TWI714258B - Preparation method for improving phycoerythrin yield - Google Patents

Abstract

The present invention provides a preparation method for increasing the yield of phycoerythrin, which comprises step one, mixing an acremonium, a fermentation broth containing Pseudoalteromonas seawater and a seawater to obtain a mixture of acremonium Step 2: Fermenting the Acremonium bacteria liquid mixture under a time condition, a temperature condition and a rotation speed condition to break the Acremonium algae to release phycoerythrin; the present invention also provides a bacterium for breaking the Acremonium algae the use of.

Description

Preparation method for improving phycoerythrin yield

The present invention relates to a method for increasing the production of phycoerythrin, and particularly relates to a method for increasing the production of phycoerythrin by using bacterial fermentation.

Phycoerythrin (PE) is a kind of phycobiliprotein (Phycobiliprotein), which is a photosynthetic pigment protein used by algae such as blue-green algae, red algae, and cryptophytes to supplement light. Due to its spectral and fluorescent properties, phycoerythrin has been widely used in applications related to fluorescent labels, such as biological indicator reagents, chemical preparations, in vitro diagnostic labels, fluorescent detection probes, etc. In recent years, phycoerythrin has also been used in food, cosmetics and health products.

However, although phycoerythrin has high industrial availability, it has a high production technology threshold and low output, which leads to the hidden concern of high cost of related application products. Therefore, how to reduce the production cost of phycoerythrin and increase the yield of phycoerythrin has become an urgent problem in related application fields.

Chinese patent CN104130319B discloses a method for extracting phycoerythrin, which includes adding red algae to ice powder at -50°C~-10°C, and performing wall-breaking treatment at a stirring speed of 1000r/min~3000r/min To obtain a phycoerythrin-containing wall-breaking liquid; then the obtained phycoerythrin-containing wall-breaking liquid is separated, purified and dried to obtain a phycoerythrin product.

At present, the acquisition of phycoerythrin is mainly based on large red algae such as laver and celestial algae as raw materials. The red algae is physically treated first, including the release of phycoerythrin by repeated freezing and thawing, ultrasonic breaking, etc. , And then use ammonium sulfate precipitation, ion exchange chromatography and other methods to separate and purify phycoerythrin. However, because the algae of large red algae are mostly fronds, it is difficult to break them to release phycoerythrin. In addition, the large red algae have a high sugar content, which makes subsequent separation and purification procedures more difficult. Therefore, how to effectively break red algae to increase the production of phycoerythrin is another problem faced by the technical field of the present invention.

The inventors of the present invention found that although Acrochaetium sp. belongs to red algae, it is different from the traditionally used large red algae. Acrochaetium sp. has less sugar content and the algae is filamentous, which is very It is suitable as a raw material for obtaining phycoerythrin. Using Acremonium as a raw material to prepare phycoerythrin has the advantage of simpler separation and purification. If it can further increase the yield of phycoerythrin that can be prepared, it will be of great help to the market application of phycoerythrin.

Based on the above findings, the purpose of the present invention is to provide a preparation method for increasing the yield of phycoerythrin, which comprises: step one, mixing an acremonium, a fermentation broth and a seawater to obtain a acremonium broth mixture; Step 2: Ferment the Acremonium broth mixture under a time condition, a temperature condition and a rotation speed condition to break the Acremonium algae to release phycoerythrin; wherein the fermentation broth contains Pseudoalteromonas marine ( Pseudoalteromonas haloplanktis ); wherein the time condition is 6-78 hours, the temperature condition is 20-25°C, and the speed condition is 100-500 rpm.

In order to achieve the purpose of the foregoing invention, the OD ₆₀₀ value of the fermentation broth is 0.9-1.1.

In order to achieve the purpose of the foregoing invention, the OD ₆₀₀ value of the fermentation broth is 1.

In order to achieve the purpose of the foregoing invention, the temperature condition is 22°C.

In order to achieve the purpose of the foregoing invention, the speed condition is 300 rpm.

In order to achieve the purpose of the foregoing invention, the time condition is 6 to 36 hours.

In order to achieve the purpose of the foregoing invention, the time condition is 24-30 hours.

In order to achieve the aforementioned purpose of the invention, the mixing ratio of the Acremonium: the fermentation broth: the seawater is 2 (g):1 (mL):1 (mL).

In order to achieve the purpose of the foregoing invention, the seawater is artificial seawater or natural seawater.

