WO2021134108A1

WO2021134108A1 - Basic medium for cultivating microorganisms

Info

Publication number: WO2021134108A1
Application number: PCT/VN2020/000012
Authority: WO
Inventors: Nguyen Phu
Original assignee: HANG, Nguyen Tuy; HUNG, Nguyen Thuan; VINH, Nguyen Khai
Priority date: 2019-12-25
Filing date: 2020-11-24
Publication date: 2021-07-01

Abstract

The invention relates to basic media for cultivating mixtures of microorganisms of EM probiotic preparations.

Description

BASIC MEDIUM FOR CULTIVATING MICROORGANISMS

Field of the Invention

The invention relates to the field of biotechnology, in particular the invention relates to basic media for cultivating microorganisms and some EM probiotic preparations made from the same.

Background of the Invention

1. Prior art EM probiotic preparations and their derivatives:

- EM probiotic preparations means effective microorganisms which were invented and applied in practice by Professor Dr. Teruo Higa, Japan in early 1980.

In the EM preparations, there are more than 80 species of anaerobic and aerobic microorganisms belonging to the groups of photosynthetic bacteria that synthesize organic maters from carbon dioxide (CO₂) and water (H₂O), nitrogen fixing bacteria (using organic matters from photosynthetic bacteria to convert nitrogens in the air into nitrogen compounds), actinomycetes (producing antibiotics that inhibit pathogenic microorganisms and decomposing organic maters), lactic acid bacteria, yeasts (producing vitamins and amino acids).

- From the original EM preparation, it can be activated to form derivatives for convenience in use.

The main and popular liquid EM derivative preparations in the fields of agrochemicals and chemical products comprise: a) EM-1 preparations, which are commonly used to made other microbial derivatives. b) From the EM-1 preparations, continuing to activate to made liquid derivatives, among which, some derivatives are popular due to their functionality and effectiveness in use, as follows: EM-2 (secondary EM) is an EM solution that has effects of decomposing organic matters, disinfecting, cleaning the environments, improving physicochemical properties of the soil, enhancing growth of pets, etc.

EM-5 (alcohol EM) is an EM solution that has effects of limiting, preventing diseases, enhancing the resistance and sustainability of plants.

EM-FPE (plant EM) is an EM solution made from plants with EM (for example, using neem leaves due to their ability to repel and kill insects, pests), that contains useful organic acids, bioactive ingredients, minerals and organic compounds.

2. Culture medium for microorganisms:

2.1. General requirements for culture media for microorganisms:

Culture media for microorganisms are nutrient substrates which are artificially prepared to meet the demands of growth, development and production of the metabolic products of microorganisms.

In general, the nutrient environment needs to meet the demands of microorganisms for carbon sources (C sources), nitrogen sources (N sources), inorganic salts (mineral salts), sources of growth factors and water (H₂O), in which: a) Carbon source (C source):

C sources, which are mainly used by microorganisms, comprise sugars, organic acids, alcohol, lipids, hydrocarbons, carbon dioxide, carbonates, etc. Among them, sugar is a good C source and energy source for microorganisms. b) Nitrogen source (N source):

N sources, which are often used to cultivate microorganisms, include peptones, fish flour, silkworm pupae flour, soybean flour, peanut flour, meat concentrates, yeast concentrates, corn concentrates, etc. c) Inorganic salt sources:

Inorganic salts are an indispensable source of nutrients for the growth of microorganisms. The inorganic salts have the main physiological functions of participating in the composition of active centers in enzymes of microorganisms, maintaining structural stability of macromolecules and cells, moderating and maintaining balances of the osmotic pressure of the cells, controlling the oxidation-reduction potentials of the cells, and they are the source of energy-generating matters for some microorganisms. d) Trace elements:

In the process of growth, the microorganisms also need a number of trace elements which participate in the components of enzymes and activate enzymes.

Microorganisms often receive trace elements from natural organic nutrients, inorganic chemicals, tap water or even from glass culturing equipments.

