TWI432146B - Strain of picochlorum and application thereof - Google Patents

Description

Picochlorum genus strain and its application

The present invention relates to a strain of algae and its use, in particular to a strain of Picochlorum and its use.

Microalgae, also known as microalgae, is a unicellular algae microorganism that has been widely used in aquaculture, nutritious food, animal feed, medicine, beauty, biomass energy, water treatment and natural pigments for nearly 20 years. a variety of fields.

Specific microalgae have the function of producing functional feeds for aquaculture. Especially for the grouper breeding industry, it is extremely important to pass through closed cultured microalgae to provide a clean and pollution-free source of feed because of the need to avoid pathogens or chemical insults.

However, when the grouper is still in the fry period, although the mouth has formed and started to eat, but because its digestive and sensory system is still not fully developed, and the mouth is still small, it is necessary to feed the animal feed food through digestion. Such as very small rotifers to help their nutritional intake, and this phenomenon of selective eating is collectively referred to as palatability. At present, animal bait organisms are mainly fed by algae. However, the size and type of microalgae also affect the palatability of very small rotifers. However, not all microalgae can be used to supply specific animal feed organisms, mainly because of the size and type of microalgae.

In addition, specific microalgae can provide high-unit protein as the main source of nutrients for animal growth. The demand for protein in biological larvae is high. For example, when the grouper grows to adult fish, the demand for protein will decrease from 50%. Up to 20%, in which the high unit of protein needs to be combined with the amino acid composition required by the organism to contribute to growth, although it has been well studied in the past, protein nourishment suitable for the amino acid composition of the grouper has not been found.

In the past, Isochrysis galbana or Nannochloropsis sp. (Nanno) was used as a green water system to provide fry DHA and EPA, but these algae still have no rotifer proliferation stage and light. The problem of shadowing effect is not a good source of nutrition. Although it is possible to solve the above problems by changing to a mixed-stage Chlorella or a heterotrophic yeast two-stage culture, it is not possible to simultaneously satisfy the problem of essential amino acids required for fish. All in all, there is currently no protein source of nutrients that can meet the needs of fry and at the same time be used for animal feed.

Therefore, how to provide a nutrient-rich algae that can be used to produce edible products. In addition, the algae can be used as a bait animal, and at the same time, it provides an amino acid required for fish growth, and can be applied to seedling raising and rearing of adult bodies, and has become an important subject.

In view of the above problems, an object of the present invention is to provide a nutrient-rich algae which can be used for the production of edible products, and which can be used for feeding animals and providing essential amino acids for fish growth, and can be applied to seedlings and Feeding adult and other uses.

To achieve the above object, the present invention provides a Picochlorum genus strain for producing an edible product.

In an embodiment of the present invention, described above for the production of food products Picochlorum strains of algae of the genus Picochlorum sp. S1b algal strains.

In an embodiment of the present invention, the Picochlorum genus strain for producing an edible product is the bacterium strain BCRC980021 or its derived algae strain or mutant algae strain.

In an embodiment of the invention, the edible product is a feed, an additive or a health food.

In an embodiment of the invention, the feed is a feed for poultry, livestock or aquatic animals.

In one embodiment of the invention, the additive is a feed additive for poultry, livestock or aquatic animals.

In an embodiment of the invention, the form of the edible product comprises a powder, a lump, a milk or a concentrate.

In accordance with the purpose of the present invention, the present invention provides a Picochlorum genus strain for use in seedling or rearing.

In an embodiment of the present invention, described above for adult Picochlorum nursery or breeding strains of algae of the genus Picochlorum sp. S1b algal strains.

In one embodiment of the present invention, the above-mentioned Picochlorum genus strain for seedling or rearing adult is a plant strain No. BCRC980021 or a derivative thereof or a mutant strain of the bacterium.

In an embodiment of the present invention, the Picochlorum genus strain used for seedling or rearing adult is a natural bait.

In one embodiment of the invention, the Picochlorum genus strain is used to culture or raise animal feed organisms.

