WO2014148943A1

WO2014148943A1 - Use of a chlorella plankton strain as dietary biologically active supplement

Info

Publication number: WO2014148943A1
Application number: PCT/RU2013/000894
Authority: WO
Inventors: Mikhail Vladislavovich KUNITSIN
Original assignee: Kunitsin Mikhail Vladislavovich
Priority date: 2013-03-22
Filing date: 2013-10-10
Publication date: 2014-09-25
Also published as: RU2013112810A; RU2550954C2

Abstract

The invention relates to the food industry, particularly to dietary biologically active supplements. As claimed, suspension of a chlorella plankton strain is used as a dietary biologically active supplement. The suspension may, before use, be concentrated by a special technique. The biologically active supplement has a beneficial effect on human health and can be stored in normal conditions for a considerable time.

Description

USE OF A CHLORELLA PLANKTON STRAIN AS A DIETARY BIOLOGICALLY ACTIVE SUPPLEMENT

The invention relates to the food industry, in particular, to dietary biologically active supplements.

A large number of dietary biologically active supplements containing various natural biologically active compounds have been developed and are used on a wide scale in practice today.

Dietary biologically active supplements based on micro-algae, and particularly chlorella, hold a special place among these dietary supplements. Chlorella is the most valuable supplement because of its unique natural makeup and high concentration of biologically active substances that give it an advantage over higher plants [1].

Chlorella is an active biomass producer having large percentages of wholesome protein, fats, carbohydrates, vitamins, and so on, a total of over 650 substances in concentrations exceeding significantly their content in traditional foodstuffs such as meat, milk, fruits, and vegetables.

Chlorella is one of "superfoods" that also include spirulina, barley and wheat green, beebread (a beekeeping product), propolis, and royal jelly that contain absolutely all nutrients essential for embryos of future life and maintain healthy existence of all biological species [2],

There is today a broad range of dietary supplements based on chlorella alone and supplements in which chlorella is combined with other micro-algae and components.

All dietary supplements based on chlorella, however, use only dry biomass of the micro- alga or infusions and extracts of dry chlorella. This preference is explained, in the first place, by the properties of the chlorella strains that are used in dietary supplement production. The reason is that the so-called soil chlorella strains that have low bactericidal capacity and cannot be used in suspension form are used around the world in dietary supplement production. After soil strain chlorella has been used to cultivate a suspension, chlorella cells are separated from the culture medium, and the paste obtained in suspension form is dried to yield dry micro-algal biomass that is used for producing dietary supplements. In this case, no use is made of the chlorella culture medium.

It has been proved in science that many important and especially valuable biologically active substances produced in plants can only preserve their properties in living cells. And, conversely, heat treatment of plants causes such substances to break up and lose their active properties. Further, many biologically active substances cannot be stored for a considerable time after a plant cell has been dried; they decay, and gradually lose their properties in storage.

Another method for preparing medications from medicinal plants and chlorella uses alcohol-base tinctures or extracts. Even though no heating is required, the preparations are practically sterile, and yet some medicinal active substances cannot be extracted from plants [3].

The best way out of this situation is eating natural products like herbs, vegetables, and fruits having medicinal properties. As a rules, natural products are not available for much of the year, and when they are, they have to be processed and stored in advance. Plants can be dried, cut up, and consumer forms prepared as finely ground powders, tablets, and capsules (to which mineral substances are added in need). In these cases, too, however, medications prepared from natural materials cannot be used internally without additional sterilization because of possible microbial semination of the natural materials [3].

It is common knowledge [4] that chlorella accumulates through photosynthesis and releases into the culture medium a wide range of substances having pronounced biological activity. Moreover, the biochemical mixtures making up the biomass of chlorella cells (355 substances) and chlorella culture medium (312 substances) contain approximately the same number and makeup of biologically active substances.

It is possible, by changing the chlorella cultivation conditions and the nutrient medium composition, to alter at will the makeup of the main components of the chlorella biomass [4],

Numerous experiments involving the use of chlorella on animals in Russia and other countries have demonstrated convincingly the substantial advantages and efficiency of chlorella suspension as compared to dry chlorella or chlorella paste [5, 6].