Trace elements can be added to the culture media for microorganisms only in the special cases. Since many trace elements are heavy metals, they will harm microorganisms if being present with excess amounts. When it is necessary to add trace elements to the culture media for microorganisms, attention should be paid to control precisely the dosages. e) Growth factors:

Growth factors are organic compounds that contain necessary microorganisms for their growth although these microorganisms are present in very small quantities and do not self-synthesize enough compared with demands. Usually, adding to the medium organic substances such as yeast concentrates, meat concentrates, and animal and plant concentrates (pupae, bean sprouts, etc.) can meet the demand for growth factors. g) Water Water is an indispensable ingredient for microorganisms to grow. Physiological functions of water in cells are dissolving and transporting substances, supporting the absorption of nutrients, releasing metabolic products; participating in a series of chemical reactions in cells; maintaining stable natural configurations of macromolecules such as proteins, nucleic acids; having good thermal conductivity, well-absorbing the heat generated in metabolisms and timely diffusing it to the outside to maintain the stability of the temperature inside the cell; maintaining conventional the cell morphology; controlling the structure of cells (enzymes, microtubules, capillaries, etc.) and the disassembling and assembling of viruses through the hydrolysis or dehydration.

2.2. Prior art culture media for microorganisms EM and current disadvantages:

- As a common practice in the world and in Vietnam, for cultivating, increasing biomass, activating microorganisms from original EM preparation (producing EM-1 preparations), or from EM-1 preparation (producing EM-2, EM-5, EM-FPE preparations), culture media such as molasses of cane sugars, beet sugars and grape sugars can be used.

Molasses can be replaced with compact-concentrated sugar (raw cane sugar obtained by heating to concentrate the molasses), powder sugar, brown sugar, etc., because these are also products made by concentrating various juices of plants such as sugarcanes, coconuts, and jiggery similar to the way of producing molasses, or by adding molasses to crystalline sugars (brown sugars). However, molasses is the most commonly used kind, because it is a major byproduct of the sugar industry (currently, cane sugar accounts for about 75% of the sugar yields in the world), with advantages such as cheap price, organic and nutritional ingredients are suitable for growing and stabling environments for various microorganisms in EM preparations.

- Regarding the molasses, which is prior art mainly popular material for cultivating, increasing biomass, activating mixtures of microorganisms in EM preparations:

As a byproduct of the sugar processing industry, including cane sugar, beet sugar, etc., the molasses is a treacly liquid remaining after removing sugars by concentration and crystallization. Usually, molasses has dry matter in range of 80-85%, in which mainly including sucrose; reducing sugar, total nitrogen and minerals. The organic substances in molasses are acids, alcohols, amino acids and vitamins.

The standard composition of molasses is usually divided into 3 parts: sugar, sugar-free substances (non-sugar substances) and minerals, in which:

Sugar: Various soluble carbohydrates (double sugar and simple sugar) are the main nutritional constituents of molasses, in which mainly including the sucrose.

Sugar-free substances: These substances determining many of physical properties of molasses, especially the adhesive viscosity. They consist mainly of various carbohydrates such as starches, nitrogenous compounds and organic acids. Nitrogen-free compounds include arabane, galactane or their hydrolysis products, coloring and flavoring agents. The coloring agents of molasses include caramel, melanoidin (which is a condensation product of reducing sugars and amino acids, that increasing the color level of molasses) and Fe²⁺complex, which is yellow-green and cannot be eliminated in the producing process. Colloids, including pectin, waxy, and mucus, greatly impacts the growth of microorganisms because they form a membrane around the cell that inhibits the processes of nutrient absorption and the discharge of metabolic products of cells into the environment. The mainly inorganic sugar-free substances are various potassium salts, calcium salts and sulfur dioxide (SO₂) residues.

Minerals: Molasses contains many mineral elements such as sodium, potassium, magnesium, sulfur (S) and also contains a very small amount of trace elements such as copper (Cu, about 4.9 mg/kg), zinc (Zn, about 34 mg/kg), iron (Fe, about 115 mg/kg), manganese (Mn, about 18 mg/kg), etc.

