TWI654296B - Preparation method of fresh algae liquid and use thereof - Google Patents

Abstract

The invention provides a preparation method of fresh algae liquid, which is capable of shortening the culture days by sterile culture and rapid cooling after standing, and the algae cells of the fresh algae liquid obtained by the above preparation method are not produced. Variation and heavy metal content, it can achieve effective anti-cancer use.

Description

Preparation method of fresh algae liquid and use thereof

The invention relates to a preparation method, in particular to a preparation method for obtaining fresh algae liquid by aseptic culture. The invention further relates to a fresh algae liquid obtained by the above preparation method, in particular to a fresh algae liquid containing no variation of algae cells. The invention further relates to the use of the aforementioned fresh algae liquid, in particular to the use of fresh algae liquid for anti-cancer.

Chlorella is a plankton, and when the concentration ratio of the algae population is too high, the algae cells will self-digest, in order to maintain the excellent survival and breeding environment of the algal group, and produce self-body. Digested algae cells produce substances that are harmful to the organism, and the algae also produce odors. Because Chlorella is an extremely nutritious natural substance for other species of organisms, it is often a biological parasite in its natural environment. However, when algae cells are parasitized, it causes unpredictable cell variability in the algae. . In addition, Chlorella can effectively adsorb toxins such as heavy metals, pesticides, chemical toxins, polychlorinated chlorin, dioxin, radiation, etc., but when the chlorella adsorbs a certain amount of toxin, it will also cause algae cells to mutate.

The existing research shows that the phytoplankton can not be seen and survived in the rapid water. As shown in Fig. 3, the culture mode of Chlorella in the prior art relies on strong water flow stirring, fermentation culture and open culture (ventilation). Culture), in order to prevent algae cells from precipitating, as long as the water flow is stopped, the algae cells are precipitated and die. Therefore, the existing commercially cultivated and produced Chlorella products are all algae bodies whose cells have been mutated, and non-primitive chlorella algae bodies. In addition, if the Chlorella cells are more than 50 degrees Celsius above thermal destruction, in the anhydrous state (spray drying, freeze-drying), high temperature wall breaking treatment, high concentration and temperature is higher than 0 degrees Celsius for more than 6 hours and at Resting for 2 hours will cause algae cells to precipitate or mutate the cells. Furthermore, the current standard for the determination of heavy metals in the CNS test for the detection of Chlorella products has been changed from 5 ppm to less than 20 ppm. It can be seen that the Chlorella algae produced by the prior art Chlorella cultivation method has not only been mutated, but also It is a mutant algae cell filled with environmental pollution toxins.

In view of the shortcomings of the prior art cultivation of Chlorella, not only the long cultivation time, high temperature and destruction of the cell wall treatment, but also the degeneration of the algae cells of the Chlorella, detoxification, pollution of the toxin and the death of the Chlorella cell precipitation, the present invention The purpose of the invention is to provide a method for preparing a fresh algae liquid, which can be rapidly heated by aseptic culture and chilling, thereby achieving the effects of shortening the culture days, not causing the algae cell variation of the chlorella and the low heavy metal content.

In order to achieve the above object, the present invention provides a method for preparing a fresh algae liquid, which comprises the steps of: cultivating Chlorella in a sterile apparatus for no more than 3 days, wherein the concentration is more than 5 parts per million, and the culture residence time is not more than 6 Hour, to obtain a culture mixture; wherein the Chlorella is Chlorella; the culture mixture is rapidly cooled to below 1 ° C but does not freeze and centrifuge, and sterile water is added to obtain algae, wherein The concentration of the algae liquid is between 2.5 mg (mg/mL) and 12.5 mg/mL per ml; the algae liquid is frozen at 0 ° C or less and is fully frozen and allowed to stand for 12 hours to 24 hours, and then heated to 10 minutes. 40 ° C to 50 ° C to form a fresh algae solution.

Preferably, the Chlorella sp. is Chlorella pyrenoidosa .

Preferably, when the chlorella is cultured in a sterile apparatus, the flow rate of the stirred culture solution is from 3 meters per minute (m/min) to 20 meters per minute.

More preferably, when the chlorella is cultured in a sterile apparatus, the chlorella absorbs the carbon source under illumination; when the pH of the mixed culture is less than 6.8, the stirring is stopped while maintaining the The mixed culture solution is in a static suspension state (no precipitation), and after the pH value of the mixed culture liquid is 7 or more, stirring is started, and the mixed culture liquid absorbs the carbon source.

