HU213164B - Process for the preparation of one or more microelements riched yeasts and compositions containing them - Google Patents

Abstract

The present invention relates to a process for the preparation of yeast enriched with one or more microelements in a fermentation broth containing the water-soluble salt of one or more microelements by proliferation of yeast and by separating, washing, optionally drying, inactivating the components of the fermentation before fermentation of the yeast. with the exception of yeast - pretreated in a high voltage electrostatic field of 5-30 kV, the yeast is fermented in their presence, if necessary, a pre-pretreated solution of additional microelements in the exponential phase of yeast slurry is added, and the intensive fermentation is continued until the yeast element saturation under the given conditions at least 50% of its value. The resulting one or more micronutrient-containing yeast can be used alone or as a component of pharmaceutical compositions and as a food additive. * EN 213164 B Λ description: 8 pages

Description

The present invention relates to a process for preparing yeast enriched with one or more trace elements, wherein the trace elements are organically bound to the yeast by incorporation into the cell. Elements that are present in the living organism in mg / kg or less are called micronutrients. Yeasts containing trace elements in the biotransformed form are marketed as such, without additives or as a nutritional supplement, as an additive in food products, or in combination with other active ingredients in medicinal products or pharmaceuticals.

Of the 88 elements that make up the Earth as a constant component, 11 macronutrients and 65 micronutrients are involved in the formation of living organisms, of which 18 are now considered to be of general or partial vital importance and 7 have been shown to have beneficial physiological effects. The beneficial physiological effects of the 25 microelements taken together are valid only in the appropriate concentration range, so it is essential for us to consume sufficient amounts of them. Domestic nutrition surveys show that the population is "malnourished" from important nutrients (selenium, zinc, iron, etc.), given the recommended daily intake (RDA) values for the US Academy of Sciences:

Recommended Daily Intake (RDA) values Element Child Adult Iodine 0.12 mg 0.15 mg Iron 10 mg 10-15 mg Zinc 12 mg 15-20 mg Selenium 0.03 mg 0.05-0.07 mg Copper 1-2 mg 1.5-2.5 mg Chromium 0.05-0.20 mg 0.05-0.20 mg Manganese 2.0-3.0 mg 2.0-3.0 mg Fluorine 1.5-2.5 mg 1.5-4.0 mg

The reason for this is primarily the lack of micronutrients in the soil, which, along the food chain, leads to micronutrients in the human diet, and secondly to the use of 'refined' foods consumed and prepared according to today's dietary habits. Over the last 10 years, there has been a gradual increase in scientific publications showing that micronutrient supply significantly influences the development of oxidative diseases (cancer, cardiovascular disease, diabetes, etc.) and immune deficiency diseases. Therefore, worldwide attention is being paid to the replacement of micronutrients in the diet. Since it is known that trace elements in the form of inorganic compounds are much less useful to the human body, there is a constant need for these elements in organic compounds, e.g. consumption in biotransformed form bound to yeast cells. Thus, the bioavailability of the micronutrients is 40-50%, while the inorganic compound has a bioavailability of ca. one tenth of this.

It deals with the enrichment of yeast with chromium No. 4,348,483. U.S. Patent No. 4,530,846 to Selenium Enrichment. U.S. Pat. The incorporation of the other 17 trace elements into yeast cells is described in Hungarian Patent Application No. 205,379, entitled "Process for the production of yeast fortified with trace elements". In these patents, a water-soluble inorganic salt of a single micronutrient to be incorporated is added to the medium during fermentation and reaches concentrations many times, even thousands of times higher than the normal micronutrient content of yeast. up to 5 mg / g for zinc.

ADE-OS 3 441 085 The German Patent Publication discloses a method for increasing the biological activity of microorganisms by introducing electricity into the fermentor. Alternating or direct current can be applied to the treatment either continuously or pulsed (0.01-100 A, 11000 V).