In addition to the substances according to the above chemical composition, molasses also contains a lot of microorganisms, most of which come from materials, a few come from the air, water, soil that mixed into the sugar solutions. It can be divided into 3 categories: bacteria, yeasts and molds, in which bacteria are dangerous due to including many species capable of producing spores. Because molasses is an quite ideal nutrient, microorganisms are easy to penetrate and grow, the quality of the molasses is therefore easy to change over the storage time.

The limitations of the culture media for microorganisms in the prior art EM preparations:

Due to being byproducts of the sugar industry from the natural materials of sugarcane, sugar beet, etc., the molasses has unstable quality, in addition to depending on growing conditions, plant species, soil, climate, it also depends on the technical levels, methods and scales of production, and the limitation or elimination of impurities, contaminating microorganisms in the sugar production process and the recovery process of molasses byproducts. This is a factor that causes the quality control difficult in the industry of producing EM preparations.

The use of molasses as a culture medium for microorganisms also leads to the derivatives of the EM preparation which often have a poor sense level, have a dark brown color, and still have a characteristic flavor of molasses:

In general, the above liquid EM preparations and other similar products such as garlic EM preparations, banana EM preparations, and herbal EM preparations all have the common disadvantage of having a dark to very dark golden brown color, with residual flavor of molasses or sugar of various kinds used in the producing process. To reduce the color density or to clear molasses, some treatment methods have been applied such as the addition of acids (such as hydrochloric acid, sulfuric acid) to molasses to react with inorganic substances, or method of using polymers of various kinds such as polymer anions to precipitate impurities, deposit sediment and filter to separate the sediment. However, it can be seen that the above methods have not yet met the product sense requirements, as well as not being able to limit and control well impurities or microorganisms available in plant materials (such as sugarcane, cane juice) for producing molasses.

- Currently, in the world as well as in Vietnam, there is no information about new publications on media that are different from molasses media in cultivated liquid, increasing biomass, and activating EM probiotic preparations in producing the above popular EM derivative preparations.

- Current EM derivative preparations are usually only suitable for use in producing products that do not require high microbiological quality and the sense of color and flavor such as in deodorizing bams, treating waste odors, treating shrimp and fish ponds, etc., and it is very difficult, or evenly impossible, to add the EM probiotic preparations in the manufacturing of household products, detergents, body cleaning products or plant protection products.

Due to the above main disadvantages, it is still necessary to study to find out culture media for mixtures of microorganisms in the EM probiotic preparations to ensure the efficiency, the quality and can improve the general sense of the preparations derived from original EM or EM-1 probiotic preparations.

Summary of the Invention

In order to overcome the above disadvantages and the limitations of the medium using water-soluble molasses (H₂O) have been indicated in the Background of the Invention, it is an objective of the invention to provide a medium for cultivating a mixture of microorganisms in various EM probiotic preparations that has the ability to improve the sense of color, flavor and limit impurities and contaminating bacteria in the manufacturing of preparations derived from EM.

Specifically, probiotic preparations derived from EM preparations making EM-1; or from EM-1 preparations making EM-2, EM-5, EM-FPE preparations and other similar derivatives such as herbal EM, various Bokashi fertilizers, etc. which are made according to the invention while still ensuring the growth of microorganisms and yeasts as cultivated in conventional medium of molasses, but with good sense due to no sugar/molasses odor, with pale yellow color, and minimizing of the existence (to the extent that it can be considered insignificant) of impurities, yeasts, and harmful microorganisms available in materials and in production process.

To achieve the above objective, according to an embodiment, the invention provides a basic medium for cultivating of mixtures of microorganisms of EM probiotic preparations, in which this basic medium is obtained by blending an industrial syrup with water in a volumetric ratio of the industrial syrup/water in the range of 1/15-1/7, at a temperature in the range of 30°C-40°C.

According to another embodiment, the invention provides the said basic medium, in which this basic medium made a semi-synthetic medium by blending the basic medium with molasses, protease, minerals, papaya resins or pineapple peels.

