TWI463012B - Duckweed hydrolysate and use thereof - Google Patents

Description

Duckweed hydrolysate and its use

The present invention relates to a hydrolysate of duckweed which can be used as a nutrient for cultivating microorganisms such as Escherichia coli to increase cell biomass and production of cell products.

It is known that duckweed (a plant containing the family Lemnaceae) has a very fast doubling time and has been widely used as a feed for ducks, chickens, fish and shrimps. Duckweed can also be used in wastewater treatment and converts carbon dioxide into biomass. Therefore, it is believed that duckweed has considerable potential to solve serious water and weather problems. Cheng et al. (Growing Duckweed to recover nutrients from wastewaters and for production of fuel ethanol and animal feed. Clean. 37(1). 17-26 (2009)) reveal that duckweed can remove nitrogen and phosphorus substances from wastewater, and float Ping Biomass can be used as an alternative source of carbohydrates for fermentation in the manufacture of fuel ethanol.

Microorganisms, whether naturally occurring or recombinant, have been widely used to produce peptides (such as pharmaceutically active proteins) and secondary metabolites (such as vitamins and carotenoids). However, many problems are still encountered when manufacturing such products using microorganisms. For example, the ingredients used in the medium are expensive and the yield under large scale fermentation may be poor. US 7,122,341 B1 (Liao, 2000) reveals metabolites Process control mode. The method comprises obtaining E. coli with high lycopene production.

However, there is still a need in the art to develop inexpensive methods that can increase large-scale fermentation yields.

The present invention provides an inexpensive supplement that increases the biological quality of cells and the yield of cellular products in microbial fermentation.

Accordingly, the present invention provides a duckweed hydrolysate obtained by a method comprising the steps of: (a) obtaining duckweed juice from duckweed; (b) at a temperature of from about 25 ° C to about 75 ° C, The duckweed juice is hydrolyzed with one or more proteases for about 6 to about 48 hours; and (c) the duckweed hydrolysate is collected.

The present invention further provides a method for preparing a carotenoid comprising the steps of: cultivating a carotenoid producing microorganism under a suitable condition in a medium comprising the duckweed hydrolysate of the present invention; collecting cells; and from the cell Carotenoids are isolated.

The present invention can be understood from the various aspects of the invention, the embodiments and the description of the embodiments disclosed herein. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with the present invention shall have the meaning as understood by those of ordinary skill in the art. And, as will be appreciated, unless the definitions provided herein are otherwise specified, in the case of any potential ambiguity, the definition of terms should be consistent with such commonly used terms (as defined in the dictionary). Can learn more The terminology used in the present invention is for the purpose of describing particular embodiments and not for the limitation.

It must be noted that the singular forms "a" and "the" are used in the s Therefore, unless the context requires otherwise, the singular terms shall include the plural and the plural terms also include the singular.

The scope of the invention is generally indicated by the <RTI ID=0.0>" </ RTI> </ RTI> <RTIgt; When the range is expressed in the above manner, it includes a range from a particular value and/or to another particular value. Similarly, when a value can be approximated by the term "about", it will be understood that it is another aspect of a particular value. It can be further appreciated that when referring to other endpoints and other endpoints themselves, the endpoints of each range are meaningful. According to the invention "about" means ±10%.

The present invention provides a duckweed hydrolysate obtained by a method comprising the steps of: (a) obtaining duckweed juice from duckweed; and (b) extracting the duckweed at a temperature of from about 25 ° C to about 75 ° C. The juice is hydrolyzed with one or more proteases for about 6 to about 48 hours; and (c) the duckweed hydrolysate is collected.

The term "duckweed" in the present invention means a plant of the family Lemnaceae, which includes Lemna, Spirodela, Wolffia and Wolfiella. The duckweed which can be used in the present invention can be, but is not limited to, Lemna aequinoctialis , Lemna gibba , Lemna disperma , Lema Xiaoqingping ( Lemna minor ), Lemna ecuadoriensis , Lemna japonica , Lemna obscura , Spirodela punctata , Spirodela polyrrhiza and Wolffia arrhiza .

The term "duckweed juice" means the liquid portion of the duckweed. However, the liquid portion does not exclude small particles derived from duckweed, such as particles less than about 0.25 mm.