If half of the recommended daily allowance for zinc (10 mg) is taken in the form of yeast fortified with zinc, it is necessary to consume 2 g of dry yeast. For the intake of other micronutrients, additional yeast should be consumed. However, it is well known that an upper limit should also be set for yeast consumption due to the risk of overdose of the vitamin B family and other ingredients. There is a need for the production of yeast containing micronutrients, wherein several micronutrients are simultaneously enriched and contain the desired amount of micronutrients.

The present invention relates to a fermentation process in which it is possible to incorporate one or more microelements into yeast cells.

Surprisingly, it has been found that increased intake of a micronutrient or simultaneous incorporation of two or more micronutrients can be accomplished by pretreating a solution of the water-soluble inorganic salt of the micronutrient (s) in a high-voltage electrostatic field and simultaneously adding to the fermentation broth. Since each micro-nutrient promotes or inhibits the incorporation of each other, the time and amount of administration of the micro-nutrients depending on the biological state of the yeast cell must be precisely determined. It has been found that when multiple micronutrients are added, the pretreated solution of the additional micronutrient (s) should be added during the exponential phase of the fermentation.

Compared to a non-electrostatic charge curve, the charge curve becomes much steeper, which means that the desired enrichment can be achieved in less time. (Elemental values refer to the time-course of the content of yeast micronutrients for a given fermentation condition. If these related concentration-time data are plotted, we obtain an elemental absorption curve that flattens after a certain steep period and reaches a saturation value. cell density, aeration rate, nutrient2

EN 213 164 Β concentration etc. - it can be varied to make a different curve, but the ability to increase the saturation value is limited by the physiological limits of the yeast cell's microelement capacity.) In industrial applications, shortening the fermentation time becomes essential for economic reasons.

In the process of the invention, unlike the above-mentioned patents in the art, the incorporation of one or more trace elements into yeast cells has been accomplished by:

- the simultaneous incorporation of several trace elements into the yeast cell;

- unlike those described in German Patent Publication No. 3,441,085, no direct flow is made to the fermentor, i.e., microorganisms are not exposed to the current, but high-voltage electrostatic treatment of water, saline, nutrient solutions used outside the fermentor. In this case, contrary to the description in the FR, the effect of current is not to increase the biological activity of the microorganisms (which does not directly influence the uptake of the microelements), but to make the microelements more suitable for incorporation into the cell;

- Starting from the yeasts of Saccharomyces cerevisiae or Candida utilis, the progressive development of resistant micro-nutrient variants.

Furthermore, it has surprisingly been found that the amount of trace element incorporated into the cell is significantly increased by electrostatic treatment and / or by the use of micronutrient resistant yeast.

Starting from a small amount of pure yeast (a few grams), using known yeast growth stages, unlike previous patents (where in the final stage of growth the micronutrient-containing nutrient feed is added), fermentation on a medium containing a steadily increasing amount of micronutrients to form a resistant variant which has grown at a much higher concentration of micronutrients in the industrial scale compared to the non-resistant variant. Thus, yeast with a higher trace element content can be produced.

SUMMARY OF THE INVENTION The present invention relates to a process for the production of yeast enriched with one or more trace elements, in particular Saccharomyces cerevisiae or Candida utilis, by growing the yeast in a fermentation broth containing water soluble salts of one or more trace elements, separating, washing, optionally drying, inactivating the cell. all components except yeast are pretreated in a 5-30 kV high-voltage electrostatic field, in the exponential biological phase of yeast growth, one or more additional micronutrient solutions or solutions of different micronutrients are added and vigorous fermentation is continued until at least 50% of its value.

Preferably the yeast is Saccharomyces cerevisiae or Candida utilis, which are commercially available. As a trace element, enrichment with zinc or at the same time with iron and zinc or at the same time with iron and copper is carried out. The process of the present invention can similarly produce yeast enriched with Cr, Mn, Se, Mo, Ni, Si.

For the enrichment with zinc and / or copper, it is preferable to use a micronutrient resistant Saccharomyces cerevisiae which, despite the micronutrient inhibitory effect of the micronutrients, is well reproduced and can be produced by the process of the invention:

With the use of known yeast growth stages, fermentation on medium containing increasing amounts of micronutrients from the first stage allows the yeast to grow well (accustomed to it) at high micronutrient concentrations, despite the inhibitory effect of the individual micronutrients.

