DE1517083A1 - Process for the production of wort by means of the enzymatic decomposition of yeast cells - Google Patents

Description

LEOSCHMETZ AACHEN Γ _τ . Vr ₀ I.

Heinrichsollee 2 1 yj \., ■ * - »-" ■ £ \ ~

om Kai se r ρ latz L— ""

Fernspn 34731

No da Shoyu Kabushiki Kaisha in Noda, jtfoda-shi, Ghiba-ken (Japan) Patent application

Process for the production of condiments by means of the enzymatic decomposition of hot cells

Yeast cells contain considerable amounts of valuable components, such as proteins, x'ettes, nucleic acids, lasers, various types of vitamins, acids, etc., and numerous attempts have been made to separate specific components from the yeast by means of extraction or decomposition thereof . For the purpose of using the proteins, for example, amino acid mixtures are produced by decomposing the nefe cells by means of hydrochloric acid or certain enzymes. 1 ' ^{1 For} the useful application of Hue linic acid derivatives v / earth cells or ribonucleic acid, which is separated from the yeast cells, decomposed by incubation with a culture broth of various Liikroorganisms, such as Actinomycetes Penicillia etc., or enzymes are added the specified microorganisms are formed, whereby 5'-ribonucleotides as Y / shorten and occasionally other types of 5'-ribonucleotides are formed by means of enzymatic deamination. So far, however, no process has become known, on the basis of which the mixture of important components and / or abbreviations by means of enzymatic decomposition of yeast cells, such as amino acids, sugars, 5'-ribonueleotides, organic acids and other un-

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known substances can be produced in high yields while obtaining the same.

• The production of such a mixture takes a long time Investigation work succeeded.

To achieve the stated objective, it is necessary as a prerequisite to find microorganisms which are able to form sufficient quantities and many types of effective enzymes, such as protease, 5'-nuclease, 5'-adenylic acid deaminase, the cell wall dissolving enzyme, etc! times repeated working through the appropriate for this purpose micro- organisms ψ was inventively discovered a previously unknown type microorganisms, which the name Streptomyces satsumaensis nov. sp. was given. If boiled yeast cells are mixed with the culture filtrate of the specified microorganisms, the same will be liquefied within 4-5 hours, the solution becomes transparent and finally a larger amount of amino acids, including glutamic acid, 5'-iibonucleotides including inosinic acid and guanylic acid and a suitable amount of unknown seasonings is formed. In this case the used ratio of the total nitrogen of the prepared yeast as starting material was over 94>, and the theoretical yield of 5'-hibonucleotides was 25 - 78> depending on the different types of 5 '-iiibonucleotides . The reaction product contains suitable amounts of glucose and other sugars, acetic acid and other organic acids.

Due to a treatment to remove malodorous substances can use the developed solution as rfürziquid- with delicate taste in this form and by means of concentration or pulverization, if necessary after adding further seasonings

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be turned. The solution can optionally be used as the only flavoring component or additional seasoning component for various Find food application.

The microorganisms used according to the invention are from the soil have been isolated, and the essential microbiological characteristics thereof are given below.

The characteristics of the various culture media are summarized in Table I. The aerial mycelium is straight or slightly wavy, forms irregular clusters and no spirals, the spores are oval or long-elliptical with a smooth surface. λ

Based on the identification according to Bergey's Manual of Determinative Bacteriology 7th Edition and SA Waksman: Actinomycete.s Volume 2, 1961, it is of the opinion that the specified microorganism used according to the invention is similar to S treptomyce s globJ8Porue. Due to the remarkable differences between these two microorganisms under the same growth conditions, the color of the Luftmycels on Czapek agar, the color of the Luftmycels on potatoes and the pigmentation of the potato plug, it has been concluded that the microorganism used according to the invention belongs to a new variety and the same is the name Streptomyce s satsumaensis nov.sp. g was ESpecify.

Table I.

Morphological and physiological properties of Streptomyoee satsuinaensis nov.sp. on different media «

Culture medium Yfachstura aerial mycelium soluble pigment other _; Own sch.