Obviously, for the entire complex of biologically active substances synthesized by chlorella to be preserved, use must be made of the living micro-algal cell and all exo-metabolites released by chlorella into the culture medium during its photosynthesis. It is possible in this case only to preserve all biologically active substances in their entirety and to achieve maximum possible effect of its application as a dietary supplement.

The main reason why soil chlorella strains cannot be used as biologically active supplements in suspension is that they lack high bactericidal qualities that could suppress or halt development of accompanying microflora in storage. For this reason, cells of soil chlorella strains are separated immediately after cultivation from the culture medium and dried. The resultant dry powder is, most frequently, compressed into tablets and used as dietary supplements.

Dietary supplements derived from dry chlorella cells are deficient in several critical respects: 1. As chlorella cells are processed and dried, they lose many important biologically active substances that can only subsist in a living chlorella cell;

2. The remaining biologically active substances in a dry chlorella cell have a significantly lower assimilability than they do in a living cell;

3. No use is made of a broad range of biologically active substances that chlorella releases into the culture medium during photosynthesis; and

4. Long storage life of dietary supplements derived from the dry chlorella cell leads inevitably to depletion and deterioration of the biologically active substances because of their gradual oxidation and decay.

Ready dry chlorella dietary supplements have a significantly lower value than the chlorella suspension from which they are produced.

It is an object of this invention to develop a biologically active supplement on the basis of living cells of chlorella micro-alga and exo-metabolites it releases into a culture medium, which is a living chlorella suspension.

This object is achieved by using a plankton strain of Chlorella vulgaris IFR No. C-1 1 1 (SU 1751981 , C 12N 1/12, Feb. 10, 1997) or chlorella daughter strains having high bactericidal qualities. The Chlorella vulgaris IFR No. C-1 11 strain is the mother strain for the strain BIN (RU 2192459, C 12N 1/12, C02F3/34 April 18, 2001) and the strain Parachlorella nurekis 1904 KIEG (301670, C 12N 1/12, May 19, 2010 ). Both daughter strains are in the plankton category having a high bactericidal capacity. Chlorella vulgaris BIN is suitable for producing biomass and treating sewage and Parachlorella nurekis 1904 KIEG is used to kill green-blue algae, bacteria, and fungi in bodies of water overgrown with algae. Both strains may be used as biomass producers and, therefore, as a biologically active supplement in the claimed suspension form because they have just as high a bactericidal capacity as their mother strain IFR No. C-11 1.

The properties of plankton chlorella strains differ significantly from all soil strains known in the prior art:

1. They can be stored in suspension form only in ordinary non-sterile conditions thanks to their high bactericidal qualities.

Soil strains can only be stored on solid nutrient media on agar-agar in a special environment that must be sterile when they are reseeded.

2. They can be either in the plankton state or in levitation in a culture medium during cultivation and at rest or in storage because of the low negative charge of the micro-algal cells.

Soil strain cells do not carry any electrical charge; they rapidly settle on the bottom of the container and must be stirred all the time as they are cultivated. 3. Plankton strains are cultivated in a monoculture and do not tolerate the presence of other species of micro-algae, and even other chlorella strains.

Soil strains of chlorella are always cultivated in the presence of other species of algae and chlorella strains in the culture. In practice, several chlorella strains are frequently cultivated simultaneously.

4. Plankton strains are significantly more effective in use than soil strains. For example, in cattle farming, suspension feeding rates are only 10 percent of what they are for soil strains, with an identical cell density in the suspension and identical results achieved.

5. Plankton strains are much less demanding than soil strains in cultivation as regards cultivation conditions and equipment design, and also the nutrient media.

When plankton chlorella strains are cultivated correctly and a strong culture is maintained, their pronounced bactericidal qualities may contribute to stabilization of the dietary supplements meeting the requirements made on the number of mesophyllic aerobic and facultative aerobic microorganisms in SanPin 2.3.2.1078-01 standards for dietary supplements based on micro-algae and having none of the characteristics of E. coli (coliform) bacteria, and pathogenic microflora, including salmonella, yeasts, and mold.

