DK154863B - Method for production of an element-rich preparation - Google Patents

Description

in

DK 154863 B

The present invention relates to a process for preparing an elemental composition for use in biological and physiological systems.

Supply of minerals to humans and animals is desirable as the intensive use of the cultivated areas results in the soil being depleted.

The plants that grow in this soil gradually become poor on trace elements that are absorbed by the plants, but which are not supplied by fertilization. This depletion of the soil can result in the lack of certain minerals in the plants grown in the soil, and thereby also in the food chain, in the animals that eat the plants and in the people who consume the plants directly and partly consumes the animals.

The usual process for preparing mineral preparations containing a large number of mineral salts consists of reacting each mineral, for example in the form of an oxide, with a suitable acid and after isolating the individual mineral salts to mix them to form the final preparation. This approach is time consuming and costly.

Fly ash from power plants using hard coal as a powder in powdered form contains, like the fuel from which it is formed, a very rich elemental spectrum, including a large number of 20 trace elements. Furthermore, the spectrum of elements present in the fuel and, although modified by combustion at approx. 1000 ° C, thus also in the fly ash, necessarily reflect and be correlated with the spectrum of elements in the plants which, hundreds of millions of years before our time, constituted the essential origin of the fossil fuel.

The terms "rich in elements" and rich in element "denote a large number of elements, especially a wide range of trace elements. The term" mineral "denotes a material of inorganic origin and containing a number of elements.

It is known that the supply of elements and especially trace elements to humans and animals can be conveniently made in the form of mixtures of mineral compounds which are readily dissociable in the organism. It is

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2 is particularly well suited to administer compounds in which the anion can be readily converted into the organism, e.g., citrates, tartrates, ascorbates, lactates, acetates, propionates, gluconates, chlorides, sulfates, carbonates or phosphates.

5 It is known that many minerals are absorbed only as chelates or as complexes. Once transformed into chelates or complexes, they can enter the intestinal cells. In the food field, natural chelates and complexes, e.g., chelates and complexes with amino acids and / or proteins and / or polypeptides, play a greater role in mineral metabolism. Absorption depends on the substance with which the minerals are chelated or complexed.

According to an article presented at the International College of Applied Nutrition Los Angeles, California, April 26, 1974,

Rubin [Rubin, M., "Chelation and Iron Metabolism", Proc. AFMA

15 Nutrition Council, November, 1967) found that neither iron citrate nor iron fructose are as effective as certain other iron chelates in transferring iron to the fetus by placental diffusion.

Danish Patent Application No. 578/77 discloses a process for preparing an elemental composition by contacting an elemental material with water, optionally in the presence of an acid such as hydrochloric acid, sulfuric acid, acetic acid or ascorbic acid. By this method a dissolved form of the elements is obtained. The resulting solution may be optionally neutralized and / or optionally concentrated or evaporated, and / or optionally applied or mixed with a carrier and then optionally dried.

British Patent Specification No. 1,460,044 discloses the use of fly ash as an additive to animal feed, possibly together with preparations based on alkali metal and / or alkaline earth metal tartrates - citrates, lactates, glycerophosphates, gluconates and phosphates .

The compositions described in this patent may be administered in any suitable form for oral administration.

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In the methods described in this part of the prior art, various mineral substrates can be used which have the common feature that they contain a large number of different minerals, for example, fly ash from power plants using as hard fuel as coal in powdered form , mineral-containing ores such as copper ore, volcanic ash, finely ground lava, finely ground dolomite or naturally occurring salts, where desired minerals not present in the mineral substrate may be added, for example in the form of oxides or carbonates, prior to treatment with the reagent which gives the easily soluble 10 forms. In other words, the mineral substrate need not directly contain all the desired minerals, since minerals which are found to be absent in analysis may be added prior to processing to produce the readily available forms.

West German Laid-Open No. 2,034,692 describes the preparation of a composition with important trace elements by simply dissolving the trace element-containing material (e.g., CuSO4, FeSO4 .Th2 O or a water-soluble mixture of salts of trace elements) in whey. The whey may be concentrated whey, partially sugared whey, whey which has been subjected to partial protein degradation, and partially sugared whey which has been subjected to proteolytic degradation. The trace elements mentioned are capable of forming complexes which lead to better physiological utilization of the trace elements according to the West German publication. Examples of trace element-containing materials are oxides, hydroxides and salts, which water-soluble salts are preferred. The concentration of the trace element-containing material is 1-8% by weight, calculated on lactose-free dry matter in the whey. In this prior art, elemental compositions are prepared which contain only a limited number of elements or which contain several elements in a less suitable form.

30 West German Publication No. 2,412,602 discloses the use of fly ash for soil improvement or fertilization, but in that publication no description of the fly ash is treated with acid, let alone treatment of the fly ash with in situ formed lactic acid, as is the case according to the method of the Of the present invention.

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U.S. Patent No. 3,421,897 discloses a method of preparing a feed supplement comprising a carrier and whey having an exploitable iron component extracted from the carrier, the method comprising heating the cultivated whey until the whey by evaporation is 5 condensed to such an extent that it contains a predominant proportion of solid component and only a minor proportion of liquid component and an increased lactic acid content, the condensed whey is mixed with an ferrous carrier and the resulting mixture is treated without further drying until a sufficient amount of iron is 10 extracted from the carrier to form a feed supplement which has a significant content of utilizable iron extracted from that carrier. The resulting free-flowing particulate product contains up to 0.6% soluble ferric iron.

According to the present invention, it is now possible to prepare an elemental supplementary composition containing several elements in biologically assimilable form for use in biological and physiological systems in a very simple manner from very inexpensive waste products.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for preparing an elemental composition which can be used as a supplement in biological and physiological systems, characterized in that lactic acid-producing bacteria are grown in an aqueous fermentation liquid containing fly ash from power plants, whereby a majority of the components of the fly ash are transformed into a biologically assimilable form which may be chelates, complexes and lactic acid salts, and the resulting liquid containing these chelates, complexes or lactic acid salts after cultivation is isolated.