Czapek agar bad know not spent Glucose asparagine Agar good white to not spent

gray-white

Calciutnuialat-Agar well whitish - not displayed.

gray-light gray

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Culture medium growth aerial mycelium

soluble pigment further properties

π • J η

Starch agar good tyrosine agar good

Glucose stock-good agar

Potato wad

Well

Carrot grafts fen

white-light gray not developed
white-light gray not developed

white-light-not trained
greenish gray
or slightly light brown

white-gray

white-gray-white

fr_äbt from brown to black borrowed

not pigmented

Cellulose

not grown

Gelatin media grows lengthways

the surface of the Hohrs

does not know formed liquefies or slightly ■ liquefies light brown quickly

milk

grows along the surface of the pipe

do not know trained

Ability to decompose on starch

nitrate
Hydrosulfide

peptonized and quickly becoming alkaline

slightly

reduced

not trained.

The invention is carried out on an industrial scale as follows: Aqueous media are inoculated with 3 creptomyces' satsumaeneis nov, ep and cultured for 2 to 5 days with continuous shaking and aeration. The broth is then used as an enzyme solution, optionally after separating the microorganisms by means of filtration and centrifugation.

The culture broth has the following Beatand parts: As organic Source of nitrogen 0.5 - 3 »0 ^ defatted soybean powderj fish meal and corn syrup are suitable when 0.1-0.2 $ or more yeast than Nitrogen source is applied. The ZeIlwantiung is in the Kulturbrüne Dissolving enzyme introduced, giving the culture broth a remarkable ability to dissolve the cell walls.

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Furthermore, the addition of a small amount of hazardous nitrogen such as ammonium sulfate or ammonium chloride are often cheap Results relating to the formation of the specified enzymes in the culture broth ,, The carbon source is 1.0-4 »0 / o starch, glucose or waste molasses and as inorganic salts 0 - Ο, Ι, ό magnesium, - · sulfate, magnesium chloride, ferrous sulfate, sodium chloride and Calcium carbonate available.

The formation of phosphatase by the specified kikrοorganisms, both the actual formation or the corresponding activity, are suppressed or inhibited by the presence of inorganic phosphate and by the addition of a sufficient amount of inorganic phosphate. The formation of phosphatase can be suppressed while at the same time maintaining the activity of the S'-nuclease. The amount of inorganic phosphate added for the stated objective is approximately Q.1 - 0.6 / ό calculated as KH ₂ PO ₄ . After the p ^ value has been regulated to about 7.0, the medium is sterilized for 10-15 minutes by means of a water vapor treatment under a pressure of 1.7 to 2.05 kg / cm. Is then inoculated with the microorganism and 2 to 5 'Hunt at a temperature of 25 - 35 ⁰ C grown with continuous shaking and aerating.

As soon as the culture broth or the supernatant liquid contains the 5 'nucleotidase contains, the medium is 5 to 20 minutes long to one Heated to a temperature of 50 - 60 ° 0 in order to deactivate the enzyme, without harming the other useful enzyme systems will.

Jitwa 20 - 40 ^ · yeast suspension under one for 5 to 15 minutes Pressure of 1.7 to 2.05 kg / cm is heated, is with the above ϋίη-zymlösung mixed, the p ^ -v / ert set to 7 * 0 to 9.0, if necessary Toluene, ethyl acetate, nitrofurazone or other antiseptics

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added and then 1 to 4 days at a temperature of 30 ° - 50 ⁰ G incubated. After filtering off or centrifuging, the p ^ value is adjusted to 5.0, if necessary by adding sodium chloride, then substances unsuitable for the wort are removed and, if necessary, the remaining solvent is separated off by distillation or in another suitable manner. The desired reaction product can be obtained without any further treatment, or the same can additionally be purified by using adsorbents or ion exchange resins and treated further by concentration or drying.

The product obtained after these treatments does not have an unpleasant smell, has a delicate taste and in this state is suitable as a seasoning, or the same can be used as an additional seasoning component can be used for various foods.

It has also been shown that this product has no toxicity whatsoever after repeated experiments with hats and mice have been carried out.

example 1

For breeding Streptomyces satsumaensis nov.sp. a Ledium with the following components is used:

Baker's yeast (commercially available) l ^v / °

soluble suark 2>

K ₂ HPO ₄ 0.5>

MgSO ₄ -7 H ₂ O 0.05> i

ilaCl

The medium is adjusted to a pu value of 7.0, 50 ml of this iuediums poured into a 500 ml shake flask,

> ζ

steam sterilized under a pressure of 2.05 kg / cm for 10 minutes, after cooling with a small amount of the spores of Btreptomyces satsumaensis nov.sp _y inoculated, for 24 hours at a temperature of 33 ° C with shaking at 120 U / min, bred, and then

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Prepared culture broth. 3 drops of this broth are used as an inoculation ungsmateri al added to the same culture medium and for 3 days grown under the same conditions.

