A nisin protectant containing ikkduin and application thereof

Abstract

The application discloses a nisin protective agent containing ectoine and application, and belongs to the technical field of nisin storage and application. The nisin protective agent containing ectoine comprises ectoine, pullulan and 2-hydroxybutanedioic acid in a mass percentage ratio of 20-70%, 10-50% and 10-40% respectively. The preparation method comprises the following steps: ectoine, pullulan and 2-hydroxybutanedioic acid are weighed according to the mass percentage ratio, uniformly mixed in a mixer, and then ground in a grinder at 20-30 DEG C to a particle size of 100-200 nm, so that the nisin protective agent containing ectoine with uniform quality is obtained. The application can significantly improve the stability of nisin in the storage and application process.

Description

Technical Field

[0001] This invention relates to a nisin protectant containing ectoine and its application, belonging to the technical field of nisin storage and application. Background Technology

[0002] Nisin is a polypeptide antibiotic secreted by lactic acid streptococci. It can inhibit most Gram-positive bacteria and has a strong inhibitory effect on Bacillus spores. After consumption, it is quickly hydrolyzed into amino acids in the human body and is harmless. It is widely used as a food preservative.

[0003] Nisin's potency decreases over time during room temperature storage. Generally, it drops to 80-90% of its initial potency after 3 months, 60-70% after 6 months, and only 20-30% after 2 years. The stability of nisin is closely related to pH and temperature. Nisin can tolerate temperatures up to 120℃ at pH 2.0 with only a slight decrease in activity, but its activity rapidly declines at temperatures above 60℃ and above pH 4.0. When used in canned meat, sausages, bread, pastries, and dairy products, the high temperatures and near-neutral pH during processing lead to a significant decrease in nisin activity, resulting in poor preservative effects. Increasing the dosage to maintain effectiveness increases costs and limits the application of nisin.

[0004] Ectoin is a compatible solute produced by salt-tolerant and halophilic microorganisms. It is a cyclic amino acid derivative that can mitigate the toxic effects of hyperosmolarity, high temperature, freeze-thaw cycles, drying, and radiation on biomolecules and even the entire cell. Pullulan is an extracellular water-soluble mucopolysaccharide produced by the fermentation of *Brachystomata*, possessing excellent film-forming, gas-barrier, and plasticity properties. 2-Hydroxybutyric acid is an intermediate product of human internal circulation, easily absorbed by the human body, and is highly acidic. The synergistic effect of these three components can significantly improve the stability of nisin during storage and application.

[0005] Chinese patent CN 109627300 B provides a long-lasting stabilizer for the production and storage of Nisin solutions. The obtained stabilizer has a high cost-performance ratio and can be used to maintain the activity and long-lasting effect of Nisin solutions in the food, cosmetics and other fields. At the same time, it can be used in the Nisin production process to reduce the damage of Nisin during acidification. However, the patent has the following main problems: (1) the protective effect of the protectant on nisin is low; (2) the operating temperature is low, and the protective effect of the protectant is not good at high temperatures. Summary of the Invention

[0006] The purpose of this invention is to provide a nisin protectant containing ectoine, which can effectively improve the stability of nisin during storage and application.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] A lactic acid nisin protectant containing ectoine, comprising, by weight percentage, 20-70% ectoine, 10-50% pullulan, and 10-40% 2-hydroxysuccinic acid.

[0009] Furthermore, by weight percentage, it includes 50% ectoine, 30% pullulan, and 20% L-hydroxysuccinic acid.

[0010] Furthermore, by weight percentage, it includes 40% ectoine, 40% pullulan, and 20% L-hydroxysuccinic acid.

[0011] Furthermore, the viscosity of the pullulan polysaccharide is 150-180 mm. 2 / s, wherein the 2-hydroxysuccinic acid is L-hydroxysuccinic acid.

[0012] Furthermore, the preparation method of the ectoine-containing nisin protectant is as follows: Weigh 20-70% ectoine, 10-50% pullulan, and 10-40% 2-hydroxysuccinic acid according to the mass percentage ratio, mix them evenly in a mixer, and then grind them at 20-30℃ using a grinder until the particle size is 100-200nm, thereby obtaining a uniform ectoine-containing nisin protectant.

[0013] Ectoin is a compatible solute produced by salt-tolerant and halophilic microorganisms. It is a cyclic amino acid derivative that can mitigate the toxic effects of hyperosmolarity, high temperature, freeze-thaw cycles, drying, and radiation on biomolecules and even the entire cell. Pullulan is an extracellular water-soluble mucopolysaccharide produced by the fermentation of *Brachystomata*, possessing excellent film-forming, gas-barrier, and plasticity properties. 2-Hydroxybutyric acid is an intermediate product of human internal circulation, easily absorbed by the human body, and is highly acidic. The synergistic effect of these three components can significantly improve the stability of nisin during storage and application.