According to the present invention, duckweed juice can be obtained by any conventional method. For example, the duckweed can be ground or treated with a juice machine, after which the liquid portion is filtered and/or centrifuged to separate the duckweed juice from the solid residue.

The term "protease" is intended to be derived from a microorganism, animal or plant that hydrolyzes proteins and polypeptides into any product or enzyme of a peptide, oligopeptide and/or amino acid. The protease of the present invention may be endoprotases, exopeptidases or a combination thereof. Proteases useful in the present invention include, but are not limited to, thermolysin, pepsin, trypsin bromelain, Alcalase (Novozyme), flavor protease (Flavorzyme). , Esperase, PTN 6.0S, Acid Protease, Protamex, Protease A, Protease M, Protease N, Protease NL Protease NL), Protease P, Protease S, Protin SD, Thermoase, Flavorpro, and Promod. The protease is preferably a propodase.

According to the present invention, one or more proteases may be added to the duckweed juice simultaneously, sequentially or separately, and the concentration of the protease added to the hydrolysis reaction may be adjusted depending on the type of protease used. For example, the amount of one or more proteases may be from about 1 A.U. to about 150 A.U. per liter of duckweed juice. The amount of one or more proteases is preferably from about 2 A.U. to about 50 A.U. The amount of one or more proteases is more preferably from about 3 A.U. to about 15 A.U.

According to the present invention, the temperature and time of the hydrolysis reaction may depend on the protease used. The type is adjusted. The temperature of the hydrolysis reaction may range from about 25 ° C to about 75 ° C, preferably from about 35 ° C to about 60 ° C, and more preferably from about 35 ° C to about 45 ° C. The hydrolysis reaction may range from about 6 to about 48 hours, preferably from about 12 to about 36 hours, and more preferably from about 12 to about 24 hours. At the end of the hydrolysis reaction, the protease may be deactivated by any conventional method as needed.

According to the method of the present invention, it further comprises the step (d) of concentrating and/or drying the duckweed hydrolysate collected by the step (c), thereby obtaining a concentrated and/or dried duckweed hydrolysate. The concentration and drying process is a conventional technique including, but not limited to, heating, lyophilization, spray drying, roller dryer drying, fluid bed drying, and any combination thereof.

The invention further provides a method for the preparation of carotenoids comprising the steps of: cultivating a carotenoid producing microorganism in a culture medium comprising an effective amount of the duckweed hydrolysate of the invention under suitable conditions; collecting Cells; and separating carotenoids from the cells.

The term "carotenoid" is intended to mean any of the different pigments having the chemical characteristics of a long chain aliphatic polyene (C40) chain composed of 8 isoprene units, and usually yellow to red, which pigments can be widely used by microorganisms, Found in plants and animals. Examples of carotenoids include beta-carotene, zeaxanthin, canthaxanthin, beta-cryptoxanthin, astaxanthin, lycopene, and lutein.

The term "carotenoid producing microorganism" means any microorganism capable of producing carotenoids. The microorganism can be any host cell that has been transformed with one or more exogenous genes encoding an enzyme involved in carotenoid synthesis, and which can express the enzyme and synthesize carotenoids under suitable conditions. Such host cells include, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, Blakeslea trispora , Agrobacterium aurantiacum , Haematococcus pluvialis , and Xanthophyllomyces dendrorhous . A preferred host cell is E. coli. The microorganism can be constructed by any of the materials and techniques well known in the art, such as those disclosed in US 7,122,341 B1, US 5,429,935, Misawa et al. (Elucidation of the Erwinia uredovora Carotenoid Biosynthetic Pathway by Functional Analysis of Gene Products Expressed in Escherichia coli ; Journal of Bacteriology, Vol. 172, No. 12, pp. 6604-6712 (1990)), Wang et al. (Engineered Isoprenoid Pathway Enhances Astrxanthin Production in Escherichia coli ; Biotechnology and Bioengineering, Vol. 62, No. 2 , pp. 235-241 (1999)), Farmer et al. (Improving lycopene production in Escherichia coli by engineering metabolic control; Nature Biotechnology, Vo. 18, pp. 533-537 (2000)), Scaife et al. (Characterization of Cyanobacterial) β-Carotene Ketolase and Hydrolase Genes in Escherichia coli , and Their Application for Astaxanthin Biosynthesis; Biotechnology and Bioengineering, Vol. 103, No. 5, pp. 944-955 (2009)) and Scaife et al. (Comparative Analysis of β-Carotene) Hydrolase Genes for Astaxanthin Biosynthesis; J. Nat. Prod., 75, 1117 -1124 (2012)). These general references are incorporated into this document as part of the specification.