The method for cultivating plankton chlorella strains comprises dispensing the nutrient medium into containers, inoculating the suspension with a strain, illuminating the culture container for as long as the micro-algae grow, and maintaining the suspension at the required temperature. The micro-algae are cultivated in special chlorella cultivating units designed as containers made of a transparent material (glass or Plexiglas) set in a metal frame and provided with outside and inside artificial illumination sources, and having devices to maintain the preset temperature of the suspension automatically.

Upon completion of cultivation and buildup of chlorella cells to a desired density, the bacterial background in the suspension holds in norm over a prolonged storage time under normal conditions, without requiring preservation or freezing of the suspension. The high bactericidal properties of the suspension are attributed to chlorella cells releasing antibiotic substances into the culture medium. The antibiotic effect of these substances is most pronounced on a broad range of pathogenic microflora only, and does not affect the good microflora of the intestines.

Example 1. A Method for Producing a Biologically Active Supplement

A nutrient medium for cultivating micro-algae on the basis of the Chlorella vulgaris IFR No. C-l 11 strain was prepared from: Ammonium nitrate 0.2-0.4 g

Superphosphate, 10% solution 0.3-0.5 ml

Ferrous chloride , 1% solution 0.15 ml

Cobalt nitrate, 0.1% solution 0.1 ml

Copper sulfate, 0.1% solution 0.1 ml

Carbon dioxide, solution (pH 3.0-5.0) 5-10 ml

Tap water 1,000 ml

Commencement of chlorella cultivation: The nutrient medium held at a temperature of around 30°C was poured into a chlorella cultivation unit to fill 80% of the container volume and a suspension of the Chlorella vulgaris IFR No. C-l 11 strain was added to take up the remaining 20% of the container volume until the required density of cells (2 million per liter) in the culture was achieved. Next, a carbon dioxide solution equivalent to between 0.5% and 1.0% of the chlorella suspension volume was introduced and the unit illumination sources were turned on. As chlorella cultivation continued, carbon dioxide solution was added every day as back-suspension (pH 3.0-5.0) once every 24 hours at a rate of 0.5% to 1.0% at the moment when the unit lamps were turned on. Chlorella cultivation continued for four full days until the required density of cells (8 million to 10 million per milliliter) was achieved. The culture was exposed to lamp illumination for 12 to 14 hours a day, the lamps being turned off for the remaining 10 to 12 hours. The temperature of the suspension was maintained at 30°C for the duration of the cultivation process. At the end of four-day cultivation, the density of cells in the suspension reached 8 million to 10 million per milliliter. The chlorella suspension was drained from the unit and was ready for use as a biologically active supplement.

Carbon dioxide can also be added as a mixture with air at a rate of 1 % to 2% of the air volume through a compressor and sprayers provided on the bottom of the unit container at a rate of 10 to 20 liters per 1 mVmin.

Ready chlorella suspension may also be thickened to give a biologically active supplement of concentrated chlorella with a cell density between 20 million and 1,000 million per milliliter.

Example 2. A Method for Concentrating Chlorella Suspension

Stage 1

According to a prior art method (SU No. 1091890), starting chlorella suspension was subjected to electroflotation, and positively charged oxygen bubbles produced by water electrolysis stimulated flotation and concentration of the biomass on the surface whereupon the biomass was transferred to storage. We have established that addition of the culture medium or chlorella suspension to the highly concentrated biomass in a specified proportion sufficient to achieve a cell density of 30 million to 1,000 million per milliliter in the resultant concentrated chlorella biomass yields a product - concentrated chlorella cell biomass that can be stored for at least 30 days in normal conditions.

In practice, this effect is attained by removal of froth from the suspension surface during electroflotation together with small quantities of chlorella suspension, that is, froth is skimmed together with chlorella suspension rather than alone. Chlorella suspension has high bactericidal qualities and performs as a preservative even if cell density increases 100-fold from that of the starting chlorella suspension.

The resultant purified chlorella culture medium is also a valuable biological product that has a broad range of dissolved biologically active substances, particularly, chlorella cell exo- metabolites ("Chlorella, Physiologically Active Substances, and Their Applications," S.S. Melnikova and E.E. Manankina, Photobiology Institute, Academy of Sciences, BSSR, 1991).

