阅读说明：本技术 一种抑制赭曲霉生长和产毒的肉桂醛皮克林乳液及其制备方法 (Cinnamaldehyde pickering emulsion for inhibiting aspergillus ochraceus growth and producing toxin and preparation method thereof ) 是由 王龑 林威 孙培龙 管乐 蔡铭 杨开 于 2021-01-29 设计创作，主要内容包括：本发明公开了一种抑制赭曲霉抑生长和产毒的肉桂醛皮克林乳液及其制备方法。所述乳液中肉桂醛含量为5～30g/L,壳聚糖含量为0.125～0.625g/L,玉米蛋白含量为2.5～12.5g/L,乙醇含量为125～250mL/L,乙酸含量为0.5～1.25mL/L。本发明肉桂醛生物资源在我国来源广、生产工艺简易、成本低、具有挥发性；对人畜安全性好,已被列入食品添加剂目录(GB 2760-2014食品安全国家标准-食品添加剂使用标准)；本发明思路新颖,乳液制备方法简单、快捷、高效,制备的肉桂醛皮克林乳液在较长时间内能保持良好的稳定性,实用性强；本发明作为乳化剂的固体颗粒(壳聚糖、玉米蛋白)来源广泛,安全无毒,可降解。本发明制备的肉桂醛皮克林乳液应用于抑制赭曲霉生长和产毒的效果好,可直接应用于粮食谷物的防霉保藏。(The invention discloses a cinnamaldehyde pickering emulsion for inhibiting aspergillus ochraceus from inhibiting growth and producing toxin and a preparation method thereof. The emulsion contains 5-30 g/L of cinnamaldehyde, 0.125-0.625 g/L of chitosan, 2.5-12.5 g/L of zein, 125-250 mL/L of ethanol and 0.5-1.25 mL/L of acetic acid. The cinnamaldehyde biological resource has wide source, simple production process, low cost and volatility in China; the food additive has good safety to human and livestock and is listed in the food additive catalogue (GB 2760-; the invention has novel thought, the emulsion preparation method is simple, quick and efficient, and the prepared cinnamaldehyde pickering emulsion can keep good stability for a long time and has strong practicability; the solid particles (chitosan and zein) used as the emulsifier have wide sources, are safe, nontoxic and degradable. The cinnamaldehyde pickering emulsion prepared by the invention has good effects of inhibiting aspergillus ochraceus growth and producing toxin, and can be directly applied to mildew-proof preservation of grain grains.)

1. A cinnamaldehyde pickering emulsion for inhibiting aspergillus ochraceus growth and producing toxin comprises cinnamaldehyde, an emulsifier and water, wherein the emulsifier is colloid particles prepared from chitosan and zein by an ultrasonic method, and the emulsion is prepared from the following main raw materials:

2. the cinnamaldehyde pickering emulsion for inhibiting aspergillus ochraceus growth and producing toxins as claimed in claim 1, wherein the emulsion is prepared from the following raw materials: 29.3g/L of cinnamaldehyde, 0.125g/L of chitosan, 2.5g/L of zein, 175mL/L of ethanol, 0.75mL/L of acetic acid and the balance of water.

3. A method of preparing the aspergillus ochraceus growth and toxin producing cinnamaldehyde pickering emulsion of claim 1, comprising:

(1) weighing chitosan according to the formula ratio, placing the chitosan in a container, adding 1% acetic acid aqueous solution, and stirring the mixture by using a stirrer until the mixture is transparent to obtain chitosan solution;

(2) weighing zein according to the formula ratio, adding the zein into the chitosan solution obtained in the step (1), adding absolute ethyl alcohol, and stirring uniformly by using a stirrer to obtain zein-chitosan composite liquid;

(3) performing ultrasonic treatment on the zein-chitosan composite liquid obtained in the step (2) for 5-10 min by using a probe type ultrasonic extraction apparatus to obtain a clear and transparent zein-chitosan solution;

(4) and (3) adding the cinnamic aldehyde into the solution obtained in the step (3), immediately shaking and uniformly mixing, quickly pouring a proper amount of water into the solution, and stirring the solution uniformly by using a stirrer to obtain the cinnamic aldehyde pickering emulsion for inhibiting the growth of aspergillus ochraceus and producing toxin.