[0014] Furthermore, the application of the ectoine-containing nisin protectant:

[0015] (1) Weigh out 1-10% nisin protectant, 1-50% sterile water and 45-98% nisin slurry by mass percentage. First, mix the nisin protectant and sterile water evenly, and then mix them evenly with the nisin slurry to obtain a mixture.

[0016] (2) The mixture was spray-dried to obtain nisin powder;

[0017] (3) Collect the above-mentioned nisin dry powder, mix it evenly, divide it into portions, and seal it.

[0018] Furthermore, in step (1), the lactic acid nisin protectant is 5% by mass percentage, sterile water is 30% and lactic acid nisin slurry is 65%.

[0019] Furthermore, the preparation method of nisin slurry in step (1) is as follows: First, adjust the pH of nisin fermentation broth to 2.0~3.0 with 30% (m / m) hydrochloric acid, keep it at 70~90℃ for 2h, then filter it with a plate and frame filter to remove insoluble matter, add 10~30% sodium chloride and 1~5% flocculant to the clear liquid for flocculation, and then filter it with a plate and frame filter to remove the clear liquid. The wet material after removing the clear liquid is nisin slurry.

[0020] Furthermore, the moisture content of the nisin slurry is 45-55%, and the nisin potency is 4000-4500 IU / mg.

[0021] Furthermore, in step (2), the inlet temperature of the spray dryer is 110~120℃ and the outlet temperature is 70~80℃.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention provides a nisin protectant containing ectoine. It utilizes the protective effect of ectoine on biological macromolecules under strong acid, strong alkali, and high temperature environments, pullulan's excellent film-forming and gas-barrier properties, and the safety and strong acidity of 2-hydroxysuccinic acid. After mixing, the mixture is ground to a particle size of 100-200 nm, resulting in good dispersibility and solubility. Adding it before spray drying of nisin effectively mitigates the impact of high temperatures during spraying on nisin activity. The resulting nisin product has a long shelf life and good application performance. Attached Figure Description

[0024] Figure 1 The trends of potency changes in different samples of Examples 1-2 and Comparative Examples 1-3. Detailed Implementation

[0025] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents, materials, and apparatus are all prior art and can be obtained commercially.

[0027] In the following examples, the ectoine content used was 99.5%; the pullulan viscosity was 150-180 mm. 2 / s; L-hydroxysuccinic acid content 99.8%; nisin slurry potency 4342 IU / mg, moisture content 52%.

[0028] Example 1

[0029] S1. Weigh 50 parts of ectoine, 30 parts of pullulan, and 20 parts of L-hydroxysuccinic acid by weight and mix them initially in a mixer. Then grind them in a grinder with the particle size controlled at 150 nm and the grinding temperature controlled at 25℃ to obtain a homogeneous nisin protectant.

[0030] S2. Weigh 5 parts of the lactic acid nisin protectant prepared in S1 and 30 parts of sterile water by mass, mix them evenly, and then mix them evenly with 65 parts of lactic acid nisin slurry.

[0031] S3. Spray-dry the nisin slurry containing the lactic acid styrax protectant added in S2, controlling the inlet air temperature at 115℃ and the outlet air temperature at 75℃. Collect the above-mentioned lactic acid styrax powder, mix it evenly, and package it to obtain the finished lactic acid styrax product containing the lactic acid styrax protectant.

[0032] Example 2

[0033] S1. Weigh 40 parts of ectoine, 40 parts of pullulan, and 20 parts of L-hydroxysuccinic acid by weight and place them in a mixer for initial mixing. Then grind them in a grinder with the particle size controlled at 150 nm and the grinding temperature controlled at 25 °C to obtain a uniform nisin protectant.

[0034] S2. Weigh 5 parts of the lactic acid nisin protectant prepared in S1 and 30 parts of sterile water by mass, mix them evenly, and then mix them evenly with 65 parts of lactic acid nisin slurry.

[0035] S3. Spray dry the lactic acid nisin slurry containing lactic acid nisin protectant added in S2, controlling the inlet temperature to 115℃ and the outlet temperature to 75℃. Collect the above lactic acid nisin dry powder, mix it evenly, and package it to obtain the finished lactic acid nisin product containing lactic acid nisin protectant.

[0036] Example 3

[0037] S1. Weigh 20 parts of ectoine, 40 parts of pullulan polysaccharide, and 40 parts of L-hydroxysuccinic acid by weight and place them in a mixer for initial mixing. Then grind them in a grinder with the particle size controlled at 100 nm and the grinding temperature controlled at 30 °C to obtain a uniform nisin protectant.