In an embodiment of the invention, the animal feed organism comprises a rotifer animal door, an arthropod door.

In an embodiment of the invention, the rotifer animal door comprises a rotifer, and the arthropod door comprises a lame, a branch, or a brine shrimp.

In accordance with the purpose of the present invention, the present invention provides a use of a Picochlorum genus strain for breeding or rearing an adult.

In an embodiment of the invention, the algal strain is a Picochlorum sp. S1b strain.

In an embodiment of the present invention, the above-mentioned algal strain is a strain of BCC 980021 or a derivative thereof or a mutant strain of the bacterium of the Food Industry Development Institute.

In an embodiment of the invention, the above-mentioned algal strain is a natural bait or used for cultivating or raising animal bait organisms.

In summary, the Picochlorum genus strain of the present invention is rich in nutrients such as protein, amino acid and fatty acid, and is therefore suitable for the production of edible products, such as additives, feeds, or health foods, in order to supplement animal nutrition. Insufficient and enhance its growth and health. In addition, the algae strain has good taste for animal bait, and can supply nutrients of animal seedlings such as fry, and has the characteristics of being suitable for large-scale growth under mixed culture, and more importantly, its amino group. The acid composition is similar to the essential amino acid composition required for some animal seedlings. Therefore, animal seedlings can obtain sufficient amino acid nutrients therefrom, which proves that it is suitable for seedling or animal breeding.

Hereinafter, a nutrient-rich Picochlorum genus strain according to the present invention, which can be used for the production of edible products, or for use in seedling raising and rearing, etc., will be described with reference to the related drawings.

The invention extracts a strain of algae from the Yanzhe area of Tainan, Taiwan, firstly cultures it by appropriate methods, observes its type and biochemical characteristics, and then analyzes the evolution tree according to its 18S rDNA sequence to determine that it belongs to the genus Picochlorum . And named it Picochlorum sp. S1b. Currently registered in the Food Industry Development Institute, the registration number is BCRC980021.

Since the algae in the genus Picochlorum have close biological and biochemical characteristics to each other, the present invention is represented by the Picochlorum sp. S1b strain as an example, and of course other Picochlorum algae having the same or similar characteristics as those described below. It is also within the scope of the invention.

The Picochlorum sp. S1b strain referred to in this embodiment comprises a derived algal strain or a mutant algal strain. "Derived algae strain" refers to the progeny of a strain of algae that undergoes proliferation and division into a normal culture environment, and its genetic material such as deoxyribonucleic acid (DNA) is very close to or different from that of the parent. The phenotype is consistent with the parent and has the same physiological function. The "mutant algae strain" refers to a mutant or a progeny thereof that occurs when a strain of algae proliferates or divides in a normal culture environment or is stimulated by any mutant substance. However, the mutant strains referred to herein contain genetic material such as DNA, but can still not affect the main features of the present invention such as the amino acid, fatty acid composition and content, appearance, growth Features such as cycles, or mutations, can enhance the above characteristics, or more suitable for the use of the present invention.

The Picochlorum sp. Slb strain of the present invention can be cultured in Walne's medium diluted twice in natural seawater, and added with 6 g of glucose, 4.5 g of yeast extract, and agarose. 8 grams was made on a solid medium suitable for the growth of the strain. The Picochlorum sp. S1b strain of the present invention was subjected to plate culture in this solid medium as shown in FIG. Under the light microscope, the appearance of a single Picochlorum sp. S1b cell showed an approximate spherical shape and a green color with a diameter of about 2 μm. In addition, Picochlorum sp. Slb strain is rich in a variety of pigments, including chlorophyll a, chlorophyll b, lutein, carotenoid alpha, carotenoid beta, lipidated astaxanthin and the like.