The elemental composition of the invention can be obtained in liquid form by separating the aqueous solution from the solids. The aqueous solution can be used per se, or it can be evaporated to give a concentrated liquid or dry product. The separation step can be carried out by filtration, centrifugation or decanting.

The reagents capable of dissolving a majority of the components of the fly ash into a biologically assimilable form are present in the fermentation medium.

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5 in which the lactic acid producing bacterium is grown.

It is not known for sure how exactly the dissolution of the fly ash components takes place in this case; however, the mechanism is likely to comprise either an initial solution of the fly ash components caused by the lactic acid formed in situ, with subsequent or simultaneous complexation or chelation with other components of the fermentation liquid which may be amino acid, peptides or proteins, or the mechanism may include an initial solution of the fly ash components in chelated or complexed form caused by components present in the fermentation liquid, without the lactic acid playing any major role in the solution of the fly ash components.

The fermentation can be carried out with a lactic acid producing bacterium of the family Streptococcaceae or Lactobacillaceae, for example of the genus Streptococcus, Leuconostoc, Pediococcus or Lactobacillus. A preferred genus is Lactobacillus, a preferred species is Lactobacillus plantarum, and a particularly preferred strain is Lactobacillus plantum CH-1. It is preferred to carry out the fermentation at an optimum temperature, preferably a temperature of approx. 30 ° C or slightly above, 20 which is the optimal fermentation temperature for this bacterium. The fermentation takes place in the presence of usual sources of nitrogen, for example normal sources of vegetable origin or of fish or animal protein origin, preferably milk protein.

For the cultivation of the lactic acid producing bacterium, any substrate comprising fermentable sugar, e.g. whey or molasses, may be used. The sugar substrate solution may contain from ca. 1/2% sugar solids for over approx. 30% of sugar solids, the upper limit of which is determined by the conditions under which the microorganism can grow. To make the polysaccharides more easily digestible to the bacterium, a di- or polysaccharide-degrading enzyme may be added. In addition to fly ash and the sugary substrate, organic nitrogen compounds and nutrients needed for lactic acid fermentation can be added.

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Thus, it is clear that one embodiment of the present invention comprises the use of lactic acid which is formed in situ during the reaction by fermentation of a sugary substrate with a lactic acid producing bacterium as a reagent to form a dissolved form 5 of the elements in the fly ash. In such a lactic acid fermentation in the presence of fly ash, the lactic acid, as it is formed, is reacted with the minerals, and when the lactic acid is thus removed from the reaction mixture it will not have any inhibitory effect on the further fermentation.

It is advantageous to use whey in the fermentation liquid in which the lactic acid producing bacterium is grown, since whey is a waste product which is produced in large quantities in the dairy industry. This waste product causes serious problems for the dairy industry as there are many restrictions on its disposal for the environment. Therefore, many attempts have been made to find a use for this product. Until now, the most frequent use has been as a feed additive for piglets.

In a preferred aspect of the invention, wool retentate is used as the sugary substrate. Whey retentate is the product formed when whey has been subjected to ultrafiltration. In this process, the whey is concentrated to a sugar content of up to 18%. The whey concentrate may be used as such, but it is preferred to add lactase to increase the conversion rate of lactose to mono-saccharides (glucose and galactose) and nutrients, eg yeast extract.

The whey or whey retentate is advantageously used in dilute form since the sugar concentration and thus the osmotic pressure is essential for the growth of the bacteria.

In a preferred embodiment of the invention, the fermentation liquid comprises whey retention diluted with water in a ratio of 1: 4-1: 9, 30 proteins, other suitable nutritional factors and fly ash. It is believed that the successive addition of whey or whey retentate so that a constant sugar content of approx. 1-5%, e.g. 2-3%, maintained in the fermentation liquid will lead to maximum conversion to lactic acid and thus to a maximum dissolution of the components of the fly ash.

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In a preferred aspect of the present invention, a slurry containing fly ash and optionally additional desired minerals, preferably in the form of oxides or carbonates, e.g. iron, manganese, zinc, cuprous and cobalt oxides or carbonates, is prepared in a rolling retentate solution containing from approx. 3% sugar to over 25% sugar, the solution being seeded with a lactic acid bacterium, for example Lactobacillus plantarum or Lactobacillus delbrueckii (previously referred to as Thermobacterium cereale) and the fermentation is carried out at ca.

30 ° C with stirring.

By this fraction, the lactic acid is formed simultaneously with its use to form lactates, and thus, as in the prior art, it is not necessary to produce and isolate the lactic acid and the desired mineral ions separately and then react with them.

In a further aspect, the fly ash is treated with a reagent which forms easily soluble mineral chelates with the minerals in the fly ash to form chelated mineral mixtures, chelates being formed with raw fly ash minerals and not, as in the prior art, with minerals separately. In this way, it is possible to prepare chelated minerals with the easily dissociable mineral compounds prepared according to the present invention.

The chelation can take place between the ions released in the solution and the amino acids derived from the proteins. It is believed that further chelation or complexation can be achieved by adding amino acids, polypeptides and / or proteins to the liquid culture fluid. It is preferred to add additional protein components to the whey retentate, and a preferred protein source is pasteurized, dried skim milk (e.g., UHT treated 9% NFDM (non-fat dry milk)).

Thus, a particularly preferred embodiment of the invention is a process for preparing an elemental composition in complexed or chelated form wherein the fly ash is treated with a liquid comprising peat retentate, an additional protein source, e.g., milk and yeast extract, and a lactic acid forming bacterium. .

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In such a combined culture fluid there are various buffer systems, namely proteins and salts. In the presence of fly ash, part of the lactic acid, as it forms, will react with the minerals, and when the lactic acid is thus partially removed from the reaction mixture, it will have less inhibitory effect on the further fermentation.