According to further another embodiment, the invention provides the said basic medium, in which this basic medium made a semi-concentrated or concentrated medium by blending the basic medium with agars, gelatins, xanthan gums, or guar gums.

According to still yet another embodiment, the invention provides a number of EM probiotic preparations obtained from a process comprising steps: a) blending a EM-1 preparation into the basic medium obtained above at a temperature in the range of 30°C-40°C in a volumetric ratio of the EM-1 preparation/basic medium in the range of 1/22-1/8 to obtain a homogeneous, clear liquid with pale yellow color; b) obtaining an EM probiotic preparation by anaerobic incubating of the liquid obtained from step a) for at least 3 days.

Detailed Description of the Invention

Here, the invention will be described in detail with particular examples and embodiments, but these examples and embodiments are only intended to clarify the nature of the invention and should not limit the claim scope of the invention. Accordingly, the invention will be described on the culture medium for cultivating mixtures of microorganisms of an EM probiotic preparation, in which this medium is obtained by blending an industrial syrup with water in a volumetric ratio of an industrial syrup/water in the range of 1/15-1/7 and depending on the type of EM probiotic preparation to be made, the temperature in the range of 30°C-40°C.

1. Regarding the basic medium:

1.1. Ingredients:

Industrial syrups made from various starches such as high fructose com syrup - HFCS, which is a natural sweetener made from corns, glucose syrup (prepared from tapioca (cassava) starches, including industrial malt or liquid natural malt that prepared by natural fermentation of rice and glutinous rice with germ flour), etc.

The above substances are selected because they meet all of the above general requirements: (i) Being the main products of the food sugar industry from natural plant materials (com, cassava, rice, glutinous rice); (ii) The quality and content of sugar are standardized, do not contain impurities and contaminating bacteria; the sugar content is determined depending on the products (such as the fructose/glucose ratio in HFCS 55 or HFCS 90 products; the % criteria of reducing sugar content; etc.); (iii) Easily soluble in water to form a homogeneous solution with a determined pH according to the ratio of the solute and water; (iv) The formed solution is a slightly viscous, clear, almost colorless liquid with no characteristic flavor of molasses/sugar; (v) Popular industrial products with low cost.

In principle and through experiments, the use of HFCS or other industrial syrups such as glucose syrup, natural liquid malt is not significantly different through the actual test results of cultivation of the mixtures of microorganisms in EM probiotic preparations, because the differences in sugar contents between these products are not much when comparing with the amount used in the blending ratio with water to meet the requirements of microorganism cultivation, it is can be adjusted through the Brix degree (°Bx, which is a common scale showing the sugar density in a solution) according to the bending ratio of industrial syrup to water.

The differences in the chemical compositions of the above industrial syrups mainly are the simple sugar components they contain, such as between natural malt and other industrial syrups, in particular:

Natural liquid malt: The sugar component in the malt is a maltose sugar, which consists of 2 molecules of glucose bonded to each other.

Other industrial syrups: The sugar component consists of fructose and glucose molecules that are not bond together

Because the insignificant differences in the nutrient demands of the culture media for the mixtures of microorganisms in the EM probiotic preparations, HFCS can be considered as a representative product of industrial syrups for researches and experiments due to the popularity and manufacturing industry have stabilized from planting and harvesting of com materials. - Regarding the high fructose com syrup - HFCS:

Manufacturing process: Com (maize) is ground to get starches; com starches are processed through two stages: (1) chemically hydrolyzing to produce millions of glucose molecules, and (2) converting glucose into fructose by metabolic enzymes. The final product is sweet liquid, liquid sugar with a very large concentration of fructose according to the consumer requirements.

Different types of HFCS have different ratios of fructose. For example, HFCS 90 is the most concentrated syrup, with up to 90% fructose; HFCS 55 (most popular and commonly used) has 55% fructose and 45% glucose.

HFCS 55 has a similar composition to conventional granulated sugar - sucrose (which is a sugar obtained from sugar beet or sugarcane, made up of one glucose and one fructose bonded to each other by 1,2-glucoside bonds). The difference between the HFCS 55 and the granulated sugar is very small.