Preferably, when the algae liquid is frozen and allowed to stand for temperature rise, the temperature can be raised by microwave or infrared ray.

Preferably, the algae solution is frozen in a fully frozen state below 0 ° C and allowed to stand for 12 hours to 24 hours, and the freezing temperature is between -1 ° C and -21 ° C.

Preferably, the algae liquid is frozen at 0 ° C or less in a fully frozen state and allowed to stand for 12 hours to 24 hours, and in the step of heating up, the temperature rise time is from 3 minutes to 10 minutes.

The invention further provides a fresh algae liquid obtained by the method as described above, which can maintain the health function of the animal originally contained in Chlorella, and has obvious differences from the products produced by the prior art, and the differences are as follows: 1. The taste is delicious, sweet and odorless; the products produced by the prior art have many bad odors. 2. Without artificial wall breaking treatment, the human body can be completely digested and absorbed by the human body (the products obtained by the prior art are not broken and the human body cannot be effectively digested and absorbed). 3. The content of the chlorella growth factor (CGF) obtained by the method of the present invention is extremely high, and generally can be more than twice as high as that of the products produced by the prior art. 4. The chlorophyll function in the fresh algae liquid can be completely preserved, and the chlorophyll obtained by the method described in the prior art can reduce the functional efficacy of the chlorophyll by high temperature treatment.

According to the present invention, the content of the green algae growth factor is determined by the method for determining the index value of the 4.5 green algae hot water extract in the green algae of CNS No. 4202, No. 4202, and the method of the present invention can be obtained. The index value of the hot water extract of the algae liquid was 3.8 to 4.9.

The fresh algae liquid obtained by the method according to the present invention is detected by the chlorophyll detection method of CNS4202 type N5134 edible green algae from 3800 mg% to 4900 mg%.

The present invention further provides a use of the fresh algae liquid as described above for the preparation of a medicament for preventing cancer, wherein a medicament containing a fresh algae solution is administered to a recipient to achieve an anti-cancer effect.

According to the present invention, "anti-cancer" means effectively inhibiting or soothing cancer.

Preferably, the effective dose of the pharmaceutical product is based on the solid content in the fresh algae liquid, that is, the dose is 10 mg (mg/kg) to 200 mg/kg per kg body weight.

According to the present invention, "effective dose" means an amount effective to achieve a desired or inhibited cancer result at a dose and for a desired period of time; according to the present invention, it means that the growth of a breast cancer tumor can be slowed down, stopped, In the case of death, as exemplified in the present invention, the dose effective to inhibit or soothe a breast cancer tumor can be obtained by administering a specific amount of a drug containing fresh algae solution and measuring a change in tumor volume within a specific time range.

Preferably, the dosage form of the pharmaceutical product includes, but is not limited to, a liquid.

The fresh algae liquid obtained by the preparation method described in the present invention can not only shorten the culture days, but also can reduce the heavy metal content contained in the fresh algae liquid obtained by the fermentation culture or the outdoor culture. In addition, the preparation method of the present invention can be borrowed. The low flow rate makes the Chlorella do not precipitate and does not cause the algae cell variation of Chlorella; the preparation method of the present invention does not destroy the cell wall at high temperature, nor spray-dry at high temperature or freeze-dry at a very low temperature, so it does not The chlorophyll cell variability is caused, so that the fresh algae liquid obtained by the preparation method of the present invention can not only retain high green algae growth factor and chlorophyll, but also has better anti-cancer effect.

The invention is exemplified by the following examples, which are not to be construed as limiting the scope of the invention.

Preparation Example 1 Culture method of Chlorella

Referring to FIG. 1 , the method for preparing Chlorella vulgaris according to the present invention comprises the following steps: (1) preparing a Chlorella sp., and selecting Chlorella pyrenoidosa in the preparation example.

(2) Aseptic culture, wherein the required carbon source is filtered sterile air, the water flow rate of the stirred algae solution is not more than 20 meters per minute, and the green source absorbs the carbon source under light conditions; when the mixed culture solution is When the pH value is lower than 6.8, the stirring is stopped and the mixed culture liquid is maintained in a static suspension state (no precipitation). After the pH value of the mixed culture liquid is 7 or more, stirring is started, and the mixed culture liquid absorbs carbon. source. The sterile culture is cultured for no more than 3 days to obtain a culture mixture. When the concentration of the culture mixture (ie, the solid matter of the algae in the culture solution) is more than 5 parts per million, the culture residence time may not exceed 6 hours, otherwise the algae body It will produce autologous digestion.