The "suitable conditions" for cultivating microorganisms in the present invention mean that conditions such as temperature and culture time allow the microorganism to grow, multiply, and exhibit enzymes related to carotenoid synthesis and synthesize carotenoids. The exact conditions required will vary depending on the species and culture mode of the microorganism. According to the present invention, the culture method can be any conventional fermentation method such as batch fermentation, feed batch fermentation, and continuous fermentation.

In the present invention, "the medium contains an effective amount of duckweed hydrolysate" Nutrients, in addition to containing duckweed hydrolysate, may also include any conventional components for culturing microorganisms, such as nitrogen sources (such as yeast extract, peptone and amino acids); carbon sources (such as glucose and glycerol); Salts (such as potassium and magnesium salts) and buffers (such as phosphate buffers). The term "effective amount" means that the amount of the duckweed hydrolysate is effective to increase cell quality and carotenoid production.

Such methods for collecting cells and isolating carotenoids from cells are well known in the art, as disclosed in EP 2 088 199 A1. This general reference is incorporated into this case as part of the specification.

Example Example 1 Preparation of duckweed hydrolysate

Take 100 kg of fresh duckweed (green duck), wash it with water, grind it with a grinder or treat it with a juice machine. The liquid portion was collected and filtered to obtain duckweed juice. The duckweed juice (60 L) obtained was treated with 450 A.U. of Protamex at 40 ° C for 20 hours to obtain a duckweed hydrolyzate.

Example 2 Preparation of lycopene without using duckweed hydrolysate

(1) Microorganisms:

Escherichia coli CCRC 940321 for the preparation of lycopene is obtained according to the method disclosed in US 7,122,341 B1. Briefly, plastids pCW9 and p2IDEI were introduced into E. coli JCL1613 to obtain lycopene production host cells. E. coli CCRC 940321 is available from the Food Industry Development Institute of the Foundation (No. 331, Food Road, Hsinchu City, Taiwan).

(2) Medium:

Inoculum culture medium:

2% (w/v) yeast extract (Difico)

2% (w/v) tryptone (Difico)

2% (w/v) glycerol (Sigma)

Batch medium: (2L)

3.6% (w/v) yeast extract (Difico)

0.54% (w/v) dipotassium hydrogen phosphate (J.T. Baker)

1.07% (w/v) monopotassium hydrogen phosphate (J.T. Baker)

1% (w/v) glycerol (Sigma)

Feed medium: (1L)

10% (w/v) monosodium glutamate

1.3% (w/v) yeast extract (Difico)

2.5% (w/v) amino acid mixture (Sigma) comprising: 20% (w/w) alanine, 10% arginine, 20% aspartic acid, 20% glycine, 10% A Thiamine and 20% lysine

1% (w/v) magnesium sulfate (J.T.Baker)

60% (w/v) glycerol (Sigma)

(3) Method:

Frozen E. coli CCRC 940321 was added to a 250 mL culture shake flask containing 50 mL of inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 108 hours, the cell density (OD ₆₀₀₎ of 140 (total equivalent per liter culture containing 46.2g stem cells) cells were collected upon. 1 gram of stem cells can obtain 16 mg of lycopene.

Example 3 Preparation of Lycopene using Duckweed Hydrolysate

(1) Microorganisms:

E. coli CCRC 940321

(2) Medium:

Inoculum culture medium:

2% (w/v) yeast extract (Difico)

2% (w/v) tryptone (Difico)

2% (w/v) glycerol (Sigma)

Batch medium: (2L)

3.6% (w/v) yeast extract (Difico)

0.54% (w/v) dipotassium hydrogen phosphate (J.T. Baker)

1.07% (w/v) monopotassium hydrogen phosphate (J.T. Baker)

1% (w/v) glycerol (Sigma)

20 mL of duckweed hydrolysate obtained from Example 1

Feed medium: (1L)

10% (w/v) monosodium glutamate

1.3% (w/v) yeast extract (Difico)

2.5% (w/v) amino acid mixture (Sigma) comprising: 20% (w/w) alanine, 10% arginine, 20% aspartic acid, 20% glycine, 10% A Thiamine and 20% lysine

1% (w/v) magnesium sulfate (J.T.Baker)

60% (w/v) glycerol (Sigma)

330 mL of duckweed hydrolysate obtained from Example 1

(3) Method:

Frozen E. coli CCRC 940321 was added to a 250 mL culture shake flask containing 50 mL of inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 108 hours, the cell density (OD ₆₀₀₎ up to 190 (total equivalent per liter culture containing 62.7g stem cells) cells were collected upon. 1 gram of stem cells can obtain 21 mg of lycopene.