In terms of chemical structure, the fructose and glucose molecules in high fructose corn syrup do not have the chemical bonds to stick together like in granulated sugar, they only exist side by side in syrups. This difference has no effect on the nutritional value (with equal calories).

1.2. Basic medium for cultivating EM microorganisms and general manufacturing process:

- The basic medium for cultivating microorganisms, yeasts to produce EM probiotic preparations and their derivatives is produced by stirring evenly industrial syrups in water.

In which:

Industrial syrups consist of various syrups made from various starches, such as high fructose com syrup - HFCS (which is a natural sweetener made from corns), and syrup of glucose (made from tapioca starches) or malt (made from rices, glutinous rices), etc.

The water is water that has removed various impurities, bacteria, yeasts and molds, and can use domestic water supply containing a residual amount of chlorine during the sterilization process, but it takes storage time in the tank to evaporate the chlorine.

Identification signs of the bacteria activating-culture medium according to the invention are colorless, transparent, slightly viscous liquid, Ddd ratio depends on the type of the industrial syrup used and the ratio of the industrial syrup/water: a) Using high fructose com syrup (HFCS, from com starches) or glucose syrup (from tapioca (cassava) starches):

b) Using natural malt (dilute, fermented from glutinous rice, rice):

2. The derived media and the EM preparations made from the basic medium according to the invention: 2.1. According to an embodiment of the present invention, other semisynthetic media can be created according to usage requirements from the above basic medium. Accordingly, blending basic medium with other substances such as molasses, protease enzymes, minerals or plants such as papaya (stem resin, leaves, fruits), pineapple (peels, fruits), etc. to create a semi-synthetic medium for activation of EM probiotic preparations.

2.2. According to another embodiment of the invention, depending on the usage requirements, the above liquid basic medium can be easily converted to the semi-concentrated or concentrated medium by further adding of agar, gelatin (gelatine), or thickeners such as xanthan gums, guar gums.

2.3. According to further another embodiment, the present invention relates to a number of EM probiotic preparations obtained from a process comprising the steps: a) blending a EM-1 preparation into the basic medium obtained in the above item 1 at a temperature in the range of 30°C-40°C in a volumetric ratio of the EM- 1 preparation to the basic medium as determined by the type of EM preparations to be made (see the table below), to obtain homogeneous, clear, pale yellow liquid;

b) obtaining an EM probiotic preparation by anaerobic incubating of the liquid obtained from step a) for at least 3 days.

Examples The following examples are intended to further clarify the nature of the invention, hut are not limited to products made from the basic medium of various forms (liquid, semi-concentrated, concentrated) and the semi-synthetic media formed from it.

Example 1: Making 20 liters of EM probiotic preparation

Blending evenly 2 liters of industrial syrup into 16 liters of water at a temperature in the range of 30°C-40°C to obtain 18 liters of a basic medium. Continuing to stir well 2 liters of EM-1 into 18 liters of the basic medium at a temperature in the range of 30-40°C, extracting to obtain liquid extract. Incubating anaerobic the liquid by covering the incubator and avoiding direct sunlight for 3 days to obtain 20 liters of EM preparations.

Example 2: Making 20 liters of EM probiotic preparation in a concentrated form

Blending evenly 2 liters of industrial syrup into 16 liters of water at a temperature in the range of 30°C-40° C to obtain 18 liters of a basic medium. Continuing to add a thickener, which is a xanthan gum, in a weight ratio of xanthan gum/ water in the range of 1-10. Then stirring well 2 liters of EM-1 into 18 liters of the basic medium at a temperature in the range of 30-40°C, incubating anaerobic the liquid by covering the incubator and avoiding direct sunlight for 3 days, extracting to obtain 20 liters of EM probiotic preparation in a concentrated form.

Example 3: Making 20 liters of EM-5/HF (EM alcohol, which is an EM solution that has the effects of limiting, preventing pests and diseases, enhancing the resistance of plants).