(3) The culture mixture is rapidly cooled to below 1 °C (on the principle of no ice), and the culture solution is removed by centrifugation in a sterile environment, and then washed with the same low temperature sterile water and then dehydrated, and then quantified with sterile water to the algae. 5 percent solids to obtain algae fluid.

(4) The algae liquid is frozen at -20 ° C for 24 hours, and then the frozen algae liquid is heated to 40 ° C to 50 ° C in 10 minutes by microwave or infrared ray, so that the algae body no longer has the ability to reproduce, and It is not necessary to use other high temperature, physical machinery, chemical and other methods to break the wall, that is, the human body can digest and absorb the most complete degree of drinking, and then quantify to the appropriate concentration with sterile water to form fresh algae liquid, and quickly freeze to -20 ° Celsius. C preservation (through 4 years of actual human consumption, it is found that in the non-frozen state, the functional effect of Chlorella will gradually decrease to disappear).

Comparative Example 1 Chlorella cultivation method of the prior art

As shown in FIG. 3, the prior art preparation of the Chlorella liquid system is to ferment culture or outdoor culture of Chlorella to form a Chlorella solution, wherein the fermentation culture uses glucose as a fermentation carbon source, and the stirring rate is 50 rpm to 100. Rpm, and need to be cultured for 5 days to 10 days, the algae cells obtained by fermentation culture can be said to be completely non-planned algae, as long as the stirring will precipitate the mutant algae cells; wherein the outdoor culture needs to supply carbon source with acetic acid, stirring rate The water flow rate per second is 10 meters (m) or more, and it needs to be cultured for 2 weeks to 8 weeks. The algae cells obtained by outdoor culture are also the mutant algae cells which are precipitated when the stirring is stopped, and the accumulated environmental toxins are caused by the outdoor culture. Miscellaneous bacteria contamination.

The green algae solution fermented or cultured outdoors is concentrated by centrifugation and the cell wall is destroyed to form a green algae concentrate. The disrupted cell wall system is treated at a temperature of 115 ° C to 125 ° C, and this temperature range causes the functional function of the effective anticancer effect of Chlorella to be completely destroyed.

The cell wall-depleted Chlorella concentrated solution is further spray-dried or freeze-dried to form a Chlorella powder. The temperature of the spray drying is 155 ° C to 180 ° C, and this temperature range causes the functional function of the effective anti-cancer effect of Chlorella to be completely destroyed.

This comparative example is collected from spray-dried or freeze-dried Chlorella powder after outdoor culture, wherein the green algae powder obtained by spray drying is referred to as group a, and the freeze-dried green algae powder is referred to as group b. The fresh algae liquid obtained in Preparation Example 1 was referred to as group c.

Example 1 Use of fresh algae liquid as anti-tumor

Twenty-four BALB/c mice (week 5 weeks old) were divided into four groups: control group, group A, group B and group C, and breast cancer cell line MDA-MB-231 was implanted into each group of mice. In the mammary gland, breast cancer cell lines (1×10 ⁶ cells/100 μL) were diluted in PBS and applied to each group as day 0, and the tumor volume grew to 58 mm ^{3 on} the fourth day. The control group was administered twice a day. The weight of the mouse was 1 mL per kilogram (1 mL / kg) PBS, and the A group was fed twice a day. The group obtained in the control group 1 was 10 mg/kg each, group B. For the feeding of the control group 1 obtained twice a day, the b group was 10 mg/kg each, and the C group was fed twice a day to prepare the group 1 obtained in the group 1 1 mL/kg each time (the fresh algae solution concentration was 10 The converted amount of mg/mL) was mixed in the feed, administered at intervals of six hours, and the tumor volume was measured on days 15 and 22, respectively.

As shown in Fig. 2A, the growth of tumor cells in group C was significantly slower than that in the control group and groups A and B. As shown in Fig. 2B, it can be seen that the formation of tumor cells in the C group reduced the atrophy state, while the tumor cells in the control group and the A and B groups all increased.

Example 2 Chlorella growth factor (CGF) content test

The content of the green algae growth factor is determined by the method for determining the index value of the 4.5 green algae hot water extract of the CNS No. 4202 type N5134 edible green algae, and then compared with the label and the test value of the commercially available related products.