[0038] S2. Weigh 5 parts of the lactic acid nisin protectant prepared in S1 and 30 parts of sterile water by mass, mix them evenly, and then mix them evenly with 65 parts of lactic acid nisin slurry.

[0039] S3. Spray dry the lactic acid nisin slurry containing lactic acid nisin protectant added in S2, controlling the inlet temperature to 115℃ and the outlet temperature to 75℃. Collect the above lactic acid nisin dry powder, mix it evenly, and package it to obtain the finished lactic acid nisin product containing lactic acid nisin protectant.

[0040] Example 4

[0041] S1. Weigh 70 parts of ectoine, 10 parts of pullulan polysaccharide, and 20 parts of L-hydroxysuccinic acid by weight and place them in a mixer for initial mixing. Then grind them in a grinder with the particle size controlled at 200 nm and the grinding temperature controlled at 20℃ to obtain a uniform nisin protectant.

[0042] S2. Weigh 5 parts of the lactic acid nisin protectant prepared in S1 and 30 parts of sterile water by mass, mix them evenly, and then mix them evenly with 65 parts of lactic acid nisin slurry.

[0043] S3. Spray dry the lactic acid nisin slurry containing lactic acid nisin protectant added in S2, controlling the inlet temperature to 115℃ and the outlet temperature to 75℃. Collect the above lactic acid nisin dry powder, mix it evenly, and package it to obtain the finished lactic acid nisin product containing lactic acid nisin protectant.

[0044] Comparative Example 1

[0045] The method is the same as in Example 1, except that in S1, 50 parts of sodium chloride are used instead of 50 parts of ectoine.

[0046] Comparative Example 2

[0047] The method is the same as in Example 1, except that in S1, 30 parts of sodium chloride are used instead of 30 parts of pullulan.

[0048] Comparative Example 3

[0049] The method is the same as in Example 1, except that in S1, 20 parts of sodium chloride are used instead of 20 parts of L-hydroxysuccinic acid.

[0050] Comparative Example 4

[0051] The method is the same as in Example 1, except that the protective agent in S1 is not ground in a grinder after initial mixing in the mixer.

[0052] Comparative Example 5

[0053] The method is the same as in Example 1, except that in S2, 5 parts of nisin protectant, 30 parts of sterile water, and 65 parts of nisin slurry are mixed simultaneously.

[0054] Comparative Example 6

[0055] The method is the same as in Example 1, except that the air inlet temperature of S3 is 130°C and the air outlet temperature is 80°C.

[0056] Experimental Example 1

[0057] Take three portions of each of the packaged nisin products from Examples 1-4 and Comparative Examples 1-6, and perform three replicates for each portion. Calculate the average potency of each example or comparative example. The potency determination method refers to GB 1886.231-2016 "National Food Safety Standard - Food Additives - Nisin". The results are shown in Table 1:

[0058] Table 1. Effects of protectants and application methods on nisin potency

[0059]

[0060] As shown in Table 1, the protective agents of ectoin, pullulan, and L-hydroxysuccinic acid in Examples 1, 2, 3, and 4 resulted in Streptococcus lactis potencies of 7657 IU / mg, 7602 IU / mg, 7532 IU / mg, and 7541 IU / mg, respectively. These potencies were all higher than those of the combination of pullulan and L-hydroxysuccinic acid in Comparative Example 1 (6643 IU / mg), higher than those of the combination of ectoin and L-hydroxysuccinic acid in Comparative Example 2 (6885 IU / mg), and higher than those of the combination of ectoin and pullulan in Comparative Example 3 (7196 IU / mg). This indicates that the Streptococcus lactis protective agent containing ectoin, pullulan, and L-hydroxysuccinic acid of the present invention can effectively reduce the loss of nisin during spray drying and protect the activity of nisin.

[0061] Example 1 showed a nisin potency of 7657 IU / mg, which was significantly higher than that of Comparative Example 4 (7368 IU / mg), Comparative Example 5 (7313 IU / mg), and Comparative Example 6 (7072 IU / mg). This indicates that the application method of the nisin protectant of the present invention can effectively reduce the loss of nisin during the spray drying process and protect the activity of nisin.

[0062] In summary, the lactic acid nisin protectant and its application method of the present invention can effectively reduce the loss of lactic acid nisin during the spray drying process and protect the activity of lactic acid nisin.

[0063] Experiment Example 2

[0064] Thirty samples each of the packaged nisin products from Examples 1-2 and Comparative Examples 1-3 were placed in a 30°C incubator. Three samples were randomly selected from each treatment at 0, 90, 180, 360, and 720 days, and the nisin potency of each sample was measured three times. The average potency of each example or comparative example was calculated. The results are shown in Table 2.

[0065] Table 2. Effects of preservatives on the potency of nisin during storage.