Example 4 Preparation of β-carotene without using duckweed hydrolysate

(1) Microorganisms:

The β-carotene producing cell line was constructed according to the method disclosed by Misawa et al. (1990). Briefly, the crtY gene (encoded lycopene cyclase) in Erwinia uredovora (ATCC 19321) was amplified by PCR, and the PCR fragment was transfected into SacI in plastid pCW9. The plastid pCW9Y was obtained at the position, and the plastids pCW9Y and p2IDI were introduced into Escherichia coli JCL1613 to obtain a β-carotene production host cell.

(2) Medium:

Same as the user in Example 2

(3) Method:

The frozen β-carotene producing cells were added to a 250 mL culture shake flask containing 50 mL of the inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 132 hours, the cell density (OD ₆₀₀₎ reached 135 (corresponding to a total liter culture containing 44.6g stem cells) cells were collected upon. 1 gram of stem cells can obtain 15 mg of β-carotene.

Example 5 Preparation of β-carotene using duckweed hydrolysate

(1) Microorganisms:

Same as the constructor of Example 4.

(2) Medium:

Same as the user in Example 3.

(3) Method:

The frozen β-carotene producing cells were added to a 250 mL culture shake flask containing 50 mL of the inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 132 hours, the cell density (OD ₆₀₀₎ 185 (equivalent per liter of the total culture of stem cells contained 61.1g) when the cells were collected. One gram of stem cells can obtain 22 mg of β-carotene.

Example 6 Preparation of Zeaxanthin without Using Duckweed Hydrolysate

(1) Microorganisms:

The zeaxanthin producing cell line was constructed according to the method disclosed by Misawa et al. (1990). Briefly, the crtZ gene (encoding β-carotene hydrolase) in Erwinia uredovora (ATCC 19321) was amplified by PCR, and the PCR fragment was transferred into plastid pCW9Y to obtain The plastid pCW9YZ, and the plastids pCW9YZ and p2IDI were introduced into Escherichia coli JCL1613 to obtain zeaxanthin production host cells.

(2) Medium:

Same as the user in Example 2

(3) Method:

The frozen zeaxanthin-producing cells were added to a 250 mL culture shake flask containing 50 mL of inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 132 hours, the cell density (OD ₆₀₀₎ reached 122 (corresponding to a total liter culture containing 40.3g stem cells) cells were collected upon. 10 g of zeaxanthin can be obtained from 1 gram of stem cells.

Example 7 Preparation of Zeaxanthin Using Duckweed Hydrolysate

(1) Microorganisms:

Same as the constructor of Example 6.

(2) Medium:

Same as the user in Example 3.

(3) Method:

The frozen zeaxanthin-producing cells were added to a 250 mL culture shake flask containing 50 mL of inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 132 hours, the cell density (OD ₆₀₀₎ 175 (equivalent per liter of the total culture of stem cells contained 57.8g) when the cells were collected. 1 gram of stem cells can obtain 13 mg of zeaxanthin.

Example 8 Preparation of canthaxanthin without using duckweed hydrolysate

(1) Microorganisms:

The canthaxanthin producing cell line is according to Misawa et al. (1990) and Misawa et al. (Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level; Journal of Bacteriology, Vol. 177, No. 22, pp. 6575-6584 (1995)) The method disclosed is constructed. Briefly, the crtW gene (encoding β-carotene ketolase) in Brevundimonas aurantiaca (ATCC66152) was amplified by PCR, and the PCR fragment was transferred to The ApaI position in the plastid pCW9Y was used to obtain the plastid pCW9YW, and the plastids pCW9YW and p2IDI were introduced into Escherichia coli JCL1613 to obtain a canthaxanthin production host cell.