4. The method according to claim 3, wherein in the step (1), the mixture is stirred with a magnetic stirrer at a speed of 800 to 1000r/min for 1 to 2 hours.

5. The method according to claim 3, wherein in the step (2), the mixture is stirred with a magnetic stirrer at a speed of 800 to 1000r/min for 1 to 2 hours.

6. The method as claimed in claim 3, wherein in the step (3), the probe type ultrasonic extractor adopts a 20mm titanium alloy probe, and the power is 600-1200 w, and the ultrasonic treatment is carried out for 3-5 min.

7. The method according to claim 3, wherein in the step (4), the mixture is stirred by a magnetic stirrer at a speed of 200 to 600r/min for 5 to 10 min.

(I) technical field

The invention relates to a cinnamaldehyde pickering emulsion for inhibiting aspergillus ochraceus growth and toxin production and a preparation method thereof, and belongs to the technical field of emulsion embedding and antibiosis.

(II) background of the invention

Cinnamaldehyde (english: Cinnamaldehyde) is an aldehyde organic compound, is a yellow viscous liquid, and is abundantly present in plants such as cinnamon. Cinnamaldehyde, which naturally occurs in nature, is a trans-structure, and the molecule is an acrolein to which a phenyl group is attached, and thus can be considered as an acrolein derivative. The cinnamaldehyde is a synthetic spice for food which is allowed to be used and specified in GB2076-2014, can be used for preparing meat, seasonings, oral care products, chewing gum and essence for candies, and can also be used as a preservative for preserving fresh fruits subjected to surface treatment. The molecular formula is: c₉H₈O, molecular weight: 132.16.

aspergillus ochraceus (Aspergillus ochraceus) is a filamentous fungus belonging to Aspergillus of the family Tricholomataceae of the order Ascomycota and is widely distributed in agricultural products such as corn, peanuts, coffee, cottonseed, nuts, fruits and the like. The aspergillus ochraceus causes the food to mildew and deteriorate, greatly reduces the nutritional quality and the processing quality of the food, and causes great economic loss. Aspergillus ochraceus has strong spore-forming capability, and spores of Aspergillus ochraceus scattered in the air can induce childhood asthma and human lung diseases. The secondary metabolite, ochratoxin A (OTA), is widely distributed in agricultural products, and pollutes grains, grapes, coffee beans, feed, spices and the like, and OTA can finally enter human bodies along with food chains and seriously harm human health. Toxicology studies show that OTA mainly damages liver and kidney, has strong hepatotoxicity and kidney toxicity, and a large amount of toxin can cause intestinal mucositis and even necrosis; OTA is considered one of the most carcinogenic natural substances at present and is identified as a secondary carcinogen by IARC.

Because of the toxicity of ochratoxin A (OTA), the limit standards of OTA in food and feed are published at home and abroad to standardize production, control the pollution of OTA and ensure the edible safety of human and animals to the maximum extent, and the limit standards of ochratoxin in European Union and China are listed in Table 1.

Table 1: european Union and China advanced OTA (over the air) limited standard for food

Cinnamaldehyde is a safe, non-toxic, natural active substance that can efficiently inhibit the growth of aspergillus ochraceus. However, because cinnamaldehyde is volatile and oxidized, the cinnamaldehyde has too short a retention time in the environment to exert its biological activity, and the control effect and the effective period of cinnamaldehyde are greatly reduced.

Therefore, the emulsion with the advantages of high coalescence stability, strong bearing capacity, strong protection capacity of the encapsulated substance, low release rate, good safety, good biodegradability, good biocompatibility and the like is developed to encapsulate the cinnamaldehyde, and the emulsion has very important economic value and social significance.