An advantage of the process according to the invention is that:

- performing simultaneous uptake of multiple micronutrients;

- the incorporation of one or more micronutrients into the yeast cell is accomplished in a substantially shorter time (about half) due to more intensive fermentation. In the case of an industrial implementation, this allows multiple products to be manufactured on the same equipment.

The yeast containing the micro-nutrient or micro-nutrients in the biotransformed form produced by the process of the present invention is a nutritional supplement in itself but can be used as an additive in dairy (eg Rudi Túró, processed cheese), confectionery (eg Napoli), bakery (eg cakes, cakes) also for the preparation of meat products (eg sausages, cold cuts, ham).

Accordingly, a further object of the present invention is to provide a process for the preparation of a foodstuff composition comprising admixing the fortified yeast of the present invention as an additive to foodstuff raw materials. A preferred amount of yeast in the compositions is 0.01 to 5.0% by weight.

The possibility of using micronutrient yeast to replace the loss of minerals and vitamins from wheat milling into flour is of great importance. Although the minerals and vitamin B content of cereals is significant, only a fraction of this mineral is added to low-milled (white) flour, which is most commonly used in mill milling, as follows:

Micronutrient content (mg / kg) of wheat flour of different degree of wholeness:

Mineral components of cf. Complete wheat- eye flour Type BL55 BL 80 BL112 BL160 Ash (%) 2.0 0.55 0.80 1.12 1.60 Iron (mg) 54.1 12.0 21.0 39.0 47.3

HU 213 164 Β

Zinc (Mg) 26.1 7.0 10.2 18.5 20.0 Copper (Mg) 4.1 1.0 1.8 2.9 4.0 Manganese (Mg) 37.4 5.7 11.2 14.7 24.6

Reformulation of micronutrients (remineralization) is already mandatory in the US A, Canada and other countries of the world, which require the use of inorganic salts. In Hungary, the use of well-utilized micronutrients bound to yeast protein is a novelty in remineralisation, at the same time replacing a significant part of the vitamin B loss in milling.

The invention further relates to a process for the remineralization of flour comprising mixing the ground flour with the yeast of the invention, preferably enriched with zinc and / or iron and / or copper.

The present invention also relates to the manufacture of a pharmaceutical composition containing yeast fortified with a micronutrient or micronutrients, which contain as active ingredient the yeast according to the invention, alone or with other active ingredients which do not exhibit synergism.

The preferred amount of yeast in the composition is 0.01 to 60% by weight.

The invention is illustrated by the following non-limiting examples.

Example 1

Application of an electrostatic force field to enhance the incorporation of iron and zinc.

The process according to the invention is a so-called baking yeast Saccharomyces cerevisiae. Becker strain, but the method can be applied to other yeasts. The fermentation was carried out in a 20 L laboratory fermenter, which was equipped with a recirculating pump, in which the medium was treated with a 15 kV high-voltage electrostatic field.

To study the effect of the electrostatic force ion on the incorporation of ferric ammonium citrate and zinc sulphate solutions, the same concentration was added to the medium in the control and in the field-treated fermentations.

Preparation of the experimental medium:

Ion exchanged water: 11.61

Phosphorus saline solution: 0.21

Iron + Zinc ion solution: 0.41

The prepared aqueous solution was passed through the condenser plates of the experimental electrostatic field for 15 minutes by starting the recirculation pump, whereupon the molasses solution was added and the fermentation was conducted in the known manner for 12 hours. The results of the experiment are shown in Table 1 for comparing dry matter increments and Table 2 for comparing trace element incorporation into yeast cells.

Table 1

Electrostatic force treated Reference* Starting volume 13.01 13.01 Starting solids 13.4 g / l 13.37 g / l Starting cell mass 174.2 g 173.81 g Final volume 20.91 20.91 Decomposable solids 17.78 g / l 15.22 g / l Stopping cell mass 371.6 g 318,098 g population growth 197.4 g 144.3 g

* The reference experiment was performed under exactly the same conditions, but the electric field was not switched on.