When the cultivation is complete, the slurry is filtered, decanted or centrifuged and the liquid containing the minerals, optionally in complexed or chelated form, is isolated. This liquid can be used 10 per se or it can be absorbed on a carrier, possibly after evaporation, or it can be evaporated and, for example, spray-dried or lyophilized and used as a powder.

Preferred agents which form the complexes or chelates are proteins of milk origin.

The agents which form soluble complexes or chelates can be used per se or in aqueous solution. The ratio of the fly ash in the mixture to the complex or chelating agent (calculated as complex or chelating solid) is between 1: 1 and 100: 1.

Prior to the growth of the bacteria, the pH of the mixture of fly ash and aqueous substrate will be alkaline, with the pH depending on the amount of fly ash. Since lactic acid-producing bacteria only grow slowly at alkaline pHs, it is believed that better reaction time results are obtained if an acid such as lactic acid is added in an amount sufficient to change the pH to about 25%. 7th

In a preferred embodiment, fly ash is successively added to the fermentation liquid at the same rate as the acid production caused by the bacterial growth. In this way, the alkaline reaction of the fly ash can be compensated and by keeping the pH of approx. 6-7 30, maximum bacterial growth can be achieved. Such successive addition of fly ash can, for example, be regulated by means of a pH state.

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Furthermore, better results are obtained with respect to conversion of the fly ash elements into an assimilable form if the reaction period is extended. It is believed that a long reaction time, eg one used in the development of lactic acid in situ, will result in increased formation of the elements in complexed or chelated form.

After the treatment of fly ash with the fermentation liquid in which the lactic acid producing bacterium has been grown, or with a lactic acid solution, a solution of the desired minerals, optionally in complexed or chelated form, is formed and this solution is optionally neutralized by the addition of a base and the resulting liquid can be used per se or mixed with a carrier, for example, green fodder or green flour, and the mixture can be dried.

The carrier used may be a suitable feed, a feed additive or a pharmaceutically tolerable carrier. For administration to animals, it is preferred to apply the obtained, optionally neutralized, solution to a carrier in the form of a feed, which is usually a component of the feed but used in smaller quantities, thereby allowing a greater concentration of the minerals to be applied. on the carrier and thus reduce the dosage uncertainty.

Suitable carriers may be, for example, wheat bran or green flour. Green meal is dried and cut green fodder, which is dried vegetable crops such as clover, alfalfa or grass. Green meal can be used directly as a feed or can be preserved. The drying of the green flour is usually carried out in a drying drum, in which hot combustion gas from an oil burner is used as a drying medium.

It is preferable to use green flour as it is cheaper than wheat bran, which is a common carrier for mineral additives. Furthermore, green flour has a constant composition and contains per se a wide 30 mineral spectrum.

When the carrier is green flour, it will often be unnecessary to dry the mixture if it is granulated, as the heat generated thereby is sufficient to evaporate the water.

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When green fodder, which typically contains 20-30% of dry matter, is used as a carrier, the amount of mineral solution added can be reduced accordingly and the mineral solution is preferably applied immediately before drying.

It is particularly advantageous to neutralize with such a base in which the cation forms a desired portion of the mineral content. Thus, for example, potassium, sodium or calcium can be added in this way in the form of carbonates.

In one particular aspect, the acidic mineral-containing solution obtained is neutralized by being added to a partially neutralized straw mass treated with a base to improve the feed value of the straw, and the resulting mass is dried to give an improved feed product.

The neutralized solution is applied to the carrier, preferably greenish, in an amount of 30-1 wt% mineral solution of 70-99 wt% carrier. In this way, a product is obtained which is easy to handle and easy to dispense.

When the fly ash has been treated by the process of the present invention and the liquid rich element composition is applied to a carrier and then dried, it has been attached to the carrier.

The solution obtained by the process of the present invention can be concentrated, optionally after neutralization. The concentration can be carried out, for example, by hyperfiltration or mainly by partial evaporation. Concentration can be carried out by simple evaporation, by evaporation under reduced pressure and possibly by spray drying.

A won concentrate or won evaporation residue can also be used as an additive for animal feed or for drinking water. Concentration or evaporation results in element-rich preparations which are easier to transport if the compositions are to be mixed with an 11

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carrier at a place of use located at a geographical distance from the place where the mineral preparation is produced.

When the optionally neutralized solution has been applied to a carrier, the resulting preparation is dried, if not used directly, and then it is advantageous to apply the solution to a feed or feed additive which must nevertheless be dried during preparation, and thus, of course, it is most advantageous to apply the prepared solution to the carrier by spraying before drying.

The elemental composition can be used in liquid form as a medium to be sprayed onto the green fodder in order to ensilage it, possibly together with a lactic acid producing bacterium, for example Lactobacillus plantarum CH-1. This bacterium is usually marketed (by Chr. Hansen's Laboratory A / S, Copenhagen, Denmark) as a 'freeze-dried product or frozen product which can be dehydrated and / or mixed with water before use. Instead of being mixed with water, the bacterial product can be mixed with the liquid element-rich preparation prior to use. A common inoculum is 1 x 10 -1 x 10 bacteria, and this amount is intended to be mixed with 10 liters of water, some of which is the elemental preparation, after which it is sprayed on 1 ton of green fodder for silage.

Tests indicate that a liquid elemental preparation prepared under the influence of lactic acid formed in situ by fermentation has an even more positive influence in bacterial processes than a liquid elemental preparation produced under the influence of lactic acid (technical purity). , although the pH values are very similar.

There is evidence that the liquid element-rich preparation may replace or enhance the salts usually added to fermentation substrates.

It is believed that the elemental composition in liquid form can be used as an additive in other biological processes, for example as an additive to bacterial, yeast and mold cultures used in food.