In the market, I purchased three different brands of green algae products: T-green (green chlorella), G-brand (live green chlorophyll), V-brand (weidan green algae), and the fresh algae liquid obtained from the process of this case. No. 4202 N5134 edible green algae 4.5 green algae hot water extraction index value determination method Determination of green algae hot water extraction index value, the index value: T card is 1.7, G card is 2.3, V card is 1.6, The hot water extract index value of the fresh algae liquid obtained by the method of the present invention is 3.8 to 4.9. It can be seen that the green algae growth factor content of the fresh algae liquid obtained by the method of the present invention is more than twice that of the prior art.

Example 3 Chlorophyll content test

Chlorophyll is highly degradable during improper production, processing and storage. Therefore, chlorophyll has the representativeness of green algae quality stability, so it has a special representativeness as a quality control index. This example is based on the detection method of chlorophyll in CNS4202 No. N5134 edible green algae, and is compared with the labeling and inspection value of the commercially available related products. Among them, CNS4202 No. N5134 stipulates that the total chlorophyll content is 1500 mg% or more. However, the total chlorophyll content of commercially available products is less than 2000 mg%. The fresh chlorophyll solution obtained in the process of this case has a chlorophyll content of 3800 mg% to 4900 mg% as measured by the chlorophyll test method of CNS4202 N5134 edible green algae. It can be seen that the chlorophyll content of the fresh algae liquid obtained by the method of the present invention is more than twice that of the prior art.

(no)

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart showing the method of preparing fresh algae liquid of the present invention. Figure 2A is a bar graph of fresh corn juice obtained by the method of the present invention for inhibiting cancer cells and measuring tumor volume on day 15. Figure 2B is a bar graph of the fresh algae fluid obtained by the method of the present invention for inhibiting cancer cells and measuring the tumor volume on day 22. 3 is a flow chart of a method for preparing fresh algae liquid in the prior art.

(no)

Claims (8)

A method for preparing a fresh algae liquid, comprising the steps of: cultivating the Chlorella in a sterile apparatus for no more than 3 days, wherein the concentration is more than 5 parts per million, and the culture residence time is not more than 6 hours to obtain a culture mixture; Wherein the chlorella is cultured in the aseptic apparatus, the chlorella is subjected to a carbon source under a light condition; when the pH of the mixed culture is less than 6.8, the stirring is stopped and the mixed culture is maintained. The liquid is in a state of static suspension. After the pH of the mixed culture solution is 7 or more, stirring is started, and the Chlorella pyrococcus is Chlorella pyrenoidosa ; the culture mixture is rapidly cooled to below 1 ° C. But do not freeze and centrifuge, and add sterile water to obtain a algae solution, wherein the concentration of the algae liquid is between 2.5 mg (mg / mL) to 12.5 mg / mL per ml; the algae liquid is frozen below 0 ° C It is fully frozen and allowed to stand for 12 hours to 24 hours, and then heated to 40 ° C to 50 ° C in 10 minutes to form a fresh algae solution. The preparation method according to claim 1, wherein when the chlorella is cultured in a sterile apparatus, the water flow rate of the stirred culture solution is from 3 meters (m/min) per minute to 20 meters per minute. The preparation method according to claim 1, wherein when the algae liquid is frozen and allowed to stand for temperature rise, the temperature can be raised by microwave or infrared ray. The preparation method according to claim 1, wherein the algae liquid is frozen at a temperature of 0 ° C or less in a fully frozen state and allowed to stand for 12 hours to 24 hours, and the freezing temperature is between -1 ° C and -21 ° C. The preparation method according to claim 1, wherein the algae liquid is frozen at 0 ° C or less in a fully frozen state and allowed to stand for 12 hours to 24 hours, and in the step of heating up again, the temperature rise time is from 3 minutes to 10 minutes. . A fresh algae liquid obtained by the method according to any one of claims 1 to 5. A use of the fresh algae liquid according to claim 6 for the preparation of a medicament for preventing cancer, wherein a pharmaceutical containing the fresh algae solution is administered to a recipient to achieve an anti-cancer effect. The use according to claim 7, wherein the effective dose of the pharmaceutical product is based on the solid content in the fresh algae liquid, that is, the dose is 10 mg (mg/kg) to 200 mg/kg per kg of body weight.