The Picochlorum sp. S1b strain has a high protein content. In an experimental example of the present invention, when the Picochlorum sp. S1b strain was cultured in the above culture medium to a concentration of 2.5 g per liter, the total amount of protein contained was about 55% of the total dry weight. In addition, Picochlorum sp. S1b strain is rich in a variety of amino acids, among which arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine ( Lys), methionine (Met), phenylalanine (Phe), serine (Thr) and proline (Val), etc., are very similar to the essential amino acid composition required by animals, especially aquatic animal seedlings. More especially the grouper fry. Furthermore, in one embodiment, it was found by chromatography mass spectrometry that the Picochlorum sp. S1b strain of the present invention contains a plurality of fatty acids, including fatty acids of twelve to eighteen carbons which contribute to the growth of aquatic animals. Based on these advantages, Picochlorum sp. S1b strains are suitable for the production of edible products to supply animals as a source of nutrients or to supplement the nutrients that are normally consumed in food. It should be noted that the above-mentioned dry weight analysis or chromatographic mass spectrometry is a technique familiar to those skilled in the art of the present invention, and thus the details of the operation will not be described herein.

The Picochlorum sp. S1b strain of the present invention can be made into a variety of edible products, such as feed health foods, or additives. The additive may be a feed or a food additive, and may be provided alone or together with the additive, and the present invention is not limited thereto. Specifically, an example shows that the Picochlorum sp. S1b strain can be used as an additive in a culture state, that is, directly using a Picochlorum sp. S1b solution containing a culture solution; or drying the algae liquid into a powder, a block, etc. Form, and become a solid additive; or the algae solution is processed, concentrated, or extracted to prepare an additive in the form of a milk, a concentrate, or an extract.

In an embodiment of the present invention, the Picochlorum sp. S1b strain can produce animal feed, wherein the "animal" can include poultry, livestock, or aquatic animals such as chickens, ducks, geese, pigeons, horses, cows, sheep, dogs. , pig, shellfish, or fish, preferably means marine fish, more preferably grouper. Furthermore, the above animals all include their larvae, seedlings or adults. The term "larva or seedling" means the period from the time when the animal is born or hatched to the time before it has fertility. Furthermore, it means that after the animal is born or hatched, its mouthparts have developed and the digestive organs have The period of certain functions and the latter. The so-called "adult" means that the animal has developed to be fertile. In one embodiment of the invention, the Picochlorum sp. S1b strain is used to feed the fry of the grouper Epinephelus .

In an embodiment of the present invention, the feed additive produced by the Picochlorum sp. S1b strain may include other suitable ingredients such as a buffer, a filler, a fragmentation agent, a binder, a flavoring agent, an odorant, a stabilizer, A leavening agent or a coating material for enteric coating which can be passed through an acidic environment. Among the above additives, the content of the Picochlorum sp. S1b strain is less than 0% of the total composition, and up to 100% of the total composition, preferably 20% or more of the total composition, and most of the total composition. Medium is less than not equal to 100%, better is 40% or more of the total composition, and most of the total composition is less than 100%, and more preferably, the total composition is greater than or equal to 60%, and the maximum is less than 60% of the total composition. Not equal to 100%. Of course, the above data will vary depending on the feed added to poultry, livestock, or aquatic animals.

In yet another embodiment of the invention, the Picochlorum sp. S1b strain is used to produce an additive to a health food as an additional or supplemental source of nutrients for the animal to enhance its health or provide growth. In addition, in another embodiment of the present invention, when the Picochlorum sp. S1b strain and the algae liquid thereof are directly used for making a health food, the buffer may be mixed and emulsified according to the demand of the supply object, the preservation of the product, or the taste. Agents, vitamins, polysaccharides, minerals, enzymes, proteins, amino acids, cellulose, fatty acids such as saturated and/or unsaturated fatty acids, carbohydrates, odorants, flavoring agents, antioxidants, preservatives, molding agents Or the microorganisms such as probiotics, etc., the invention is not limited thereto. The prepared health food can be in the form of a dry block, a chewable tablet, a quick-dissolving tablet, a foaming ingot, a powder, a liquid, a suspension, an emulsion, a tablet, a multi-layer ingot, a double-layer ingot, a capsule, and a soft Gelatin capsules, hard gelatin capsules, buccal tablets, powders, or granules, etc., are not limited herein. The Picochlorum sp. S1b strain incorporated in the above healthy food has a specific gravity of about 1 to 80%, preferably about 10% to 70%, more preferably about 20% to 60%, and more preferably about 30% to 50%. %.