(2) Medium:

Same as the user in Example 2

(3) Method:

The frozen canthaxanthin-producing cells were added to a 250 mL culture shake flask containing 50 mL of inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 124 hours, the cell density (OD ₆₀₀₎ reached 142 (corresponding to a total liter culture containing 46.9g stem cells) cells were collected upon. 1 gram of stem cells can obtain 14 mg of zeaxanthin.

Example 9 Preparation of Canthaxanthin Using Duckweed Hydrolysate

(1) Microorganisms:

Same as the constructor of Example 8.

(2) Medium:

Same as the user in Example 3.

(3) Method:

The frozen canthaxanthin-producing cells were added to a 250 mL culture shake flask containing 50 mL of inoculum culture medium, and cultured at 32 ° C for 12 hours while shaking at 150 rpm. The activated cells were transferred to a 5 L-fermentation tank containing 2 L of batch medium, and cultured at 30 ° C with stirring at 400 to 600 rpm and aeration at 2 L/min. When the cell density (OD ₆₀₀ ) reached 15 to 25, the feed medium was added to the fermentation tank (pH was controlled at 6.8-7.0). After 124 hours, the cell density (OD ₆₀₀₎ 180 (equivalent per liter of the total culture of stem cells contained 59.4g) when the cells were collected. One gram of stem cells can obtain 18 mg of canthaxanthin.

The culture conditions and results of Examples 2 to 9 are summarized in Table 1 below.

As shown in Table 1, the addition of duckweed hydrolysate not only significantly increased the biological quality of the cells, but also increased the yield of carotenoids.

The above description and examples are merely illustrative of the invention and are not intended to limit the scope of the invention. It will be understood by those skilled in the art that the present invention may be modified and varied without departing from the spirit and scope of the invention. Changes and equivalents.

Claims (12)

A duckweed hydrolysate obtained by a method comprising the steps of: (a) obtaining duckweed juice from duckweed; (b) treating the duckweed juice at a temperature of from about 25 ° C to about 75 ° C. Or hydrolyzing a plurality of proteases for about 6 to about 48 hours; and (c) collecting the duckweed hydrolysate, wherein the protease is thermolysin, pepsin, trypsin, bromelain, alkaline protease, flavor protease, prostaglandin, PTN 6.0S, acid protease, putozyme, protease A, protease M, protease N, protease NL, protease P, protease S, protease SD, thermophilic enzyme, flavor enzyme or promozyme. The duckweed hydrolysate of claim 1, wherein the duckweed is Lemna aequinoctialis , Lemna gibba , Lemna disperma , Lema Xiaoqingping ( Lemna minor ), Lemna ecuadoriensis , Lemna japonica , Lemna obscura , Spirodela punctata , Spirodela polyrrhiza or Wolffia arrhiza . The duckweed hydrolysate of claim 2, wherein the duckweed is a green duck. The duckweed hydrolysate according to any one of claims 1 to 3, wherein the protease is a plungase. The duckweed hydrolysate of any one of claims 1 to 3, wherein the amount of the one or more proteases is from about 3 A.U. to about 15 A.U. The duckweed hydrolysate according to any one of claims 1 to 3, wherein the temperature in the step (b) is from about 35 ° C to about 45 ° C. The duckweed hydrolysate according to any one of claims 1 to 3, wherein the reaction time in the step (b) is from about 12 to about 24 hours. The duckweed hydrolysate of claim 1, wherein the method further comprises the step (e) of concentrating and/or drying the duckweed hydrolysate collected from the step (d), thereby obtaining concentrated and/or dried Duckweed hydrolysate. A method for the preparation of carotenoids comprising the steps of: cultivating a carotenoid producing microorganism in a medium under suitable conditions, the medium comprising an effective amount of the duckweed according to any one of claims 1 to 8. Hydrolysate; collect cells; and isolate carotenoids from the cells. The method of claim 9, wherein the carotenoid is selected from the group consisting of β-carotene, zeaxanthin, canthaxanthin, β-cryptoxanthin, astaxanthin, lycopene, and leaf yellow. Prime. The method of claim 9 or 10, wherein the carotenoid producing microorganism is selected from the group consisting of Escherichia coli, Saccharomyces cerevisiae, B. trispora, Agrobacterium tumefaciens, Haematococcus pluvialis and Rhodotorula . The method of claim 11, wherein the carotenoid producing microorganism is Escherichia coli.