Stage 2

A well-known reverse osmosis method or ultrafiltration is used for thickening purified chlorella culture medium by a factor of five to 100.

Reverse osmosis is used to concentrate all substances in the purified culture medium (chlorella cell exo-metabolites) and recover the pure solvent (water) from the solution, except for a quantity of univalent ions of Na, K, and CI. Reverse osmosis is practically reduced to thickening the purified chlorella culture medium and obtaining concentrated chlorella culture medium. This method for thickening chlorella culture medium has not been used by anyone. There is a great variety of reverse osmosis units in our day. Concentration level is raised at least tenfold, or, in need, 30- to 50-fold. Concentrated chlorella culture medium has a storage life of at least three months.

Concentration may be conducted by ultrafiltration. A pump forces the culture medium to be filtered under pressure from inside to outside of hollow fibers of an ultrafiltration module. Purified water passes through the porous walls of the hollow fibers and is then used as ordinary pure water. Particles of the chlorella culture medium 0.01 to 0.1 μπι in size remain inside the hollow fibers of the ultrafiltration module. After the fibers have been washed, concentrated chlorella culture medium is collected in the thickened culture medium storage.

Even though ultrafiltration is simple to perform, it has a significant deficiency because it can only concentrate a definite range of substances in chlorella culture medium having molecule sizes within 0.01 to 0.1 μπι.

Stage 3

Concentrated chlorella culture medium was mixed with concentrated chlorella cell biomass to obtain concentrated chlorella. The quantity of concentrated chlorella cell biomass and concentrated chlorella culture medium depends on the concentration (thickening ratio relative to the starting substances) of concentrated chlorella cell biomass and concentrated chlorella culture medium. These ingredients may be mixed in different concentrations. In this invention, with the maximum ratio of concentrated chlorella cell biomass and concentrated chlorella culture medium in concentrated chlorella, concentrated chlorella is a range of products having, on the one hand, a maximum concentration of chlorella cells and a minimum concentration of concentrated chlorella culture medium (in fact, concentrated chlorella cell biomass), and, on the other hand, a minimum concentration of chlorella cells and a maximum concentration of concentrated chlorella culture medium (in fact, concentrated chlorella culture medium). A biologically active supplement has a cell density of 1 million per milliliter to 1,000 million per milliliter (the minimum will be used hereafter), and the liquid medium is the concentrated chlorella culture medium at a thickening ratio of 2 to 100.

In some cases, it is reasonable to produce concentrated chlorella from the same quantity of the starting suspension, that is, a thickened concentrated chlorella cell biomass and a thickened concentrated chlorella culture medium are produced separately and then mixed together. This is, in fact, a product thickened relative to the starting commercial suspension at a multiple ratio (10, 15, 20, 25, 30, and so on).

Chlorella suspension as a biologically active supplement having a cell density of 8 million to 10 million per milliliter or a dietary concentrated chlorella supplement having a cell density of 20 million to 1,000 million per milliliter can be stored in normal condition at a temperature of 0°C to 24°C for at least 45 days from production time. While in storage, chlorella cells in the dietary supplement start gradually to settle on the container bottom within days of storage. Devoid of photosynthesis conditions, chlorella cells gradually go over into anabiosis, and all cells are deposited on the bottom within approximately a month. Chlorella cells, though, remain alive and ready to begin photosynthesis at any moment. For this reason, chlorella suspension must be shaken well before taking the dietary supplement.

Carbon dioxide may be used to extend the storage life of chlorella suspension as dietary supplement and concentrated chlorella suspension dietary supplement to as long as six months. The mass proportion of carbon dioxide in chlorella suspension used as dietary supplement is as it is for mildly carbonated drinks (under the GOST 28188 Standard) - from 0.20% to 0.30%, inclusive. Stored in this supplement form, chlorella cells take in as much carbon dioxide as they require for breathing in storage in a tightly closed receptacle.

The tests conducted by the applicants on volunteer subjects have shown the dietary supplement in suspension form to be very effective.