Disclosure of the invention

The invention aims to provide a cinnamaldehyde pickering emulsion which has high coalescence stability, strong bearing capacity, strong protection capacity of an encapsulated substance, low release rate, good safety, good biodegradability and good biocompatibility and is used for inhibiting the growth of aspergillus ochraceus and producing toxin, so that the cinnamaldehyde pickering emulsion can be used as a natural aspergillus ochraceus growth inhibitor.

The technical scheme adopted by the invention is as follows:

a cinnamaldehyde pickering emulsion for inhibiting growth of aspergillus ochraceus comprises cinnamaldehyde, an emulsifier and water, wherein the emulsifier is colloid particles prepared from chitosan and zein by an ultrasonic method, and the emulsion is prepared from the following main raw materials:

the balance being water.

Preferably, the main raw materials for preparing the emulsion comprise the following components: 29.3g/L of cinnamaldehyde, 0.125g/L of chitosan, 2.5g/L of zein, 175mL/L of ethanol, 0.75mL/L of acetic acid and the balance of water.

The average particle size of the colloidal particles is about 77nm, and the average particle size of the emulsion is about 573 nm.

The invention also relates to a method for preparing the cinnamaldehyde pickering emulsion for inhibiting aspergillus ochraceus growth and producing toxin, which comprises the following steps:

(1) weighing chitosan with the formula ratio, placing the chitosan into a container, adding 1% acetic acid aqueous solution (about 50-100 mL of 1% acetic acid aqueous solution is added to every 0.1g of chitosan), and stirring the chitosan solution by using a stirrer until the chitosan solution is transparent to obtain a chitosan solution;

(2) weighing zein according to the formula ratio, adding the zein into the chitosan solution obtained in the step (1), adding ethanol (50-100 mL of ethanol is added to every 1g of zein), and stirring uniformly by using a stirrer to obtain zein-chitosan composite liquid;

(3) performing ultrasonic treatment on the zein-chitosan composite liquid obtained in the step (2) for 5-10 min by using a probe type ultrasonic extraction apparatus to obtain a clear and transparent zein-chitosan solution (colloid can be formed after water is added for dilution in subsequent operations, and the average particle size of colloid particles obtained by analysis by a particle size analyzer is about 77 nm);

(4) and (3) adding the cinnamaldehyde into the solution obtained in the step (3), immediately shaking and uniformly mixing, immediately and quickly pouring a proper amount of water into the solution added with the cinnamaldehyde, and stirring the mixture uniformly by using a stirrer to obtain the cinnamaldehyde pickering emulsion for inhibiting the growth of aspergillus ochraceus and producing toxin.

Preferably, in the step (1), a magnetic stirrer is used for stirring for 1-2 hours at the rotating speed of 800-1000 r/min.

In the step (2), stirring for 1-2 h by using a magnetic stirrer at a rotating speed of 800-1000 r/min.

In the step (3), a probe type ultrasonic extractor selects a titanium alloy probe with the diameter of 20mm, the power is 600-1200 w, and the ultrasonic treatment is carried out for 3-5 min.

In the step (4), stirring for 5-10 min at a rotation speed of 200-600 r/min by using a magnetic stirrer.

In China, the cinnamaldehyde has wide sources, simple production process, low cost, volatility and good safety to people and livestock, is listed in the food additive catalogue (GB 2760-. Pickering emulsions are emulsions stabilized by a colloid instead of a surfactant. Surfactants generally form a fluid interface with a large surface lateral diffusion coefficient, the adsorption/desorption process of which is dynamic in a short time. Unlike surfactants, once colloids are adsorbed at the oil-water interface, they are effectively and irreversibly immobilized there, so that a major advantage of pickering emulsions over conventional emulsions is their high resistance to coalescence. The invention prepares the cinnamaldehyde into the pickering emulsion, has good safety and slow release effect, and has important significance for long-acting bacteriostasis.