The invention further provides a Picochlorum genus strain for breeding or rearing an adult. In one embodiment, the term "nurturing or rearing adult" refers to the nutrient required to grow or maintain physiological functions of a seedling or adult of an animal. Herein, the "animal" may be defined by the foregoing examples, and more particularly, the seedlings or adults of marine fish such as groupers, groupers, or shellfish such as oysters, clams, mussels, and scallops. As for other related features, feeding methods and other descriptions of the Picochlorum genus strain, reference may be made to the above, and no further details are provided herein.

However, it is worth mentioning that the Picochlorum sp. S1b strain has a wide range of salinity growth, and the salinity is between about 10 and 70 practical salinity units (psu). The Jiadi system grows in about 30-60 psu, so it can not only directly co-cultivate the algae plant with seawater animal feed organisms, or directly grow in animal seedlings or adult feeding or growing environments, and then directly or Indirect help to breed, and the scope of application is more extensive than the average seawater microalgae.

In one embodiment, the animal feed organism may be a rotifer phylum or an arthropod phylum, which is suitable as a nutrient source for seedlings or adults of aquatic fish or shellfish, particularly aquatic fish seedlings such as grouper. Among them, the rotifer animal door organism can be a rotifer, and the arthropod door organism can be an animal such as a lame, a branch, or a brine shrimp.

Hereinafter, the present invention will Picochlorum sp. S1b of several experimental examples described in detail culture characteristics Picochlorum genus algal strains and demonstrates the case by Picochlorum present invention algal strains nourished of animal feed organisms having better the growth rate and egg ratio . In addition, the fry raised with the Picochlorum sp. strain of the present invention also have a longer body length and a lower mortality rate from water.

Experimental Example 1: Cultivation of Picochlorum sp. S1b strain

In this experimental example, the Picochlorum sp. S1b strain and the algae from the Nannochloropsis oculata (Nanno) strain from the Dongkang Biotechnology Research Center of the Taiwan Fisheries Research Institute were taken from the end of the logarithmic growth phase, and Picochlorum Sp. S1b is a mixed camp and self-cultivated, while Nanno is self-cultivated.

The mixed culture method of Picochlorum sp. S1b strain was carried out in a 20-liter plastic bucket containing 10 liters of medium. The medium was formulated as two-diluted Walne's medium in seawater with a salinity of 35 psu, and glucose and yeast extract were added thereto at 6 g and 4.5 g per liter, and sterilized to be inoculated after cooling. The initial density of Picochlorum sp. S1b strain was 0.05 g per liter dry weight. During the culture, 0.1 vvm was pumped and shaken at 125 rpm while controlling the illumination at 3,000 lux for 12 hours per day at a temperature of 27 ± 1. °C. Seven days after inoculation, the culture density of S1b was about 2.5 g per liter (the total protein content was 55% of the dry weight).

In comparison with the autotrophy cultured Picochlorum sp. S1b strain and the genus Chlorella, the Chlorella and Picochlorum sp. S1b strains were separately inoculated into two 120 L acrylic tanks, among which The tank contains 100 L of sterilized Wan's medium, respectively. Then carry out photosynthetic self-cultivation. For mixed culture, the initial density of inoculation was also 0.05 g per liter of dry weight, and the culture was carried out at an illumination of 9,000 lux and a temperature of 27 ± 2 °C. Eight days after inoculation, the culture density of Picochlorum sp. S1b strain was measured to be 0.2 g per liter, which was higher than the culture density of Chlorella pyrenoids by 0.1 g per liter.