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12 commodity industry. Examples include use with lactic acid cultures in various foods for preservation or as a stimulating factor for specific cultivations.

It is further believed that the liquid-rich formulation can be used per se as a liquid substrate in the preparation of extracts to be used in the preparation of other culture media. In the preparation of a complete medium, the liquid-rich element composition can be used as a solvent for the nutrients. This solution is then dried to form a powder which is rehydrated before use.

When the elemental composition is evaporated to form a dry product, this product can be used as an additive in biological and physiological processes, for example in microbial processes, and for use in humans or animals.

Tests have shown that a difficulty in swine rearing, e.g., tail biting, has been drastically reduced when the pigs have been given as an additive to the feed an amount of the product produced according to the present invention. The product may be given as a liquid containing the optionally chelated product sprayed onto the green fodder in an amount of 1-20% liquid of 99-80% dried green fodder. This additive can be given to pigs in an amount of 0.1-2.5 g / feed unit (1 feed unit corresponds to the feed value of 1 kg of barley).

For example, when the prepared elemental composition is to be administered as an additive to the feed, it is admixed with the feed in amounts corresponding to 0.005-0.1, preferably 0.01-0.06, weight percent mineral substrate in the feed ready for consumption by small amounts. animals, eg poultry. In the case of medium-sized animals, e.g., pigs, the mineral preparation is preferably incorporated into the feed in an amount equal to 0.0015-0.025, preferably 0.0025-0.015, by weight mineral substrate in the feed ready for consumption, which feed is typically given in an amount of approx. 2 kg / day / animal with a body weight of approx. 50 kg. For larger animals, eg cows and horses, the mineral preparation is preferably incorporated into the feed in a 13

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amount corresponding to 0.003-0.06, in particular 0.006-0.04, weight percent mineral substrate in the feed, which is typically given in an amount of approx. 14 kg / day / animal with a body weight of approx. 600 kg.

Conveniently, when the mineral preparation is to be used in preparations administered to humans, the amount administered is of a similar order of magnitude to the body weight. For this use, it is preferred that the composition is administered as a tablet or a powdered mineral additive composition of the same type as the mineral preparation described in British Patent Specification No. 1,298,299, i.e.

10 is a composition containing easily soluble salts of minerals such as sodium, potassium, calcium and magnesium which have been enriched by the addition of a trace element rich composition prepared according to the invention, for example by using the elemental solution prepared by the preparation of the tablet preparation the process of the present invention as a granulating liquid. Other suitable forms of administration for humans are a mineral water-like beverage containing the minerals which are made soluble.

The present invention is further illustrated by the following Examples: EXAMPLE 1

Fly ash is used directly as an additive in a biological system. The amount added is shown in Table I, in which the fly ash has obtained fraction # 1 per se.

EXAMPLE 2 A part by weight of fly ash is added to two parts by weight of tap water with stirring. The slurry is left under stirring for 24 hours at 30 ° C. Then part of the mixture is centrifuged (8000 g for 10 minutes) and the supernatant is decanted.

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The washer is subjected to sterile filtration (0.45 u) to give Fraction 2.1.

The sediment from the centrifugation is fraction 2.2.

The amounts added to each of these fractions are shown in Table I.

EXAMPLE 3

Example 2 is repeated, however, maintaining the pH at 4.8 in a pH state by adding 80% lactic acid.

Liquid fraction 3.1 is obtained in the same way as fraction 2.1.

Sediment fraction 3.2 is obtained in the same way as fraction 2.2.

Part of the remaining slurry is adjusted to pH 6.5 by the addition of 25% NH 2 OH and the liquid is allowed to stand for 10 minutes, after which centrifugation and decanting are performed as described in Example 2.

Liquid fraction 3.3 is obtained in the same way as fraction 2.1.

Sediment fraction 3.4 is obtained in the same way as fraction 2.2.

The amounts added to each of the fractions are shown in Table I.

EXAMPLE 4

To 8 liters of spring retentate (about 22% dry matter) are added 5000 lactase units, g 40 ml of yeast extract (about 12 g) 0.5 kg fly ash and 8 x 10 bacteria 20 (Lactobacillus plantarum CH-1). The mixture is incubated with stirring under inert gas at 30 ° C until a pH of 5.0 is reached.

Part of the mixture is centrifuged and decanted as described in Example 2.

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Liquid fraction 4.1 is obtained in the same way as fraction 2.1.

Sediment fraction 4.2 is obtained in the same way as fraction 2.2.

The pH of the remaining portion of the mixture is adjusted to 6.5 by the addition of 25% NH 2 OH. The mixture is centrifuged and decanted as described in Example 2.

Liquid fraction 4.3 is obtained in the same way as fraction 2.1.

Sediment fraction 4.4 is obtained in the same way as fraction 2.2.

The amounts added to each of the fractions are shown in Table I.

EXAMPLE 5 Example 4 is repeated except that 50% of the retentate is replaced with 9% UHT-treated skimmed milk (NFDM; non-fat dry milk) and the fermentation is continued until a pH of 4 is reached. 5th

Liquid fraction 5.1 is obtained in the same way as fraction 4.1.

Sediment fraction 5.2 is obtained in the same way as fraction 4.2.

Liquid fraction 5.3 is obtained in the same way as fraction 4.3.

Sediment fraction 5.4 is obtained in the same way as fraction 4.4.

EXAMPLE 6

Example 5 is repeated except that fly ash is not used and acidification is done by adding 80% lactic acid until a pH of 4.8 is reached.

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Liquid fraction 6.1 is obtained in the same way as fraction 5.1.

Sediment fraction 6.2 is obtained in the same way as fraction 5.2.

The amounts added to each of the fractions are shown in Table I.

It should be noted that in Examples 1-3, the ratio of fly ash to liquid 5 is 1: 2 (by weight), while the ratio of fly ash to liquid in Examples 4 and 5 is 1:16 (by weight).