The present invention also provides a use of bacteria for crushing Acremonium, which comprises mixing an Acremonium and a bacterium to ferment; wherein the bacterium is Pseudoalteromonas marine.

The invention solves the problem that in the prior art, the algae is difficult to be broken The yield of phycoerythrin is not high. The method provided by the present invention can not only increase the yield of phycoerythrin, but also has simple procedures and easy implementation, greatly reduces the technical threshold of phycoerythrin production, and is beneficial to promote the application progress of phycoerythrin in the market. The present invention also solves the problem that no one has used Pseudoalteromonas parasitica as a use for breaking Acremonium in the past.

Figure 1 shows the flow chart of Acremonium fermentation experiment.

Figure 2 shows the effect of time conditions on the production of phycoerythrin.

All technical and scientific terms mentioned in this manual, unless otherwise defined, are meanings that can be commonly understood by the professionals in the field. The singular terms "one", "one", "the", "the said "Unless otherwise specified, it can refer to more than one object. In addition, the terms "include" and "include" are open-ended conjunctions; in addition, unless otherwise specified, the materials used in the present invention are commercially available and readily available .

The term "extraction" refers to the separation of an effective ingredient or active ingredient from other ingredients.

The term "breaking the wall" refers to breaking the cell wall of an organism so that the components in the organism are released outside the cell wall.

The term "broken" means that the organism will be broken up so that the components of the organism will be released outside the cell wall.

The term "fermentation" refers to the reaction process of using organisms to decompose target substances, where the biological system includes but not limited to bacteria, yeasts, plant cells or microorganisms.

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples.

The Acrochaetium formosanum used in the embodiment of the present invention is Acrochaetium formosanum , which can be obtained from natural seas by those with ordinary knowledge in the technical field of the present invention. The embodiments of the present invention are illustrated by Acrochaetium formosanum , but the present invention is not limited by Acrochaetium formosanum . Species of the genus Acrochaetium or Acrochaetium that can produce phycoerythrin should be covered by the scope of the present invention.

The bacteria used in the examples of the present invention is the standard strain Pseudoalteromonas haloplanktis (ATCC 14393), which can be purchased on the market. The strains obtained were purchased for cryopreservation, and were treated with a solid medium (Marine agar) coating plate for activation before use.

In the present invention, as shown in Figure 1, the fermentation broth is prepared by using Pseudoalteromonas parasitica first, the fermentation broth is mixed with an acremonium and a seawater to obtain an acremonium broth mixture, and then the The Acephalosporium bacteria liquid mixture is fermented under a temperature condition and a rotation speed condition to break the Acremonium, and then samples are taken to detect the phycoerythrin content to confirm the optimal fermentation time condition.

The preparation method for improving the yield of phycoerythrin provided by the present invention includes:

Step 1. Mixing an acremonium, a fermentation broth containing Pseudoalteromonas seawater and a seawater to obtain a acremonium broth mixture, wherein the OD ₆₀₀ value of the fermentation broth is 0.9-1.1, The OD ₆₀₀ value of the fermentation broth is preferably 1, wherein the mixing ratio of the Acremonium: the fermentation broth: the seawater is 2(g):1(mL):1(mL);

Step 2: Ferment the Acremonium liquid mixture under a time condition, a temperature condition and a rotation speed condition to break the Acremonium algae to release phycoerythrin, wherein the time condition is 6-78 hours, and the time condition Preferably 6-36 hours, the best time condition is 24-30 hours, the temperature condition is 20-25°C, the temperature condition is best 22°C, the speed condition is 100-500rpm, the speed condition is better It is 200-400rpm, and the best speed condition is 300rpm.

Example 1: Preparation of fermentation broth

After deicing the cryopreserved Pseudoalteromonas parasitica, take 100 μL of bacterial solution to a solid medium (Marine agar) coating plate to activate the strain. After culturing at 22°C for 12 hours, use 1 mL of Phosphate buffered saline (Phosphate buffered saline). saline, PBS) Rinse and inoculate Pseudoalteromonas parasitica in 50mL of liquid culture medium (Marine broth), carry out liquid culture at 22°C and 300rpm for 12 hours, and then use a spectrophotometer to detect the absorbance at 600nm , When the OD ₆₀₀ value is 0.9-1.1, it can be used as a fermentation broth for Acremonium fermentation.