Example 3

A male, 43, was taking 200 milliliters of chlorella suspension daily for a month. Data of the immunographs taken before and after chlorella suspension consumption are given below:

2. complexes

MIC 350 units 460 units 31.4

3.

Immunoglobulins:

4.

A g/liter 2.81 2.66 5.3

M g liter 1.63 1.12 45.5

G g liter 15.28 16.41 7.4

Example 4

A male, 44, was taking 200 milliliters of chlorella suspension daily for a month. Data of the immunographs taken before and after chlorella suspension consumption are given below:

G g/liter 14.26 16.13 13.1

Tests in Examples 3 and 4 were conducted at the Penza Regional Center for AIDS Prevention where studies were carried out into the immunostimulating effect of the Chlorella vulgaris (strain IFR No. C-l 1 1) micro-alga on human immunological, biochemical, and clinical characteristics. The studies revealed the stimulating effect of chlorella on the phagocytic system, humoral immune system, and the cell immunity system of both patients studied. Both volunteers spoke about steady improvement in their health from the early days of preparation taking. The volunteers studied registered significant improvements in immunograph parameters over a month of studies from the start of the experiment. A majority of characteristics showed normalization in both patients in the biochemical studies.

It was established through the studies that the Chlorella vulgaris IFR No. C-l 1 1 strain had an immunostimulating effect on the immunocompetent cells of both volunteer subjects participating in the studies.

Example 5

A group of three males and five females volunteered to take part in the experiment. The males were aged 43 to 45, two females were 30 and 40, respectively, and three females were over 65. One of the females, 72, suffered two strokes before she joined in the experiment. The experiment was conducted at the immunology and allergy chair of the Central Research

Laboratory, Penza Institute for Medical Practice Improvement.

Summary: Between March 21 and May 15, 2006, tests were conducted at the Central Research Laboratory of the Penza Institute for Medical Practice Improvement to study the immunostimulating effect of the Chlorella vulgaris (IFR No. C-l 1 1 strain) micro-alga on human immunological, biochemical, and clinical characteristics. All eight volunteer subjects took 100 grams of the micro-alga studied twice a day. The characteristics of interest were measured before commencement and upon completion of the experiment.

The studies established that a stimulating effect had been exerted on the phagocytic system of three patients, the humoral immune system of four patients, and cell immunity system of all eight patients studied. In biochemical studies, all the patients registered normalization of a majority of characteristics. Clinical blood analysis did not reveal any changes in the blood picture.

The studies carried out uncovered the immunostimulating effect of the Chlorella vulgaris micro-alga on the immunocompetent cells of all the subjects involved in the experiment.

Example 6

Tests were conducted at the SB SEI PFUR chair between March 24 and May 15, 2008, to study the effect of dietary suspension of the Chlorella vulgaris (IFR No. C-l 1 1 strain) micro- alga. The studies included an analysis of the immunostimulating qualities of the dietary suspension and its effects on human immunological, biochemical, and clinical characteristics. Twenty human volunteers (Group 1) and ten primates (Group 2) were given 10 grams of the dietary micro-algal suspension under study twice a day for 15 days. The characteristics studied were measured before the experiment began and upon completion of micro-alga administration. The studies included immunological blood checks - general immunograph, cell immunity, general IgE indicator, and general diagnostic biochemical analysis. Besides, Group 1 was analyzed for bioelectrography (evaluation of vegetative functions), endoecological evaluation of organism contamination through the specialized irido-prognostic Iris-Sigma study, evaluation of visual functions and neurophysiological characteristics by color campimetry, results of electrophysiological studies, subjective sensations, and the results of objective examinations.

The studies were conducted jointly with the City Center for Control of AIDS and Infectious Diseases, the Endoecological Rehabilitation Ophthalmology Center of the Oktyabrsky Resort, and the Medical Primatology Research Institute in Sochi.