Fermentation with iron and zinc ions and electrostatic field fermentation resulted in a 36.8% higher growth rate.

Table 2

Fermentation time Vas (ppm) Zinc (ppm) Reference Handled Reference Handled 0 hours 392.24 392.24 529.04 529.04 2 o'clock 2638.97 2967.71 1724.8 1906.23 12 o'clock 4437.42 4815.42 4051.46 5667.86

In the same amount of time, in the presence of iron and zinc, 45.89% more zinc incorporation and 8.63% more iron incorporation is observed using the electrostatic field than in the reference experiment.

Example 2

Application of an electrostatic force field to enhance zinc incorporation.

The procedure was carried out in the same manner as in the first example, except that ferric ammonium citrate was added to the medium, but only zinc sulfate solution. The dry matter increments are compared in Table 3 and the zinc inclusions in Table 4.

Table 3

Electrostatic force treated Reference* Starting volume 13.01 13.01 Starting solids 13.39 g / l 13.37 g / l Starting cell mass 174.07 g 173.81 g Final volume 20.91 20.91 Decomposable solids 16.88 g / l 15.22 g / l Stopping cell mass 352.8 g 318,098 g population growth 178.73 g 144.29 g

HU 213 164 Β

Electrostatic field-treated fermentation resulted in a 23.87% increase in zinc ion supplementation.

Table 4

Fermentation time Zinc (ppm) Reference Handled 0 hours 529.04 529.04 2 o'clock 1438.6 1632.07 12 o'clock 3380.73 4733.02

Zinc incorporation was 39.9% higher over the same time period using the electrostatic field compared to the reference experiment.

Example 3

Applying an electrostatic force field to enhance the incorporation of iron and copper.

The procedure was carried out as described in the previous examples except that ferric ammonium citrate (200 g) and copper (II) sulfate (10 mg / l Cu) were added to the medium. The comparison of dry matter increments is shown in Table 5 and the ion incorporation is shown in Table 6.

Table 5

Electrostatic force treated Reference* Starting volume 13.01 13.01 Starting solids 13.5 g / l 13.4 g / l Starting cell mass 175.5 g 174.21 g Final volume 20.51 20.51 Decomposable solids 16.2 g / l 14.8 g / l Stopping cell mass 332.1 g 303.4 g population growth 156.6 g 129.2 g

Due to the presence of copper, the cellular growth of the reference fermentation is lower than that of zinc and iron, since copper is known to be toxic to all living organisms, including yeast, even at lower concentrations. In spite of this, an electrostatic force field resulted in a 21.2% growth rate.

Table 6

Fermentation time Vas (ppm) Copper (ppm) Reference Handled Reference Handled 0 hours 392.24 392.24 52.1 52.1 2 o'clock 2729.5 3312.1 344.8 385.4 12 o'clock 4626.8 5644.6 810.1 988.6

In the same amount of time, iron and copper co-fermentation showed 21.9% more iron and 22.04% more copper incorporation using the electrostatic field than in the reference experiment.

Example 4

Application of an electrostatic field to intensify the iron uptake of Candida utilis.

The procedure was carried out as described in Example 1 with the following exceptions:

- yeast used: Candida utilis

- applied voltage: 25 kV

- the medium contained iron (II) sulphate equivalent to 200 mg / dm ³ Fe, the fermentation time was 12 hours under force and during the reference fermentation.

Table 7 compares the dry matter increments and Table 8 compares the iron uptake.

Table 7

Electrostatic force treated Reference Starting volume 13.01 13.01 Starting solids 12.53 g / l 12.22 g / l Starting cell mass 162.89 g 158.86 g Final volume 20.91 20.91 Decomposable solids 18.2 g / l 16.1 g / l Stopping cell mass 380.38 g 336.49 g population growth 217.49 g 177.63 g

Electrostatic field-treated fermentation resulted in 22.4% higher growth in the same iron medium without the use of a force field.