The invention has the following beneficial effects: the invention provides the cinnamaldehyde pickering emulsion which has high coalescence stability, strong bearing capacity, strong protection capacity of an encapsulated substance, low release rate, good safety, good biodegradability and good biocompatibility, can inhibit the growth of aspergillus ochraceus and produce toxin, is used as a natural aspergillus ochraceus growth inhibitor, has good effect, is safe and has no toxic or side effect.

(IV) description of the drawings

FIG. 1 is an appearance diagram of a cinnamaldehyde pickering emulsion prepared in an example in long-term preservation;

FIG. 2 is a scanning electron micrograph of a cinnamaldehyde pickering emulsion prepared in example;

FIG. 3 is a graph showing the effect of different concentrations of cinnamaldehyde pickering emulsion prepared in the examples on the growth of Aspergillus ochraceus.

FIG. 4 is a graph showing the effect of cinnamaldehyde pickering emulsion prepared in example on growth, toxicity yield and toxicity producing ability of Aspergillus ochraceus in corn.

(V) detailed description of the preferred embodiments

For the purpose of enhancing understanding of the present invention, the present invention will be described in further detail with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention.

In the examples, Aspergillus ochraceus (Aspergillus ochracea) was used in CGMCC and the strain was numbered 3.4412 in CGMCC; denoted herein in the specification as CGMCC No. 3.4412.

Example 1: preparation of cinnamic aldehyde pickering emulsion

(1) Weighing 0.025g of chitosan, placing the chitosan in a container, adding 15ml of 1% (v/v) acetic acid aqueous solution, and stirring the mixture for 2 hours by a magnetic stirrer at 1000r/min until the mixture is transparent to obtain a chitosan solution;

(2) weighing 0.5g of zein (Mecline, Z822847), adding into the chitosan solution obtained in the step (1), adding 35ml of absolute ethyl alcohol, and stirring for 2h at 1000r/min by using a magnetic stirrer to obtain zein-chitosan composite liquid;

(3) carrying out ultrasonic treatment on the zein-chitosan composite liquid obtained in the step (2) for 5min by using a probe type ultrasonic extraction instrument (20mm titanium alloy probe, 1200w power) to obtain a clear and transparent zein-chitosan-containing solution;

(4) and (3) adding 5.86g of cinnamaldehyde into the solution obtained in the step (3), immediately shaking and uniformly mixing, immediately pouring 150ml of water into the solution, and stirring uniformly by using a stirrer to obtain the cinnamaldehyde pickering emulsion for inhibiting the growth of aspergillus ochraceus and producing toxin.

Fig. 1 is an appearance diagram of the prepared cinnamaldehyde-containing emulsion in long-term storage; fig. 2 is a scanning electron micrograph of the prepared cinnamaldehyde-containing emulsion. FIG. 1 illustrates that the cinnamic aldehyde pickering emulsion has better stability after being placed for a longer time, and the color of the cinnamic aldehyde pickering emulsion becomes darker and the stability of the emulsion gradually becomes worse after 15 days; figure 2 illustrates that cinnamaldehyde pickering emulsions contain cinnamaldehyde encapsulated by chitosan and zein, which can form spherical emulsion droplets.

Example 2: determination of cinnamaldehyde fumigation bacteriostatic effect

PDA culture medium: boiling 200g of peeled potato for 30min, taking the filtrate, adding 20g of glucose and 16g of agar, diluting to 1000mL with deionized water, autoclaving at 121 ℃ for 15 min, cooling to about 55 ℃, and pouring into a flat plate, wherein each flat plate is 20 mL.

Activating strains: aspergillus ochraceus (CGMCC No.3.4412) was inoculated to PDA medium and grown at 28 deg.C for 7 days, and stored at 4 deg.C.

