阅读说明：本技术 螺旋藻培养剩余液在制备意大利青霉抑制剂中的应用 (Application of spirulina culture residual liquid in preparation of penicillium italicum inhibitor ) 是由 李博生 潘晴 于 2021-07-09 设计创作，主要内容包括：本发明属于螺旋藻培养技术领域,具体涉及一种螺旋藻培养剩余液在抑制意大利青霉中的应用。本发明首次对螺旋藻培养剩余液的相关功效进行了研究,发现其可有效地抑制意大利青霉,其抑制作用甚至要优于常见的抗生素纳他霉素。(The invention belongs to the technical field of spirulina culture, and particularly relates to application of a spirulina culture residual solution in inhibition of penicillium italicum. According to the invention, the related efficacy of the remaining liquid of the spirulina culture is researched for the first time, and the inhibition effect of the surplus liquid is even better than that of the common antibiotic natamycin, namely the penicillium italicum can be effectively inhibited.)

1. Use of the remaining culture broth of Spirulina in the preparation of Penicillium italicum inhibitor is provided.

2. The use according to claim 1, wherein the spirulina culture residue is a culture residue after removing spirulina in the logarithmic growth phase or the stationary growth phase by filtration.

3. The application of the residual liquid for culturing the spirulina comprises the steps of performing ice temperature treatment on the spirulina in the logarithmic growth phase, continuously culturing for 3-7 days, and filtering to remove the residual liquid after the spirulina is removed.

4. Use according to claim 3, wherein the temperature of the ice temperature pre-treatment is between-5 and-1 ℃.

5. The use according to claim 3, wherein the time of the ice temperature pretreatment is 10-15 h.

6. The use according to claim 2 or 3, wherein the temperature of the spirulina is 28-35 ℃.

7. The use according to any one of claims 1 to 6, wherein the culture solution used in the process of culturing Spirulina comprises NaHCO₃ 16.5～17.0g/L、NaCl 0.8～1.2g/L、K₂HPO₄ 0.4～0.6g/L、MgSO₄·7H2O 0.15～0.25g/L、NaNO₃ 2.4～2.6g/L、CaCl₂·2H2O 0.03～0.05g/L、K2_SO4 0.8～1.2g/L、EDTA-2Na 0.06～0.10g/L、FeSO₄·7H2O 0.008～0.012g/L。

8. The use according to any one of claims 1 to 6, wherein the culture residue is concentrated to 1/2 to 1/8 volume parts before use in the preparation of Penicillium italicum inhibitors.

9. A Penicillium italicum inhibitor characterized by being added with the residual spirulina culture solution according to any one of claims 1 to 8.

Technical Field

The invention belongs to the technical field of spirulina culture, and particularly relates to application of a spirulina culture residual solution in inhibition of penicillium italicum.

Background

Spirulina, an autotrophic prokaryote, is named because of its spiral form. About 38 types of spirulina are found, and are distributed in fresh water, and few spirulina are distributed in salt water or ocean. With the progress of research, spirulina has been produced on a large scale and has been applied to various fields such as food, feed, medicine, cosmetics, and the like.

After the plant is stressed by certain environmental factors, the total dry weight of the plant in the recovery period after stress relief is larger than that of a control group, the phenomenon is called compensation growth, and the phenomenon of compensation growth is widely existed in land, aquatic animals and higher plants at present and is intensively researched. Their stress factors include temperature, light, pH and salt stress. Patent CN 109722407 a discloses a related scheme for the ice temperature compensation growth of spirulina, in which the effect of ice temperature treatment on spirulina biomass is studied. The prior art focuses on increasing the biomass of spirulina by compensating the stress of the spirulina, and so far, no report on the efficacy research of residual culture solution after the spirulina is cultured is found. In fact, during the culture process of spirulina, the cells are widely exchanged with the components of the culture solution, and a plurality of bioactive substances exist in the rest culture solution, have related activity and function, and are worthy of being researched.

Disclosure of Invention

Aiming at the problem that the related activity of the remaining liquid of the spirulina culture is not researched in the prior art, the invention firstly provides the application of the remaining liquid of the spirulina culture in preparing the Penicillium italicum inhibitor.

The invention firstly finds that the spirulina culture residual liquid has obvious inhibition activity on the penicillium italicum and has important significance on the preservation of oranges and the preservation of related foods and the like.

Preferably, the remaining liquid after the spirulina is cultured in the logarithmic growth phase or the stationary growth phase is removed by filtration. The invention discovers for the first time that the spirulina in the normal growth period (including logarithmic growth period and stable growth period) can generate effective components with inhibition effect on the Penicillium italicum in the process of growth and metabolism, and the residual culture solution after filtering the spirulina can be used for preparing the Penicillium italicum inhibitor.

Preferably, the residual liquid of the spirulina culture is the residual liquid of the spirulina culture obtained by continuously culturing spirulina in logarithmic growth phase for 3-7 days after ice temperature treatment and filtering to remove the spirulina. After the spirulina is treated by the ice temperature, the rest culture solution has better inhibition activity on the Penicillium italicum.

Preferably, the temperature of the ice temperature pretreatment is-5 to-1 ℃.

Further preferably, the temperature of the ice temperature pretreatment is-3 to-2 ℃.