Table 8

Fermentation time Iron content of cell mass (ppm) Reference Handled 0 hours 418.3 418.3 2 o'clock 3216 4063 12 o'clock 5841 6685

Applying an electrostatic force field to a medium containing the same amount of iron in the same amount of time, the iron uptake is 12.62% higher than applying without a force field.

Example 5

Natural selection and propagation of zinc-resistant yeast cells.

The Saccharomyces cerevisiae strain was propagated in a shake flask culture in the first step:

The composition of the medium:

glucose 50.0 g / l ammonium sulfate 5.0 g / l potassium dihydrogen phosphate 1.0 g / l yeast extract 3.0 g / l

EN 213 164 Β Biotin 0.04 g / L Zinc Sulfate 100 mg / L Zn pH 4.5

The medium was sterilized in a known manner and inoculated with a tube of Becker culture. Cultivation was carried out at 30 ° C for 24 hours in 750 ml Erlenmayer flasks. The cultures obtained in this way are called so-called. They served as a vaccine for Johann bottles.

In order to increase resistance, the zinc concentration of the same medium in the Johann flasks was doubled (200 mg / l Zn). The propagation was continued for another 24 hours. The resulting cultures served as inoculum for the 201 laboratory fermenter. In the laboratory fermentor, feed fermentation was carried out in the flask or Yeast cell mass grown in Johann flasks. The medium used was molasses solution supplemented with 40 µg of ammonium hydroxide. The initial concentration of zinc in the fermentation broth was increased to 300 mg / L and an additional amount of 100 mg / L was added at 6 hours of the 12 hour fermentation. Gradually increasing the concentration achieved a gradual increase in zinc resistance. This amount of zinc has a strong inhibitory effect on fermentation in a strain without resistance. In our experiment, the dry matter increment was barely reduced compared to the control without zinc.

The experimental results, i.e. the increase in zinc content in the yeast cell during each stage of resistance development, are shown in Table 9.

Table 9

Phase Zn content at the end of propagation Erlenmeyer flasks 3542 ppm Johann bottle 5620 ppm Laboratory fermentor 6986 ppm

With the usual scale-up in yeast production, our experiments were carried out until the amount of seed milk needed for the 20 m ³ fermentor was produced. The fermenter inoculum was a resistant yeast containing -7000 ppm zinc. At the beginning of the 12-hour, well-guided fermentation, a zinc concentration of 300 mg / L was provided in the fermentation broth and the amount consumed by the growing yeast was replaced. This means an additional dose of 100 mg / l of zinc. The feeding time was determined by microscopic examination. The condition of the culture was monitored and replenished during the most intensive period of growth. We found that further growth was able to take up to -7000 ppm zinc. The cell mass obtained at the end of the fermentation was separated by separation from the fermentation broth and two-thirds dried as a finished product.

A third of the fermentation was started on the previously used medium. Our strategy was slightly modified based on microscopic examination. Further zinc administration was performed only after reproduction of yeast cells that had been rounded by separation and washed with water, and after the first appearance of the first daughter cells.

Surprisingly, we found that our results were reproducible even after two inoculations. Thereafter, the dry matter growth decreases due to aging. For further fermentation, a further pre-propagation had to be performed as described previously.

Thus, according to our method, micronutrient enrichment in yeast cells does not proceed in a single step, depending on the current physiological state of the strain, but is based on the development of gradual resistance from laboratory culture to industrial scale. Yeast cells metabolize and multiply over several generations by absorbing zinc at increasing concentrations. Girls' cells show greater resistance after each generation and are able to accumulate more zinc than the mother cell from which they were derived.

Example 6

Preparation of Zinc Enriched Micronutrient Resistant Saccharomyces cerevisiae.