The experimental results are shown in Figure 2. From this result, it can be seen that if the Picochlorum sp. S1b strain is cultured in a mixed manner, high yield can be obtained, and the concentrated algae liquid can be mass-produced through a professional factory, and the self-cultivation method can be directly applied to the general fish farm. The equipment is cultivated, and the culture effect is better than that of the currently used Pseudococci, whether it is cultivated by self-operating or mixed mode.

Experimental Example 2: Analysis of nutrient composition of Picochlorum sp. S1b strains

The Picochlorum sp. S1b algal body was hydrolyzed and analyzed for the composition of the free amino acid. First, 0.05 g of dried Picochlorum sp. S1b algal flour was added and hydrochloric acid having a concentration of 6N was added to a total volume of 1 mL, and treated in a vacuum reaction flask at 110 ° C for 24 hours to carry out an acid hydrolysis reaction. After hydrolysis, centrifuge at 1,000 × g for 3 minutes, take the supernatant, and dilute the supernatant 30 times with deionized water, and refer to The instructions for the amino acid analysis reaction kit are operated and analyzed.

The results of the analysis are shown in Fig. 3. The percentage of the protein hydrolyzed amino acid composition of the Picochlorum sp. S1b strain is closer to the essential amino acid composition percentage of the grouper than the amino acid composition of the Nanno strain, so it is suitable for breeding. The source of nutrient supply for grouper fry.

Fatty acid composition analysis

A gas chromatography mass spectrometer (CP-3800 & 320-MS, Varian, USA) was used with a Supelco SP-2380 (Sigma-Aldrich, St. Louis, MO, USA) column for analytical method reference (Yang et al . , 2010), the analysis results are shown in Figure 4. Where aj is a dodecane saturated acid (C12:0), a fourteen carbon saturated acid (C14:0), a sixteen carbon saturated acid (C16:0), an eighteen carbon saturated acid (C18:0), sixteen Carbatrienoic acid (C16:3), octadecadienoic acid (C18:2), octadecadienoic acid (C18:3), and octadecatrienoic acid (C18:3). Among them, the content of sixteen carbon and eighteen carbon fatty acids accounted for 94.6% of the total fatty acid content.

Combining the composition analysis of the previous experimental example, the Picochlorum sp. S1b strain is rich in various amino acids, fatty acids and high protein content, so it is suitable for use as an edible product or for breeding.

Experimental Example 3: Breeding use of Picochlorum sp. S1b strain Picochlorum sp. S1b algae breeding animal bait organism

The Picochlorum sp. S1b strain and the Nanno strain of the experiment were obtained by the culture method as in Experimental Example 1. After centrifugation at 10,000 x g for 3 minutes, it was washed with sterile seawater having a salinity of 20 psu to remove the culture solution. Next, the algae solution was diluted to a concentration of 1 × 10 ⁷ algae cells per liter in a sterilized seawater having a salinity of 20 psu for the experiment. The above two algae strains were fed with a very small rotifer ( Brachionus rotundiformis , purchased from Taiwan, Fisheries Research Institute, Donggang Biotechnology Research Center, and observed and compared their reproductive rate and egg ratio. The experimental procedure for the extremely small rotifers and the method for determining the egg-bearing rate and the rate of proliferation are based on the literature of the Donggang Biotechnology Research Center of the Aquatic Research Institute (Su et al ., 1994 and Snell et al . 1987).

The results are shown in Fig. 5. The proliferation rate of the extremely small rotifer was expressed by the relative reproductive rate based on the proliferation rate of the group feeding the Nanno strain. The relative proliferation rates of very small rotifers after feeding Picochlorum sp. S1b or Nanno were 126±7% and 100±10%, respectively. The egg-bearing rates were 51±12% and 7±8%, respectively, indicating the use of Picochlorum sp. S1b cultures extremely small rotifers have higher yields than Nanno.