On the other hand, the same yeast as that used to make the Culture medium applied, so suspended in water that 20> üige suspension is present. This suspension is heated for 10 minutes under a pressure of 2.05 kg / cm. After cooling down it becomes the p - ^ value adjusted to 8.0 by adding sodium hydroxide.

The mixture of 2 1 of this culture broth (, iSnzymlöaung) with 2 1 yeast slurry is adjusted by adding sodium hydroxide to a Ρττ value of 8.0, then 33 ml of toluene were added and incubated at a temperature of 45 ⁰ C. Instead of adding toluene, adding 20 ppm of nitrofurazone before incubation gives practically the same results. After 3 days, this semicircle is filtered through cheesecloth, the Pjr value of the filtrate is adjusted to 5.0 by adding lactic acid and the filtrate is extracted three times with 5 liters of ethyl acetate to remove the impurities. The solution treated in this way is adjusted to a p ^ -. / Ert of 6.5 and then ^{distilled under a reduced pressure at 4ο ο} ΰ. As soon as 20> of the filtrate has been distilled off or the filtrate has been kept at a temperature of 20 ° C. for 20 minutes, the remaining solvent is practically removed. The filtrate is then adjusted to a Pu value of 5-6.

The product obtained in this way is quite transparent and does not smell more uncomfortable. The analysis results are given below:

Note: The analytical values are calculated including the components of the medium. It is assumed that the product contains a number of unknown substances related to the flavoring which are not found in the analytical values. _G

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"° ~ 1 b'l / UOd

Total ticks off 0.7025 * (applied ratio of total nitrogen 93> 8 $>)

Formol nitrogen $ 0.289

Amtuonium nitrogen f $ 0.033

Titratable acid (acetic acid, lactic acid, etc.) 0.553%

Glutamic acid 4 »48 mg / ml

Extracts $ 8.86 5'-RibonucIeοtide (calculated as free acids) Cytidylic acid 43 »6 mg> (calculated yield $ 39.6)

Uridylic acid 78.7 days? «) -" "71.3 / °)

In os in acid 73.3 mg ·; * (? "61.8 ^ 0)

Guanyl acid 31.2 day% · ("" 25 »

Example 2

The following medium is inoculated with Streptomyces satsumaeneis nov.epo) and incubated for ^{4 days at a temperature of 3O 0 O.}

The incubation conditions are the same as in the example 1 with the exception that the volume of the medium (75 ml in each flask) and the incubation time of the Vaccination culture (48 hours) are different.

Baker's yeast (commercially available) (total nitrogen $ 7.14, moisture $ 8.19, fiibonucleic acid 3.58y °) l · / »

soluble starch 2yi

KH ₂ JO ₄ 0.5 '/ »

IUgCl ₂ . 6 H ₂ O 0.05 '/ »

kaismolasse '0.5 ^

This led ium is brought to a Ρττ-Wefct of 8.0.

After üntfernen of the microorganisms from the broth, the supernatant is heated to 50 ⁰ C for 15 minutes, whereby the

J ₀ 5'-nucleotidase is completely deactivated.

^ The anäfy tables values of the enzyme solution after this treatment are _o reproduced below:

σ total nitrogen O, O49O? b

^ ₃ formol nitrogen 0.0350 / 4

^m ammonium nitrogen 0.0103 '/)

' Glutamic acid negligible

5'-nucleotides negligible

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On the other hand, a 20% aqueous suspension of dry yeast (Torula utilis) (total nitrogen 7.28 ^, ribonucleic acid 8.32 / °, humidity 6.8250) is prepared and adjusted to a p ^ r value of 8 by adding sodium hydroxide , 0 set. 1 liter of enzyme solution is mixed with 1 liter of the yeast suspension, the Pjr value is adjusted to 8.0 by adding sodium hydroxide, then 10 ml of toluene are added and the mixture is ^{incubated at a temperature of 45 °} C. for 3 days. The enzymatic hydrolysis is then completed. The reaction product is adjusted to a pg value of 6.5 under one pressure

of 2.05 kg / cm for 10 minutes to deactivate the enzyme activities heated.