[0066]

[0067] The proportion of nisin potency in the examples or comparative cases after being placed in a 30℃ incubator for different times to the initial potency was calculated, with the potency at day 0 being calculated as 100%. The specific calculation method is as follows:

[0068] Remaining valence (%) = Valence after n days / Valence after 0 days * 100%

[0069] Valence decrease (%) = 100% - Remaining valence (%)

[0070] The calculation results are as follows Figure 1 As shown.

[0071] From Table 2 and Figure 1 It can be seen that after 180 days of storage in a 30°C constant temperature chamber, the potency of samples from Examples 1-2 decreased by 12-14%, while that of samples from Comparative Examples 1-3 decreased by 20-29%. After 360 days of storage, the potency of samples from Examples 1-2 decreased by 20-23%, while that of samples from Comparative Examples 1-3 decreased by 40-52%. After 720 days of storage, the potency of samples from Examples 1-2 decreased by 27-30%, while that of samples from Comparative Examples 1-3 decreased by 52-68%. It is evident that, for the same storage time, the potency decrease of the samples from the examples of this invention is significantly less than that of the comparative samples. This indicates that the lactic acid nisin protectant of this invention can effectively protect the activity of lactic acid nisin and has a long shelf life.

[0072] In summary, this invention provides a protective agent for nisin containing ectoine and its application. By utilizing the protective effect of ectoine on biological macromolecules under strong acid, strong alkali, and high temperature environments, the excellent film-forming and gas-barrier properties of pullulan, and the safety and strong acidity of 2-hydroxysuccinic acid, the mixture is ground to a particle size of 100-200 nm, resulting in good dispersibility and solubility. When added before spray drying of nisin, it can effectively mitigate the impact of high temperature during spraying on the activity of nisin. The resulting nisin product exhibits a small decrease in potency with prolonged storage time, a long shelf life, and good application effects.

Claims

1. A lactic acid nisin protectant containing ectoine, characterized in that: By mass percentage, it includes 20-70% ectoine, 10-50% pullulan, and 10-40% 2-hydroxysuccinic acid; wherein the 2-hydroxysuccinic acid is L-hydroxysuccinic acid.

2. The lactic acid nisin protectant containing ectoine according to claim 1, characterized in that: By weight percentage, it includes 50% ectoine, 30% pullulan, and 20% L-hydroxysuccinic acid.

3. The lactic acid nisin protectant containing ectoine according to claim 1, characterized in that: By weight percentage, it includes 40% ectoine, 40% pullulan, and 20% L-hydroxysuccinic acid.

4. The lactic acid nisin protectant containing ectoine according to claim 1, characterized in that: The viscosity of the pullulan polysaccharide is 150-180 mm. 2 / s.

5. A method for preparing a lactic acid nisin protectant containing ectoine as described in any one of claims 1-4, characterized in that: Weigh out 20-70% ectoine, 10-50% pullulan, and 10-40% 2-hydroxysuccinic acid by mass percentage. Mix them evenly in a mixer and then grind them at 20-30℃ until the particle size is 100-200nm to obtain a uniform ectoine-containing nisin protectant.

6. The application of the lactic acid nisin protectant containing ectoine as described in claim 1, characterized in that: (1) Weigh out 1-10% nisin protectant, 1-50% sterile water and 45-98% nisin slurry by mass percentage. First, mix the nisin protectant and sterile water evenly, and then mix them evenly with the nisin slurry to obtain a mixture. (2) The mixture was spray-dried to obtain nisin powder; (3) Collect the above-mentioned nisin dry powder, mix it evenly, divide it into portions, and seal it.

7. The application of the ectoine-containing nisin protectant according to claim 6, characterized in that: In step (1), the lactic acid nisin protectant is 5% by mass percentage, sterile water is 30% and lactic acid nisin slurry is 65%.

8. The application of the ectoine-containing nisin protectant according to claim 6 or 7, characterized in that: The preparation method of nisin slurry in step (1) is as follows: First, adjust the pH of nisin fermentation broth to 2.0~3.0 with 30% (m / m) hydrochloric acid, keep it at 70~90℃ for 2h, then filter it with a plate and frame filter to remove insoluble matter, add 10~30% sodium chloride and 1~5% flocculant to the clear liquid for flocculation, and then filter it with a plate and frame filter to remove the clear liquid. The wet material after removing the clear liquid is nisin slurry.

9. The application of the lactic acid nisin protectant containing ectoine according to claim 8, characterized in that: The nisin slurry has a moisture content of 45-55% and a nisin potency of 4000-4500 IU / mg.

10. The application of the ectoine-containing nisin protectant according to claim 6, characterized in that: The spray drying process in step (2) involves an air inlet temperature of 110~120℃ and an air outlet temperature of 70~80℃.

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