Example 2: Acremonium fermentation treatment

A piece of Acremonium is added to seawater, and then the fermentation broth prepared by the method described in Example 1 is added to obtain a mixture of Acremonium broth. Ferment the acremonium broth mixture under a temperature condition and a rotation speed condition, Acremonium was broken to release water-soluble phycoerythrin, and samples were taken at 0, 6, 12, 24, 30, 36, 48, 54, 60, 72 and 78 hours for phycoerythrin content detection. Wherein, the mixing ratio of the Acremonium: the seawater: the fermentation broth is 2(g):1(mL):1(mL); wherein the seawater is artificial seawater or natural seawater; wherein the temperature condition is 20- 25°C, the best temperature condition is 22°C; wherein the speed condition is 100-500rpm, the speed condition is preferably 200-400rpm, and the speed condition is 300rpm.

Example 3: Determination of Phycobiliprotein Content

The content of phycobiliprotein was detected by referring to the method proposed by Allen et al. (1973). At each sampling point, 1 mL of the fermented Acremonium broth mixture was taken, centrifuged to obtain the supernatant, and then detected with an enzyme immunoassay. The absorbance values of 562nm, 615nm and 652nm are substituted into the following formula to obtain different phycobiliprotein content: Phycocyanin (mg/mL)=(OD ₆₁₅ -0.474(OD ₆₅₂ ))÷5.34 Paraphycocyanin (mg/mL) )=(OD ₆₅₂ -0.208(OD ₆₁₅ ))÷5.09 Phycoerythrin (mg/mL)=(OD ₅₆₂ -2.41(PC)-0.89(APC))÷9.62

From Figure 2 and Table 1, it can be seen that the content of phycoerythrin released by Acremonium after simple physical treatment is only 0.128g, and after 6-78 hours of fermentation by the method described in Example 2, the released The content of phycoerythrin is higher than that of simple physical treatment; among them, the fermentation time is 6-36 hours, which can release more phycoerythrin content; among them, the fermentation time is 24-30 hours, which can release the most Phycoerythrin content.

The present invention provides a method for preparing high phycoerythrin production, which utilizes Pseudoalteromonas salivarius to ferment and decompose Acremonium so that the algae is broken, and the internal components of Acremonium are released so that the nutrient components of Acremonium or Active ingredients, especially phycoerythrin, are easier to be precipitated or extracted to make it easier to use. The method provided by the present invention can increase the yield of phycoerythrin, has the advantages of simplicity and easy operation, and is beneficial to industrial production applications.

As disclosed in the preferred embodiments of this specification, the present invention belongs to Those with ordinary knowledge in the technical field can clearly understand that the foregoing embodiments are only examples; those with ordinary knowledge in the technical field to which the present invention belongs can implement many changes and substitutions without any difference from the technical features of the present invention. According to the embodiments of the specification, the present invention can have many variations without hindering implementation. The claims provided in this specification define the scope of the present invention, which covers the aforementioned methods, structures and equivalent inventions.

The above-mentioned multiple functions are in fact fully in line with the statutory patent requirements for novelty and advancement. An application is filed in accordance with the law, and you are urged to approve this invention patent application to encourage invention.

Claims (10)

A preparation method for increasing the yield of phycoerythrin, which comprises: step one, mixing an acremonium, a fermentation broth, and a seawater to obtain an acremonium broth mixture; step two, the acremonium broth The mixture is fermented under a time condition, a temperature condition and a rotation speed condition to break the Acremonium to release phycoerythrin; wherein the fermentation broth contains Pseudoalteromonas parasitica; wherein the time condition is 6-78 hours , The temperature condition is 20-25°C, and the speed condition is 100-500rpm. The method described in item 1 of the scope of patent application, wherein the OD ₆₀₀ value of the fermentation broth is 0.9-1.1. The method described in item 1 of the scope of patent application, wherein the OD ₆₀₀ value of the fermentation broth is 1. The method described in item 1 of the scope of patent application, wherein the temperature condition is 22°C. The method described in item 1 of the scope of patent application, wherein the speed condition is 300 rpm. The method described in item 1 of the scope of patent application, wherein the time condition is 6-36 hours. The method described in item 1 of the scope of the patent application, wherein the time condition is 24-30 hours. The method described in item 1 of the scope of patent application, wherein the acremonium: the fermentation The mixing ratio of the bacterial liquid: the seawater is 2 (g): 1 (mL): 1 (mL). The method described in item 1 of the scope of patent application, wherein the seawater is artificial seawater or natural seawater. A use of bacteria for breaking Acremonas, which includes mixing an Acremonium and a bacterium to ferment; wherein the bacteria is Pseudoalteromonas parasitica.

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