10 g of "Celite 555" are added to this product by means of suction filtered, and then the delicate hydrolyzate obtained.

The analytical values of the hydrolysates are given below.

Total nitrogen 0 ', 667OyiJ

(applied ratio of total nitrogen = 84 ^)

Formol nitrogen 0.231255

Ammonium nitrogen $ 0.0234

Glutamic acid 3.82yo

5'-ribonucleotides:.

Oytidylic acid 169.5 mg / £ (calculated yield 81.2 ^C / O)

Uridylic acid 153.0 mg / o ("" 77.6 ^c / o)

Inosinic Acid- 164.8 mg # (""

Guanylic acid 91.9 mg / 'ό (""

AJIdP '. 5.8

Example 3

The "liquid product obtained by the treatment according to the Example 1 is dried by means of spray-drying at a temperature of 12O ⁰ C, jv.an obtained as 92.7 g of a product pulverföroiigen The analytical values of this product are shown below.:

Humidity 9.88 / 0

Total nitrogen 7.15 ^f A

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Formol nitrogen 2.94 %

Ammonium nitrogen 0.33 %

Glutamic acid. 45.5 mg / g

Titratable Acids 5.67

5'-inosinic acid (as free acid) 7.33 mg / g

5'-guanylic acid ("" ") 3.12 mg / g

5'-uridylic acid ("" ») 7.87 mg / g.

This product can be used in this form as a condiment or in the form of a mixture with other condiments for powdery ones Food or the like. Can be used.

Example 4

The liquid product obtained by the treatment according to Examples 1 or 2 is treated with sodium chloride, various amino acids, Added to sugars, various spices, soy sauce, fish extracts, poultry or meat extracts, by means of spray drying concentrated or pulverized or further treated by means of other conventional methods. One obtains in this way very tasty products.

Example 5

When an appropriate amount of the treated according to Examples 1 or 2 is used liquid product for soy sauce, miso (soy bean puree), Vinegar or other flavor enhancers are added one has superior seasoning effects compared to the cases in which 'seasonings that only consist of ribonucleotides or amino acids, can be added to other dishes.

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Claims (1)

Claims 1 “Process for producing wort by enzymatic decomposition of yeast cells, characterized in that the culture medium in which a suitable amount of inorganic phosphate is used to suppress or inhibit the formation or activity of the phosphatase is present while maintaining the activity of the 5'-iiuclease, with Streptomyces satsumaensis nov. sp. inoculated, the culture broth thus obtained was added to an aqueous yeast suspension containing antiseptics and the yeast cells, separating out a different amino acid, " 5'-nucleotides, sugars, organic acids and other seasoning ingredients containing product are decomposed. 2.Verfahren according to claim 1, characterized in that instead of the ^ entire culture broth only the protruding iliquid or the filtrate is applied as an enzyme solution will. 3. Teriahren according to claims 1 or 2, thereby / eke η η - λ ε eich η et, aas the culture brahe or the supernatant liquid to activate the reading of the b 'nucleotidase to a temperature γόη bC ° - 60 ° G heated will. 4. / experienced after speeches 1 or 2, characterized in that that as an antiseptic of the kefe suspension toluene, Ethylacetut o- j.er .iitrofurazor is added. 3. Method · -: after any: of claims 1 to 4, thereby 909840/0576 " in that the hydrolyzed LöBung adjusted to a _pH value of 5.0, extracted with Aethylaoetat adjusted to a value of 6.5 Ρττ, distilled under reduced pressure to distilled off 20% of it, and finally to 5-6 is set. 6. The method according to claim 5, characterized in that the hydrolyzed solution after setting its P _H -V / ertes to 6.5 for the purpose of removing the remaining solvent for 20 minutes at a temperature of 100 ° 0 is heated. 7. The method according to any one of the preceding claims, characterized in that the liquid obtained Reaction product is subjected to spray drying at a temperature of 90-120 ° G. Aachen, November 22, 1963 For: Noda Shoyu Kabushiki Kaisha The patent attorneys through: 909840/0576 BAD original