TABLE I

Amount of fly ash, filtrate or sediment prepared Amount of cultured according to Examples 1-6 Sample No. Fraction no

Nothing 1 100 ml

Nothing 2 100 ml 0.05 g 3 1 100 ml 0.25 g 4 1 100 ml 15 0.5 ml 5 2.1 100 ml 2.5 ml 6 2.1 100 ml 0.05 g 7 2.2 100 ml 0.25 g 8 2.2 100 ml 0.5 ml 9 3.1 100 ml 20 2.5 ml 10 3.1 100 ml 0.05 g 11 3.2 100 ml 0.25 g 12 3.2 100 ml 0.5 ml 13 3.3 100 ml 2.5 ml 14 3.3 100 ml 25 0 , 05 g 15 3.4 100 ml 0.25 g 16 3.4 100 ml 0.5 ml 17 4.1 100 ml 2.5 ml 18 4.1 100 ml 0.05 g 19 4.2 100 ml 30 0.25 g 20 4.2 100 ml 0.5 ml 21 4.3 100 ml

Table I continued 17

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2.5 ml 22 4.3 100 ml 0.05 g 23 4.4 100 ml 0.25 g 24 4.4 100 ml 5 0.5 ml 25 5.1 100 ml 2.5 ml 26 5.1 100 ml 0.05 g 27 5.2 100 ml 0.25 g 28 5.2 100 ml 0.5 ml 29 5.3 100 ml 10 2.5 ml 30 5.3 100 ml 0.05 g 31 5.4 100 ml 0.25 g 32 5.4 100 ml •. 0.5 ml 33 6.1 100 ml 2.5 ml 34 6.1 100 ml 0.05 g 35 6.2 100 ml 0.25 g 36 6.2 100 ml

The samples prepared according to Examples 1-6 are subjected to culture tests with three bacteria of the genus Lactobacillus in four culture fluids, namely culture fluid X, culture fluid Y, culture fluid Z and 20 culture fluid Q. The composition of the culture fluids is as follows:

Culture liquid X g / liter distilled water

Universal Peptone ("Bacto Peptone") 10.0 Meat Extract ("Lab Lemco") 5.0 Yeast Extract ("Difco") 5.0 D (+) Glucose 20.0

Polyoxyethylene sorbitan monooleate ("Tween" ® 80) 1.0 18

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Culturing fluid Y

Culture liquid Y consists of culture liquid X, to which inorganic salts are added in the following quantities: g / liter 5 Diammonium hydrogen citrate 2.0

Sodium acetate 5.0

Cultivating fluid Z

Z Culture liquid Z consists of culture liquid X to which inorganic salts have been added in the following quantities: 10 g / liter

Dicalcium hydrogen phosphate 2.0

Magnesium sulfate 0.1

Manganese sulphate 0.05

Cultivating fluid 15 Culturing fluid Q

Culture liquid Q consists of culture liquid X, to which are added both the organic salts mentioned under culture liquid Y and the inorganic salts mentioned under culture liquid Z.

The culture fluids are heated in an autoclave to 121 ° C for 10 minutes and cooled. Then, the culture fluids are poured into 36 test tubes, each containing 100 ml, and the fly ash fractions listed in Table I are added and mixed. 10 ml of culture liquid is poured into test tubes which are heated to 105 ° C in an autoclave for 10 minutes.

After cooling, each of the test tubes is seeded with approx. 3 x 10 bacteria except sample # 1 in each test series. The bacteria used are Lactobacillus plantarum CH-1, Lactobacillus acidophilus CH-2 and Lactobacillus acidophilus CH-5, respectively. The tubes are incubated at 30 ° C and 37 ° C, respectively, until there is 30 visible growth in one or more tubes in each test series. Then all the tubes are cooled to 5 ° C and pH measurement is performed.

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As a control, a similar number of test tubes are kept at 37 ° C without inoculation until all the inoculated tubes are incubated and cooled. The bacteria-free test tubes are used as a reference when evaluating bacterial growth.

5 The control values are shown in Table II. The pH values for Lactobacillus planar tear CH-1, Lactobacillus acidophilus CH-2 and Lactobacillus acidophilus CH-5, respectively, are shown in Tables III, IV and V, respectively. Tables III, IV and V also show the decrease in pH, which is an expression for the growth of bacteria.

Table II

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pH-control values.

5

Cultivation Cultivation Cultivation Cultivation Test No. liquid X liquid Y liquid Z liquid Q

1 6.22 5.69 6.40 6.08 10 2 6.23 5.69 6.40 6.08 3 6.23 5.73 6.44 6.10 4 6.60 5.87 6.58 6.25 5 6.22 5.73 6.43 6.04 6 6.30 5.55 6.50 6.10 15 7 6.25 5.71 6.45 6.06 8 6.44 5.81 6.49 6.07 9 5.97 5.64 6.35 5.99 10 5.40 5.50 5.62 5.72 11 6.10 5.68 6.40 6.01 20 12 6.04 5.68 6.37 6.02 13 5.97 5.65 6.30 5.98 24 5.56 5.46 5.78 5.75 15 6.14 5.69 6.43 6.02 16 6 , 04 5.69 6.40 6.01 25 17 6.12 5.68 6.42 5.99 18 6.08 5.68 6.34 5.98 19 6.10 5.68 6.40 6, 02 20 6.13 5.68 6.40 6.03 21 6.16 5.68 6.42 6.03 30 22 6.28 5.76 6.45 6.06 23 6.13 5.70 6, 42 6.08 24 6.16 5.72 6.42 6.02 25 6.08 5.67 6.40 6.01 26 5.97 5.63 6.27 5.94 35 27 6.12 5, 69 6.40 6.01 28 5.94 5.66 6.35 5.93 29 6.12 5.68 6.43 6.01 21

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30 6.15 5.70 6.38 6.02 31 6.08 5.70 6.40 6.03 32 6.12 5.67 6.37 6.00 33 6.00 5.67 6.39 5 , 98 5 34 5.68 5.58 6.26 5.90 35 6.03 5.69 6.43 6.00 36 6.03 5.68 6.39 5.98

When assessing the figures in Tables III, IV and V, note the following: 10 Sample # 1 is the check. Sample # 2 is a grafted control used as reference for evaluation of Sample Nos. 3-16 and Sample Nos.