Picochlorum sp. S1b algae breeding grouper fry

The newly hatched grouper ( Epinephelus coioides ) was reared in two six-ton tanks, and each of the six-ton tanks was fed with 1,500 grouper seedlings. The seawater used was previously sterilized with bleach and the residual chlorine was removed with Sodium thiosulphate (hyposulfite). Nanno and Picochlorum sp. S1b algae were added individually on the first day after hatching, and the density of the algal solution was maintained at 2.5×10 ⁵ cells/mL until no algae solution was added for 29 days. The feeding of the fry is to feed the rotifers nourished by commercial supplies 4 to 20 days after hatching, and to feed the brine shrimp that have been nourished by commercial supplies for 14 to 42 days, and to move the pool in 42 days. The water mortality and body length of grouper fry were recorded 24 hours after the pond was moved.

As a result, as shown in Fig. 6, in the grouper seedlings in which the Picochlorum sp. S1b and Nanno algae liquids were respectively added, the mortality rate of the water-repellent group fed the Picochlorum sp. S1b algae solution was 1.00%, which was lower than that of the Nanno algae solution. The death from water in the group was 1.32%, and the proportion of fry with a longer body length (greater than 1.5 cm) was higher.

In summary, the Picochlorum genus strain of the present invention is rich in nutrients such as protein, amino acid and fatty acid, and is therefore suitable for the production of edible products, such as additives, feeds, or health foods, in order to supplement animal nutrition. Insufficient and enhance its growth and health. In addition, the algae strain has good taste for animal bait, and can supply nutrients of animal seedlings such as fry, and has the characteristics of being suitable for large-scale growth under mixed culture, and more importantly, its amino group. The acid composition is similar to the essential amino acid composition required for some animal seedlings. Therefore, animal seedlings can obtain sufficient amino acid nutrients therefrom, which proves that it is suitable for seedling or animal breeding.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

Figure 1 is an image diagram of the Picochlorum sp. S1b strain cultured in the solid medium of the present invention;

2 is a growth data diagram of the Picochlorum sp. S1b strain of the present invention cultured in a mixed manner;

Figure 3 is a graph showing the analysis of the essential amino acid composition of the Picochlorum sp. S1b strain and the grouper seedling of the present invention;

Figure 4 is a graph showing the analysis of fatty acid composition of the Picochlorum sp. S1b strain of the present invention;

Figure 5 is a graph showing the relative proliferation rate and egg-bearing rate of the Picochlorum sp. S1b strain of the present invention in a very small rotifer;

Fig. 6 is a graph showing the body length and the mortality of water leaving after the grouper fry are fed with the Picochlorum sp. S1b strain of the present invention.

Claims (14)

A method for the production of food products Picochlorum algal strains of the genus, the strain of algae Picochlorum sp.S1b algal strains, and the article-based edible feedstuff, food additive or health. For example, the algae strain described in claim 1 is the food industry development institute registration number BCRC980021. The algae strain according to claim 1, wherein the feed is a feed for poultry, livestock or aquatic animals. The algae strain according to claim 1, wherein the additive is a feed additive for poultry, livestock or aquatic animals. The method of claim 1, wherein the edible product comprises a powder, a lump, a milk or a concentrate. A method for rearing or breeding Picochlorum adult algal strains of the genus, the strain of algae Picochlorum sp.S1b algal strains. For example, the algae strain described in claim 6 is the food industry development institute registration number BCRC980021. The algae strain described in claim 6 is a natural bait. The strain of the algae described in claim 6 is used for cultivating or raising animal bait organisms. The algae strain according to claim 9, wherein the animal bait organism comprises a rotifer animal door, or an arthropod door. The rotifer animal door according to claim 10 of the patent application includes a rotifer, and the arthropod door includes a lame, a branch, or a brine shrimp. Strains of the genus Picochlorum one kind of use for feeding into the body or nursery, the algal strain is Picochlorum sp.S1b algal strains. The use of the invention of claim 12, wherein the strain is the BSC 980021 strain of the Food Industry Development Institute. The use of claim 12, wherein the strain is a natural bait or used to culture or raise animal bait organisms.