellia saponin is the main component, and camellia saponin is a by-product of camellia oleifera processing. Camellia oleifera saponin, which is the residue of camellia oleifera cake after oil-pressing, belongs to by-product utilization, low cost, and sufficient raw materials; at the same time, camellia saponin has a very good effect on inhibiting the formation of Staphylococcus aureus biofilm significantly.

The above descriptions are only preferred embodiments of the present invention, and do not limit the technical scope of the present invention in any way, so any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention are valid. Still belong to the scope of the technical solution of the present invention.

Claims (8)

1. A method for preparing a composition for inhibiting staphylococcus aureus biofilm, which is characterized by comprising the following steps of; the method comprises the following steps:

s1: preparation of tea-oil camellia saponin: crushing oil tea cakes after oil extraction, and sieving the crushed oil tea cakes with a 50-100-mesh sieve, wherein the extracting agent is prepared according to a feed-liquid ratio of 1:10, concentrating and drying the extract, dissolving in water and filtering; eluting the filtrate by using macroporous adsorption resin, wherein the eluent is 30% -95% ethanol, performing gradient elution, respectively collecting the eluted liquid, measuring, drying and concentrating to obtain the oil tea saponin;

s2: preparation of honeysuckle extract: pulverizing flos Lonicerae, sieving with 50-100 mesh sieve to obtain flos Lonicerae powder, adding 9 times of water into the flos Lonicerae powder, mixing, leaching in 70-80deg.C water for 120min, filtering the leaching solution, and concentrating the filtrate to obtain concentrated solution; adding 80% -85% ethanol to leach the concentrated solution for 6 days, and concentrating the leached liquid to obtain slurry; by supercritical CO ₂ Extracting the slurry by a fluid extraction process to obtain a honeysuckle extract;

s3: preparation of Echinacea extract: crushing Echinacea purpurea, sieving with a 50-100 mesh sieve to obtain Echinacea purpurea powder, adding 20% -50% ethanol into the Echinacea purpurea powder, heating and refluxing for 3 times, filtering and mixing the filtrates to obtain ethanol extract and filter residue; extracting the residue with 8 times of 1% lysozyme aqueous solution again, filtering, mixing the lysozyme extract with the ethanol extract to obtain Echinacea purpurea extract, and concentrating the extract to obtain Echinacea purpurea extract;

s4: preparation of lemon extract: pulverizing fructus Citri Limoniae, sieving with 20-50 mesh sieve, reflux extracting at 50-60deg.C with 3-3.5 times of ethanol to obtain extractive solution, mixing extractive solutions, concentrating on rotary evaporator, and recovering ethanol to obtain fructus Citri Limoniae extract;

s5: preparation of punicalagin: crushing pericarpium Granati, sieving with 20-50 mesh sieve, adding water into the crushed pericarpium Granati for flash extraction, and removing polysaccharide in supernatant: concentrating the upper layer extract, adding 3.5 times of 70% -90% ethanol, standing at low temperature for 10 hr for precipitation, centrifuging, filtering, collecting supernatant, and concentrating the supernatant to obtain punicalagin concentrate;

s6: preparation of the composition: mixing the oil tea saponin obtained in the step S1, the honeysuckle extract obtained in the step S2, the echinacea extract obtained in the step S3, the lemon extract obtained in the step S4 and the punicalagin obtained in the step S5 according to a proportion, adding a preservative into the mixed solution, and regulating the pH value to 7 to obtain a composition; the composition comprises the following components in percentage by weight: 60-75% of tea saponin, 10-20% of honeysuckle extract, 7-15% of echinacea extract, 5-10% of lemon extract and 3-5% of punicalagin; the honeysuckle extract contains 50% -90% of chlorogenic acid; the content of polysaccharide in the Echinacea purpurea extract is more than or equal to 21.50%, the content of polyphenol is more than or equal to 8.70%, and the content of chicoric acid is more than or equal to 5.00%; the content of total flavonoids in the lemon extract is more than or equal to 80%.

2. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; before eluting by using the eluent in the step S1, eluting by using distilled water with the amount of 5 times of resin and ethanol solution with the concentration of 10% in sequence to remove impurities.

3. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; the extractant in the step S1 is acetone with the concentration of 70%, the extraction temperature is 65 ℃, and the extraction time is 2 hours.

4. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; leaching the above S2 in 70-80deg.C water twice, and collecting supercritical CO ₂ Fluid extraction conditions: the pressure is 20-22mpa, the fluid ratio is 5-6%, the temperature is 45-50 ℃ and the time is 120 minutes.

5. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; in the step S3, 20% -50% of ethanol is added into the echinacea powder, and then Ca (OH) is adopted ₂ The pH was adjusted to 10.

6. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; the conditions for re-extracting the filter residue in the step S3 by adding 8 times of 1% lysozyme aqueous solution are as follows: the filter residue is soaked in lysozyme aqueous solution at 38 ℃ for 10 hours.

7. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; the flash extraction conditions in the step S5 are as follows: the ratio of the pericarpium Granati powder to the water is 1:3, a step of; the extraction speed is 6000rpm; the extraction time was 100s.

8. The method of preparing a composition for inhibiting a biofilm of staphylococcus aureus according to claim 1, wherein; the S6 comprises the following components in percentage by weight when being mixed: 60-75% of tea saponin, 10-20% of honeysuckle extract, 7-15% of echinacea extract, 5-10% of lemon extract and 3-5% of punicalagin.