33-36 are references for assessing Sample No. 17-32.

TABLE III

Lactobacillus plan tear chamber CH-1 15 Cultivation Cultivation Cultivation Cultivation

liquid X liquid Y liquid Z liquid Q

Sample Decline Decline Decline Decline

No. pH in pH pH in pH pH in pH pH in pH

1 6.10 0.12 5.70 0.01 6.50 -0.10 6.00 0.08 20 2 5.50 0.73 5.60 0.09 6.03 0.37 5.71 0 , 37 3 4.90 1.33 5.20 0.53 5.47 0.03 5.32 0.78 4 4.65 1.95 4.90 0.97 4.49 2.09 4.84 1 , 31 5 5.54 0.68 5.50 0.23 5.97 0.46 5.34 0.70 6 5.63 0.67 5.44 0.11 5.31 1.19 5.29 0 , 81 25 7 5.00 1.25 5.27 0.43 5.68 0.23 5.30 0.76 8 4.75 1.69 5.05 0.76 5.19 1.30 5.07 1.00 9 4.35 1.62 4.53 1.11 4.79 1.56 4.80 1.19 10 4.09 1.31 4.48 1.02 4.32 1.30 4.48 1.24 11 5.00 1.10 5.35 0.33 5.86 0.54 4.96 1.05 30 12 4.73 1.31 5.04 0.64 5.36 1.01 5, 03 0.99

Table III continued 22

DK 154863 B

13 4.37 '1.60 4.72 0.93 4.67 1.33 4.83 1.15 14 4.12 1.44 4.53 0.93 4.31 1.47 4.51 1, 24 15 5.00 1.14 5.43 0.26 5.90 0.53 5.38 0.64 16 4.77 1.27 5.07 0.62 5.19 1.21 5.16 0, 85 5 17 5.25 0.87 5.50 0.18 5.65 0.77 5.31 0.68 18 4.87 1.21 5.20 0.48 5.10 1.24 5.07 0 , 91 19 5.20 0.90 5.36 0.32 5.77 0.63 5.30 0.72 20 4.98 1.15 5.15 0.53 5.48 0.92 5.23 0 , 80 21 5.40 0.76 5.55 0.13 5.57 0.85 5.50 0.53 10 22 5.25 1.03 5.42 0.34 4.90 1.55 5.30 0.76 23 5.05 1.08 5.43 0.27 5.87 0.55 5.44 0.64 24 4.98 1.18 5.05 0.67 5.22 1.20 5.15 0.87 25 5.50 0.58 5.55 0.12 5.99 0.41 5.52 0.49 26 5.28 0.69 5.40 0.23 4.97 1.30 5.26 0.68 15 27 5.35 0.77 5.42 0.27 5.97 0.43 5.38 0.63 28 5.10 0.84 5.20 0.46 5.70 0.65 5, 0.68 29 5.55 0.57 5.58 0.10 5.72 0.71 5.50 0.51 30 5.33 0.82 5.43 0.27 4.73 1.65 5, 80 0.22 31 5.30 0.78 5.48 0.22 5.74 0.66 5.42 0.61 20 32 5.10 1.02 5.20 0.47 5.51 0.86 5 48 0.52 33 5.53 0.47 5.55 0.22 6.00 0.39 5.38 0.60 34 5.25 0.43 5.45 0.13 5.81 0.45 5 , 28 0.62 35 5.62 0.41 5.62 0.07 6.14 0.29 5.40 0.60 36 5.55 0.48 5.55 0.13 6.11 0.28 5.45 0.53 25 30

Table IV

23

DK 154863 B

Lactobacillus acidophilus CH-2 5

Cultivation Cultivation Cultivation Cultivation

liquid X liquid Y liquid Z liquid Q

Sample Decline Decline Decline Decline

10 No. pH in pH pH in pH pH in pH pH in pH

1 6.16 0.06 5.67 0.02 6.40 0.00 6.00 0.08 2 5.67 0.56 5.65 0.04 5.62 0.78 5.63 0.45 3 5.42 0.81 5.52 0.21 5.55 0.89 5.46 0.64 15 4 6.39 0.21 5.45 0.42 5.87 0.71 5.33 0, 92 5 5.50 0.72 5.63 0.10 5.55 0.88 5.55 0.49 6 5.52 0.88 5.67 -0.12 5.75 0.75 5.55 0 , 55 7 5.42 0.83 5.57 0.14 5.50 0.95 5.52 0.54 8 5.60 0.84 5.50 0.31 5.62 0.87 5.43 0 64 20 9 5.20 0.77 5.57 0.07 5.60 0.75 5.58 0.41 ... 10 5.19 0.21 5.40 0.10 5.37 0.25 5.58 0.14 11 5.55 0.55 5.60 0.08 5.50 0.90 5.57 0.44 12 5.33 0.71 5.55 0.13 5.40 0.97 5.54 0.48 13 5.15 0.82 5.53 0.12 5.45 0.85 5.49 0.49 25 14 5.17 0.39 5.15 0.31 5.33 0, 45 5.60 0.15 15 5.53 0.61 5.62 0.07 5.60 0.77 5.58 0.44 16 5.19 0.85 5.55 0.14 5.34 1, 06 5.53 0.48 17 5.41 0.71 5.49 0.19 5.30 1.12 5.42 0.57 18 5.09 0.99 4.80 0.88 5.04 1, 30 5.16 0.82 30 19 5.43 0.67 5.56 0.12 5.40 1.00 5.47 0.55 20 5.15 0.98 5.52 0.16 5.44 0 , 96 5.46 0.57 21 5.45 0.71 5.49 0.19 5.78 0.64 5.48 0.55 22 5.10 1.18 4.93 0.83 5.15 1 , 5.27 0.79 23 5.27 0.86 5.60 0.10 5.46 0.96 5.50 0.58 35 24 5.13 1.03 5.50 0.22 5.45 0.97 5.47 0.55 25 5.32 0.76 5.52 0.15 5.36 0.96 5.42 0.59 26 5.14 0.83 5.08 0.55 5.07 1.20 5.42 0.52