Preparing ochratoxin spore liquid: washing Aspergillus ochraceus spores on plate culture medium with sterile water, dissolving in 0.01% Tween 80, and diluting the initial spore solution with sterile water to spore concentration of 1 × 10⁶cfu/mL。

10 μ L (1X 10) of Aspergillus ochraceus suspension⁶cfu/mL) was inoculated in the center of PDA medium to complete uptake. 3 sterilization filter paper sheets with the diameter of 8mm are symmetrically placed on the edge of a culture dish cover, 20 muL, 40 muL and 60 muL of cinnamyl aldehyde pickering emulsion are respectively placed in different culture dishes to be uniformly adsorbed on the filter paper sheets, no cinnamyl aldehyde is added in the blank, the concentration range of the cinnamyl aldehyde is 8 muL/L to 32 muL/L, 3 pieces of cinnamyl aldehyde are arranged in parallel, the culture dishes are sealed by sealing films and then placed in a self-sealing bag, and the culture dishes are placed in an incubator for culture at 28 ℃ for 6 days. The colony diameters were measured once a day from the next day, twice using the cross method, and the results were averaged, and the results are shown in fig. 3.

The results show that all the emulsions containing cinnamaldehyde can obviously inhibit the growth of aspergillus ochraceus, and the inhibition effect is enhanced along with the increase of the concentration of cinnamaldehyde. The inhibition rate of the cinnamaldehyde emulsion of 16 mu L/L reaches 33.9 percent, and the cinnamaldehyde emulsion of 32 mu L/L can completely inhibit the growth of aspergillus ochraceus (the inhibition rate reaches 100 percent).

Example 3: determination of growth and toxicity production effects of aspergillus ochraceus in corn by cinnamaldehyde fumigation

Corn: 50g of corn grits are taken and put into a 250mL conical flask and autoclaved at 121 ℃ for 15 minutes.

Activating strains: aspergillus ochraceus (CGMCC No.3.4412) was inoculated to PDA medium and grown at 28 deg.C for 7 days, and stored at 4 deg.C.

Preparing ochratoxin spore liquid: washing Aspergillus ochraceus spores on plate culture medium with sterile water, dissolving in 0.01% Tween 80, and diluting the initial spore solution with sterile water to spore concentration of 1 × 10⁶cfu/mL。

10 mu L of ochratoxin suspension is taken to be dissolved in 10mL of sterile water, inoculated in corn residues and mixed evenly.

Suspending 1 piece of sterilized filter paper with diameter of 16mm in the center of a conical flask, placing 300 μ L of cinnamaldehyde pickering emulsion in different conical flasks to be uniformly adsorbed on the filter paper, sealing the conical flasks with sealing film, placing in a self-sealing bag, culturing at 28 deg.C in an incubator for 14 days, wherein no cinnamaldehyde is added in the blank, and each group has 3 parallel groups.

And OTA: after the culture, 15g of corn was dissolved in 100mL of 80% (v/v) methanol, extracted by high-speed shaking, centrifuged to obtain the supernatant, concentrated by nitrogen-blowing, and subjected to High Performance Liquid Chromatography (HPLC) to determine the content of OTA, the results are shown in FIG. 4.

Ergosterol: after 14 days of culture, 15g of corn was dissolved in 100mL of 0.03mol/L KOH ethanol solution, shaken at high speed, extracted by ultrasonic-assisted saponification, centrifuged to obtain the supernatant, concentrated by nitrogen-blown method, and subjected to HPLC to determine the content of ergosterol, which was used as an indicator of Aspergillus ochraceus biomass, and the results are shown in FIG. 4. The ratio of OTA to ergosterol content in corn units was calculated as its toxigenic capacity and the results are shown in FIG. 4.

The result shows that after the cinnamaldehyde pickering emulsion is fumigated, the content of aspergillus ochraceus ergosterol in the corn is reduced by 44.9%, the content of OTA is reduced by 84.3%, and the content of OTA in the ergosterol unit is reduced by 71.6%, which shows that the cinnamaldehyde pickering emulsion can obviously inhibit the growth, the toxicity yield and the toxicity production capacity of aspergillus ochraceus in the corn.