Preferably, the time of the ice temperature pretreatment is 10-15 h. The ice-temperature pretreatment is carried out at the temperature and the time, so that the spirulina can be effectively stimulated to generate effective components for inhibiting mildew, the spirulina can generate a compensation growth phenomenon, and the formation of the output of the spirulina is promoted.

Preferably, the culture condition of the spirulina is 28-35 ℃.

Preferably, the culture solution used in the process of culturing spirulina comprises NaHCO₃ 16.5～17.0g/L、NaCl 0.8～1.2g/L、K₂HPO₄ 0.4～0.6g/L、MgSO₄·7H2O 0.15～0.25g/L、NaNO₃ 2.4～2.6g/L、CaCl₂·2H2O 0.03～0.05g/L、K2_SO4 0.8～1.2g/L、EDTA-2Na 0.06～0.10g/L、FeSO₄·7H2O 0.008～0.012g/L。

Preferably, the culture residue is concentrated to 1/2-1/8 of the original volume and then used for preparing the Penicillium italicum inhibitor. The bacteriostatic effect is more obvious after the concentration to the multiple.

In another aspect, the invention provides an inhibitor of penicillium italicum, wherein the inhibitor is supplemented with a surplus solution of spirulina culture as described herein.

1) According to the invention, the related efficacy of the remaining liquid of the spirulina culture is researched for the first time, and the inhibition effect of the surplus liquid is even better than that of the common antibiotic natamycin, namely the penicillium italicum can be effectively inhibited.

2) The invention further discovers that the inhibition effect of the obtained filtrate on Penicillium italicum is more obvious after the spirulina is subjected to ice temperature pretreatment culture.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The embodiment relates to application of residual liquid of spirulina culture under common culture conditions in bacteriostasis.

1) Culturing spirulina at 30 deg.C under illumination intensity of 4000lux for 12 hr for 3d (the spirulina is in logarithmic phase), filtering with 1000 mesh filter screen to obtain culture residual solution, and concentrating the culture residual solution to 1/5;

2) and in the bacteriostasis experiment, 200 mu L of penicillium italicum bacterial suspension is uniformly coated, a hole puncher with the thickness of 7mm is selected for operation after the surface is solidified, and the top of a corresponding culture dish is marked. The samples to be tested were filtered through 0.22 μm sterile microfiltration membranes, 50 μ L of which were added accurately to each well, 3 replicates per sample. After the sample is added, the record is sealed by a sealing film, and the culture dish is stably placed into a constant temperature incubator for culture. All the above operations are aseptic operations. Culturing at 28 deg.C for 2h to observe the bacteriostatic diameter. The positive control was 50. mu.g/mL natamycin and the negative controls were all saline.

Examples 2 to 3

The difference compared to example 1 is that in step 1) the culture retentate was concentrated to 1/2 (example 2) or 1/4 (example 3) of the original volume.

Example 4

The present embodiment relates to the application of remaining liquid of spirulina culture under ordinary culture conditions in bacteriostasis, and compared with embodiment 1, the difference is that the filtrate is the remaining liquid of spirulina culture obtained after ice temperature pretreatment.

1) Placing spirulina at about 30 ℃ under the conditions of illumination intensity of 4000lux and light dark culture for 12h respectively for culturing for 1d, then placing the spirulina at about-3 ℃ for culturing for 12h, finally placing the spirulina at about 30 ℃ under the conditions of illumination intensity of 4000lux and light dark culture for 12h for culturing for 3d, filtering the culture solution by adopting a 1000-mesh filter screen to obtain culture residual solution, and concentrating the culture residual to 1/5 of the original volume;

2) and in the bacteriostasis experiment, 200 mu L of penicillium italicum bacterial suspension is uniformly coated, a hole puncher with the thickness of 7mm is selected for operation after the surface is solidified, and the top of a corresponding culture dish is marked. The samples to be tested were filtered through 0.22 μm sterile microfiltration membranes, 50 μ L of which were added accurately to each well, 3 replicates per sample. After the sample is added, the record is sealed by a sealing film, and the culture dish is stably placed into a constant temperature incubator for culture. All the above operations are aseptic operations. Culturing at 28 deg.C for 2h to observe the bacteriostatic diameter.

Examples 5 to 6

The difference compared to example 4 is that in step 1) the culture retentate was concentrated to 1/2 (example 5) or 1/4 (example 6) of the original volume.

Comparative example 1

Compared with example 1, the difference is that 50 mug/mL natamycin is used as a positive control, and the bacteriostatic experiment operation in step 2) is the same as that in example 1.

Comparative example 2

Compared with example 1, the difference is that physiological saline is used as a positive control, and the bacteriostatic experiment operation in the step 2) is the same as that in example 1.

Comparative example 3

The difference from example 1 was that the inhibitory action of the culture residual solution on Penicillium digitatum was examined, and the procedure was the same as in example 1 except that Penicillium italicum was replaced with Penicillium digitatum.

Examples of the experiments

The results of the examples and comparative examples are shown in table 1:

TABLE 1

From the above data, it can be seen that the spirulina culture residue has a certain inhibitory effect on penicillium italicum, and when the concentration degree is proper, the inhibitory effect is even stronger than that of natamycin, and the inhibitory effect of the culture residue obtained after ice-temperature treatment is further enhanced. But not other microorganisms such as Penicillium digitatum.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.