Table IV continued

DK 154863 B

24 27 5.44 0.68 5.58 0.11 5.30 1.10 5.58 0.43 28 5.17 0.77 5.55 0.11 5.17 1.18 5.55 0, 38 29 5.30 0.82 5.53 0.15 5.50 0.93 5.51 0.50 30 4.95 1.20 5.17 0.53 4.99 1.39 5.40 0, 62 5 31 5.30 0.78 5.63 0.07 5.43 0.97 5.50 0.53 32 5.10 1.02 5.51 0.16 5.05 1.32 5.48 0 , 52 33 5.31 0.69 5.58 0.09 5.42 0.97 5.48 0.50 34 5.20 0.48 5.40 0.18 5.17 1.09 5.45 0 , 45 35 5.46 0.57 5.64 0.05 5.54 0.89 5.53 0.47 10 36 5.28 0.75 5.60 0.08 5.40 0.99 5.47 0.51 TABLE V 15

Lactobacillus acidophilus CH-5

Cultivation Cultivation Cultivation Cultivation

liquid X liquid Y liquid Z liquid Q

20 Sample Decline Decline Decline Decline

No. pH in pH pH in pH pH in pH pH in pH

1 6.18 0.04 5.68 0.01 6.43 -0.03 6.01 0.07 2 4.79 1.46 5.60 0.09 4.47 1.93 4.95 1, 13 25 3 4.47 1.76 5.32 0.41 4.38 2.06 4.94 1.16 4 4.52 2.08 5.11 0.76 4.55 2.03 5.05 1 , 20 5 4.95 1.27 5.57 0.16 4.45 1.98 5.03 1.01 6 4.75 1.55 5.54 0.01 4.43 2.07 4.95 1 , 15 7 4.57 1.68 5.40 0.31 4.45 2.00 4.95 1.11 30 8 4.40 2.04 5.79 0.02 4.35 2.14 4.90 1.17 9 4.40 1.57 5.05 0.61 4.50 1.85 4.83 1.16 10 4.55 0.85 4.80 0.70 4.58 1.04 4.78 0.94 11 4.58 1.52 5.50 0.18 4.45 1.95 4.97 1.04 12 4.45 1.59 5.30 0.38 4.53 1.84 4.80 1.22 35 13 4.52 1.45 4.95 0.70 4.55 1.85 4.85 1.13 14 4.57 0.99 4.75 0.71 4.57 1.21 4, 76 0.99 15 4.50 1.64 5.50 0.19 4.44 1.99 4.97 1.05

Table V continued

DK 154863 B

25 16 4.28 1.76 5.28 0.41 4.36 2.04 4.90 1.11 17 4.53 1.59 5.40 0.28 4.44 1.98 4.96 1, 03 18 4.38 1.70 4.86 0.82 4.35 1.99 4.80 1.18 19 4.46 1.64 5.40 0.28 4.35 2.05 4.90 1, 12 5 20 4.40 1.73 5.05 0.63 4.32 2.08 4.84 1.19 21 4.50 1.66 5.45 0.23 4.45 1.97 5.04 0 , 99 22 4.40 1.88 4.97 0.79 4.39 2.06 4.92 1.14 23 4.46 1.67 5.49 0.21 4.40 2.02 5.00 1 , 08 24 4.32 1.84 4.95 1.07 4.35 2.07 4.83 1.19 10 25 4.94 1.14 5.42 0.25 4.40 2.00 4.95 1.06 26 4.45 1.52 4.91 0.72 4.29 1.98 4.80 1.14 27 4.75 1.37 5.57 0.12 4.33 2.07 5.00 1.01 28 4.48 1.46 5.40 0.26 4.38 1.97 4.78 1.15 29 4.50 1.62 5.45 0.23 4.48 1.95 4.93 1.08 15 4.31 1.84 4.97 0.73 4.38 2.00 4.80 1.22 31 4.46 1.62 5.57 0.13 4.41 1.99 4, 92 1.11 32 4.42 1.70 5.35 0.32 4.32 2.05 4.88 1.12 33 4.48 1.52 5.53 0.14 4.37 2.02 4, 92 1.06 34 4.35 1.33 5.29 0.29 4.35 1.91 4.78 1.12 20 35 4.58 1.45 5.58 0.11 4.52 1.91 4 , 92 1.08 36 4.60 1.43 5.55 0.13 4.45 1.94 4.94 1.04

Although the initial pH values (reference values) are not identical, it is clear from the columns when an effect is clear, questionable or absent. Tables III, IV and V can be summarized in Table VI:

Table VI

26

DK 1 54863 B

Cultivation Cultivation Cultivation Cultivation Wash X liquid Y liquid Z liquid Q Sample Sample Sample Sample 5 3-16 17-32 3-16 17-32 3-16 17-32 3-16 17-32 L.p. CH-1 + + + + + + + +/- L.a. CH-2 +/- + +/- +? + /? ? + L.A. CH-5 +/- + + / - + - - 10 In the above table, "+" means positive effect, means a variable, but mostly positive effect, means absence of 'confirmed positive effect, and'? ' means variable effect.

Thus, the effect of adding the elemental preparation is seen to be positive for all the fractions of Lactobacillus plantarum CH-1.