Step 1 (Creating a semi-synthetic medium): Blending evenly (in a clean, disinfected bottle) 1 liter of industrial syrup, 1 liter of vinegar (acetic acid), 1 liter of food alcohol (45-50 degrees of alcohol) and 16 liters of water. Ratio of industrial syrup/water is 1/16. Step 2: Continuing to evenly stir 1 liter EM-1 with 18 liters of the culture medium of the Step 1 (at a temperature in the range of 30°C-40°C); extracting the stirred mixture of the EM and the culture medium into a cleaned incubator, limiting the space in the incubator. The ratio of EM-l/semi-synthetic medium is 1/18.

Step 3: Incubating anaerobic by covering the incubator and avoiding direct sunlight (usually at least 3 days, depending on the ambient temperature).

Example 4: Making 20 liters of EM-FPE/HF (plant EM, which is an EM solution made from plants with EM):

Step 1 (Creating a basic medium): Blending evenly (in a clean, sterilized bottle) 1 liter of industrial syrup (from com starch, or tapioca starch), and 19 liters of water. Ratio of industrial syrup/water is 1/19.

Step 2: Stirring evenly the mixture of the Step 1 with 1 liter of EM-1 and 2 kg to 3 kg of chopped fresh plants (such as neem leaves). Putting in a closed incubator. The ratio of EM-1 /basic medium is 1/20.

Step 3: Incubating the mixture in anaerobic conditions (covering tightly, limiting the space in the incubator) for 7 to 14 days, avoiding direct sunlight on the incubator. During incubation, it is necessary to regularly stir the liquid in the incubator to release gas.

Effectiveness of the Invention

The basic medium and semi-synthetic media in the concentrated and semi-concentrated forms from this basic medium primarily use industrial products that are thoroughly mixed with water at the ratio determined according to the usage requirements to make various preparations. Because selected substances dissolved with water are industrial products, with criteria of sugar content standardized and determined relatively accurately during the production, circulation and distribution, it is completely possible to determine the proportion of preparation according to the industrial scale for each type of derivative probiotic preparations to be made. The optimum ratio can be easily determined by the experimentation, actual production for each individual product or in the experiments of manufacturing and producing of new products.

The quality management of the medium preparations for cultivating mixtures of microorganisms is more rigorous and easier due to overcoming the quality limitations of seasonal materials.

On the other hand, impurities commonly found in molasses (such as bacteria, yeasts, molds, organic and inorganic impurities) of the selected substance are insignificant, therefore the treatment step of removing residual harmful impurities in derived EM probiotic preparations made from the basic medium according to the invention is not necessary. This can lead to the reduction of the production steps related to the removal of contaminating bacteria and impurities in the processes of feeding materials, storage and production of EM probiotic preparations, thereby, requirements for equipments and production lines is more convenient and simpler.

Limiting a contamination of harmful microorganisms and achieving good general sense (free from sugar/molasses odor, pale yellow color) of the medium for cultivating microorganisms according to the invention can help open up the possibility of manufacturing products with diverse applications in the fields of agrochemicals, chemical products, and cosmetics with high efficiency and quality at low cost.

Claims

1. A basic medium for cultivating mixtures of microorganisms of EM probiotic preparations, in which this basic medium is obtained by blending an industrial syrup with water in a volumetric ratio of the industrial syrup/water in the range of 1/15-1/7, at a temperature in the range of 30°C-40°C.

2. The basic medium as claimed in claim 1, in which this basic medium creates a semi-synthetic medium by blending the basic medium with molasses, proteases, minerals, papaya resin or pineapple peels.

3. The basic medium as claimed in claim 1, in which the semi-concentrated or concentrated medium is made from this basic medium by blending the basic medium with agar, gelatin, xanthan gum, or guar gum.

4. EM probiotic preparations are obtained from a process that comprising steps: a) blending the EM-1 preparation into a basic medium obtained in claim 1 at a temperature in the range of 30°C-40°C in a volumetric ratio of the EM-1 preparation/basic medium in the range of 1/12-1/8 to obtain uniform and clear liquid with pale yellow color; b) obtaining an EM probiotic preparation by anaerobic incubating of the liquid obtained from step a) for 3 days.