The studies established the existence of a stimulating effect on the phagocytic system in 65% of the patients, humoral immune system in 70% of the patients, and cell immunity in 84% of the patients examined. In biochemical studies, 80% of the patients showed normalization of a majority of characteristics. Improvements were registered in vegetative functions and the percentage of tissue, lymphatic, subcutaneous fat contamination decreased, ocular functions improved, and neurophysiological and electrophysiological characteristics improved by an average 15% to 25%. Clinical blood analysis revealed improvement in the blood picture, in particular, in color, thrombocytes, lymphocytes, segmented neutrophils, eosinophiles, and erythrocyte sedimentation rate (ESR) in 68% of the patients.

The studies conducted, therefore, have uncovered an immunostimulating effect of the Chlorella vulgaris micro-alga on the immunocompetent cells of all the human patents participating in the experiment.

Example 7

A male, 78, has been taking at least 1.5 liters of chlorella suspension daily over the past five years already, using it alone in place of water in his diet. Since he first began taking chlorella suspension he has felt significantly better. Gradually, he cured himself of many illnesses, including chronic diseases of the digestive system, parodontosis, and catarrhal diseases. While he was taking chlorella suspension, he had no need of seeing doctor a single time. In his own assessment, he feels like a healthy man of 50.

By our observations, 200 milliliters of chlorella suspension taken daily by all volunteer subjects made them feel substantially better already within the first week of treatment. Their digestion improved, stool normalized, and they recovered strength faster after physical strain. Chlorella improved their cerebral circulation and memory, brain got more receptive, blood pressure recovered to normal, and immunity grew stronger. While they were taking chlorella, the volunteer subjects felt a significant easing-off of the symptoms of the diseases they had before.

Reports of chlorella-based dietary supplements around the world vindicate a broad spectrum of effects the micro-alga has on human organism. An unrivaled effect of chlorella shows up when it is used to relieve endogenous and exogenous intoxications and alcoholic hangover, cure constipation, ulcers, allergies, arthritis, atherosclerosis, high cholesterol level, high blood pressure, and cardiovascular diseases, as an antiviral drug, and much else. The general positive effect of chlorella suspension on human organism is a significant and steady improvement in the quality of life.

Significant and steady improvements in the general conditions are observed after a prolonged use of dry chlorella. Certainly, dietary supplements based on dry cells have a favorable effect on human health because the proportion of biologically active substances in the cells is still high enough, even after the cells have been separated, dried or extracted. When, though, living chlorella suspension is consumed, steady improvements set in virtually during the first week of consumption. This positive effect is achieved, for the most part, thanks to the living form of chlorella suspension that retains to the fullest extent possible the entire spectrum of all biologically active substances locked up in the micro-algal cells and in the culture medium and contributes to maximum assimilation of these substances by human organism.

It is, therefore, the first time ever that a living plant in the form of micro-algal suspension is claimed for human use as a biologically active supplement based on a plankton chlorella strain having unique bactericidal properties that permit cells themselves to be preserved in suspension in a living state for a considerable time in normal conditions without addition of preservatives or freezing.

Chlorella suspension can be used as a dietary supplement as a suspension having a cell density of 8 million to 10 million per milliliter immediately upon completion of cultivation, or in a thickened form with cell concentration ranging from 20 million to 1 ,000 million per milliliter.

BIBLIOGRAPHY

1. Mike Adams, "Superfoods for Optimum Health: Chlorella and Spirulina."

2. Robert Atkins, "Vita-Nutrient Solution (Nature's Answer to Drugs)."

3. Patent RU (11) 2178660 (13) C2.

4.S.S. Melnikova, E.E. Manankina, "Chlorella: Physiologically Active Substances and Their Uses," Nauka i tekhnika, Minsk, 1991.

5. M.Ya. Salnikova, "Chlorella: A New Kind of Feed," Kolos, Moscow, 1977.

6. A.M. Muzafarov, T.T. Taubayev, R.A.Selyametov, "Chlorella and Its Uses in Cattle Farming," Tashkent, 1974.

Claims

1. Use of suspension of a chlorella plankton strain as a dietary biologically active supplement.

2. Use of suspension claimed in claim 1 , wherein a suspension at a concentration of 8 million to 10 million cells per milliliter is used.

3. Use of suspension claimed in claim 1, wherein a suspension at a concentration of 20 million to 1,000 cells per milliliter is used.