With respect to Lactobacillus acidophilus CH-2, the effect of tests 1-3 is variable and questionable, while the effect of tests 4-5 is essentially positive.

With respect to Lactobacillus acidophilus CH-5, the effect of adding the fractions prepared according to Examples 1-3 varies in the 20 tests performed with culture liquid X and Y, while the effect of adding the fractions prepared according to Examples 4-5 is positive. in culture fluids X and Y. The effect of adding the fractions prepared according to Examples 1-3 and 4-5 is not positive.

These results show that the addition of fly ash is advantageous in 25 liquids to which no inorganic salts have been added.

Table VI shows that addition of the fractions prepared according to Examples 4 and 5 (samples 17-32) appears to be equally good or 27

DK 154863 B

better than tapping the fractions prepared according to Examples 1-3 in light of the fact that the amount of fly ash in the starting material is only 1: 8 in Examples 4 and 5 compared to Examples 2 and 3.

5 It is evident from Table III-V that the addition of each fraction at the highest level results in the largest pH drop and the lowest pH value.

Therefore, it is believed that if the ratio of fly ash to liquid in Examples 2-5 was identical, an even better result could be obtained, expressed in pH decrease in the resulting liquid in Sample 17-32 (similar to Examples 4 and 5). . This assumption is supported by the fact that ash determinations in liquids prepared as described in Example 5 (fraction 5.1), assuming the fly ash volumes are 400 g and 1100 g respectively, show 1.92% and 2.49%.

EXAMPLE 7

Similar tests have been performed with Lactobacillus plantarum CH-1 without fly ash, with the same bacterium with added fly ash and with Lactobacillus delbrueckii with added fly ash.

The substrate consists of: 1500 ml of 9% NFDM ("non-fat dry milk"), 1500 ml of woolen retentate, 1875 lactase units and 15 ml of yeast extract, and, in cases where fly ash is added, 150 g fly ash, and in these substrates, the pH is adjusted to below 7 by the addition of technical lactic acid after 30 minutes. The pH values are measured 1 hour, 13 hours and 24 hours after the start of the test, and the titratable acidity (titer) (ml 0.1N NAOH / 100 ml sample) is determined after 1 hour, 13 hours and 24 hours.

The results are summarized in Table VII below:

Table VII

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DK 154863 B

Lactobacillus Lactobacillus plantarum plantarum Lactobacillus CH-1 without CH-1 with delbrueckii 5 fly ash fly ash with fly ash

titer pH titer pH titer pH

1 hour 37 6.21 19 6.80 14 7.05 13 hours 44 5.92 46 5.83 44 5.90 10 24 hours 77 4.78 113 4.45 87 4.95

Table VII shows that the addition of fly ash increases acid production, especially when using Lactobacillus plantarum CH-1, and that Lactobacillus delbrueckii, which is known to grow very slowly, even in milk enriched with yeast extract, provides a significant amount of acid when It is grown in the manner described above.

Example 8 10 liters of molasses solution containing 12% sugar solids, nutrients and organic nitrogen compounds are seeded with a lactic acid bacterial culture of Thermobacterium cereale. At the same time 0.9 kg fly-ash is added.

The fermentation is carried out with stirring at 52 ° C, i.e. 5-6 ° C above the optimum temperature of the bacteria, to avoid the development of, for example, butyric acid bacterial cultures which could convert the sugar in the molasses to butyric acid. The lactic acid formed reacts with the minerals and as the acid production increases, the pH is adjusted to approx. 5 by the addition of potassium, sodium and calcium carbonate. At the end of the reaction, liquid is decanted and magnesium phosphate is added to the mineral lactate-containing liquid which is mixed with green flour in a ratio of 10% liquid to 90% green flour.

29

DK 154863 B

By this process a mineral product is obtained in which all the minerals can easily react.

EXAMPLE 9

An elemental preparation is prepared by adding 80 g of fly ash 5 to 1 liter of spring retentate (22% dry matter) seeded with Lactobacillus delbrueckii. The mixture is left to stir for 22 hours at 48 ° C. The slurry is filtered through a filter cloth and the liquid is poured over green flour in an amount of 100 ml of liquid on 1 kg of green flour.

This elemental green meal is mixed with the normal feed for pigs in an amount of 1.5 g / feed unit (~ 1 kg barley).

698 pigs were fattened with the feed prepared in the manner described above and then slaughtered.

During the fattening period, the tail bite decreased drastically in comparison with slaughter pigs that were fattened with normal feed without the elemental supplement.

By the time the pigs were ready for slaughter, their feed consumption had been 2.92 feed units / kg weight gain.

Claims (9)

A process for preparing an elemental composition for use in biological and physiological systems, characterized in that lactic acid-producing bacteria are grown in an aqueous fermentation liquid containing fly ash from power plants, whereby a majority of the components of the fly ash are converted into a bio-assimilable. form, which may be chelates, complexes and lactic acid salts, and the liquid obtained containing these chelates, complexes or lactic acid salts after cultivation is isolated. A method according to claim 1, characterized in that the lactic acid producing bacterium is a bacterium of the family Lactobacillaceae or Streptococcaceae. Method according to claim 2, characterized in that the lactic acid producing bacterium 15 is selected from the genera Streptococcus, Leuconostoc, Pediococcus and Lactobacillus. Method according to claim 3, characterized in that the lactic acid producing bacterium is the strain Lactobacillus plantarum CH-1. Process according to any one of claims 1-4, characterized in that the fermentation liquid also comprises rolling retentate. Process according to any one of claims 1-5, characterized in that the fermentation liquid also contains a protein source. Process according to any one of claims 1-6, characterized in that the fermentation liquid also contains milk powder. DK 154863 B Process according to any one of claims 1-7, characterized in that the liquid obtained is concentrated after the isolation. Process according to any one of claims 1-8, 5, characterized in that the obtained liquid is added to a fermentation medium after the isolation.