The procedure was carried out as described in Example 1, except that the starting yeast was the trace element resistant Saccharomyces cerevisiae prepared in Example 5 and only zinc sulphate solution was added to the medium. The results of the fermentation are summarized as follows:

Starting volume 13.01 Starting dry matter: 13.8 g / l Initial cell mass: ' 180,0g Final volume: 17 g / l Stopping solids: 221 Stopping cell mass: 374g population growth: 194 yrs

Table 10

Fermentation time Zinc (ppm) 0 o'clock 6900 2 o'clock 7040 12 o'clock 7100

The experiment shows that cell growth similar to that of the non-resistant yeast mentioned in Example 2 was achieved using the micronutrient resistant Saccharomyces cerevisiae with significantly higher zinc contents.

Example 7

Use of zinc-enriched inactive yeast powder prepared as in Example 2 as an additive for the preparation of a zinc-enriched bakery product, a cake.

One pack contains 250 g of scones. 1000 Packaging Materials for Making Pasta:

BL 55 flour 158 kg

Yeast 7.90 kg

Zinc-containing yeast

HU 213 164 Β (3.5 mg zinc / g yeast) 0.79 kg

Cooking salt 5.53 kg

Margarine 56.25 kg

Powdered milk 3.95 kg

Sugar 1.58 kg

Water 47.4-55.31

After the raw materials are prepared, the dough is prepared by a direct process. The dough is processed, raised and baked. After baking, allow the patties to cool and then wrap them.

The final product cake has a zinc content of 2.4 mg / 100 g of product.

Example 8

Use of Zinc-enriched inactive yeast powder prepared in Example 2 for the preparation of a medicament.

1 tablet composition Lecithin 100.00 mg Yeast Powder (5000 mg / kg Zn) 400.00 mg Lactose 125.00 mg polyvidone 45.00 mg Tocopherol acetate 2.00 mg macrogol 10.00 mg talc 25.00 mg Magnesium stearate 7.00 mg Silica-Kollam 3.50 mg Microcrystalline. cellulose 20.00 mg Altogether: 754.00 mg One tablet of this medicinal product contains 2 mg of zinc

contain.

Example 9

The use of inactive yeast powder enriched with zinc and iron as in Example 1 for the remineralization of flour.

per kg of BL 55 flour is mixed with 2.5 g of inactive yeast powder with a iron content of 7 mg / g and a zinc content of 7 mg / g, after mixing:

29.5 mg / kg, the zinc content will be 24.5 mg / kg. In this case, 92% of the zinc loss in milling and 42% of the iron loss is replaced.

Claims (12)

PATIENT INDIVIDUAL POINTS A method of producing yeast enriched with one or more microelements at one time in a fermentation broth of a water-soluble salt of one or more microelements by proliferation of yeast and by separating, washing, optionally drying, inactivating the cell mass, characterized in that the yeast components are fermented - yeast. except in the presence of yeast growth, the pre-treated solution of additional micronutrient is added, and the intensive fermentation is continued until at least 50% of the yeast loading value under the given conditions is intensively fermented. reached. 2. The method of claim 1, wherein the yeast is Saccharomyces cerevisiae. 3. The method of claim 1, wherein the yeast is Candida utilis. 4. The method of claim 1, wherein the yeast is a microelement-resistant yeast. 5. A method according to claim 2, wherein Saccharomyces cerevisiae is produced by zinc-enriched or iron and zinc-enriched. 6. A method according to claim 2, wherein Saccharomyces cerevisiae is enriched with iron and copper. 7. The method of claim 3, wherein the iron enriched Candida utilis is produced. 8. The method of claim 4, wherein the zinc or copper enriched microelement-resistant Saccharomyces cerevisiae is produced. 9. A process for the preparation of food preparations comprising, for food ingredients, from 0.01% to 5% by weight of the food additive The yeast produced according to claim 1 is mixed. 10. A method of replenishing a meal of a micronutrient of flour, characterized in that the ground flour is mixed with the yeast which has been prepared according to claim 1. 11. The method of claim 10, wherein said yeast enriched with zinc and / or iron and / or copper is used. 12. A method for the preparation of a pharmaceutical composition comprising, as active ingredient, 0.01 to 60% by weight of the yeast prodrug as claimed in claim 1, optionally together with the other active ingredient which does not exhibit synergism with yeast. - and mixed with excipients.