CH641644A5 - METHOD FOR PRODUCING HISTAMINE ARMS, OR HISTAMINE-FREE FOODSTUFFS, FOODSTUFFS AND FEEDSTUFFS AND PRODUCTS MADE THEREFOR. - Google Patents

Description

The present invention relates to a process for the production of foods, luxury foods and animal feeds which are low in histamine or completely free of histamine. The invention further relates to the foodstuffs, luxury foods and animal feeds produced by this process.

In recent years, ever greater efforts have been made to reduce as far as possible the content of harmful substances in foods, luxury foods and animal feeds, such as pesticides, heavy metals, mycotoxins, nitrosamines and other carcinogens, and ideally the foods, luxury foods and To keep feed free of such substances at all. It is particularly important for animal feed to keep them as free as possible from pollutants, because the pollutants can be accumulated both in the tissues of the animals, i.e. in the meat, as well as an accumulation of the pollutants

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

3rd

641 644

occurs frequently in the milk of animals, in particular in cow's milk and products made from it.

It is known that the consumption of certain foods, in particular certain alcoholic beverages such as wine and beer, as well as cheese and also sauerkraut, can lead to migraines and headaches and that such foods and luxury foods are also indigestible for people who are allergic to, Diarrhea, gastric and duodenal ulcers.

Studies carried out on beer in connection with these mentioned effects seem to show that top-fermented types cause disorders more often than bottom-fermented types. Nevertheless, the severity of the disturbances depends more on the amount of beer consumed than on the type of beer chosen (Pilsener, export, etc.). Surprisingly, it has been found that the beers with the same alcohol, fusel oil and hop extract content can have a wide range of undesirable effects. However, it was shown that beers with a high protein content and amino acid content are less digestible than beers with a lower protein content.

Histamine and other biogenic amines usually only get into the food during the processing of primary agricultural products.

It has now been shown that numerous microbes are able to decarboxylate the histidine which occurs in primary agricultural products to the biogenic amine histamine.

Histamine is a substance that triggers strong physiological reactions in humans, for example headache (migraine), nausea and diarrhea. In addition, histamine is an essential factor in allergies and anaphylaxis.

Histamine is a strong base that forms salts with acids. Histamine is found in plants such as spinach and nettles, and can be found in various animal and human tissues. Basophilic leukocytes store histamine in an inactive form, but it can be released from them by allergic reactions or by ionizing radiation or in the event of tissue destruction or by liberators.

It is also believed that there is a close relationship between the histamine content of the gastric mucosa, ulcer recurrence and stomach acid. Chronic gastric ulcers can be significantly improved by administering histamine receptor antagonists. The same applies to migraines. A wholesome, histamine-free diet is of great importance for migraine prevention.

People with chronic duodenal ulcer store significantly less histamine in the body mucosa than people with healthy stomach. While healthy people break down food-borne histamine with a histaminase, the absence of this ferment leads to histamine headache (Horton syndrome), which is also called histamine encephalgia. Bing head pain syndrome and Harris neuralgia, a disorder that occurs mostly at certain times of the day, predominantly at night, is caused by semi-lateral pain attacks in the eyes, forehead, temples, with tears, reddening of the eyes and face, swelling of the nasal mucosa etc., is triggered by histamine and can be successfully treated with antihistamines.

Desensitization and generation of increased resistance to histamine is achieved by administration of hist aminazoprotein. There is usually an antihistamine factor in the serum, the absence of which is seen as a sign of a latent allergy.

Histamine is excreted in the stool and in the urine.

Histamine increases smooth muscle contraction and relaxes vascular muscles. This lowers blood pressure, but increases capillary permeability, causing the formation of pimples and edema in the skin. Human serum can bind free histamine. Only in the case of allergy sufferers the binding ability is greatly reduced, it is inhibited by a so-called antipexin.

Marquardt (see H. Marquardt, "Die Weinwirtschaft", p. 127, 1978) considers 2 mg histamine per kilogram of food or luxury foods, such as wine, to be the tolerance limit that is hazardous to health.

Various foods and luxury foods have now been checked for their histamine content. In the case of dairy products, the results summarized in Table I below were obtained:

Table I

Histamine content in various dairy products

Product histamine mg / kg

Pasteurized V-milk 0.3

Pasteurized V-milk 0.5

Pasteurized V-milk 0.7

U whole milk 0.8

U whole milk 0.8

Dikmelk 1,2

Yogurt 2.1

Yogurt 1.7

Tilsiter cheese Vollf. 50.0

Tilsiter cheese Vollf. 60.2

Gouda cheese complete 54.0

Gouda cheese complete Austrian 41.0

Gouda cheese complete holl. 180.0

Camembert Vollf. 35.0

Camembert Vollf. 55.0

Stilton Vollf. Engl. 158.0

Harzer cheese 390.0

Harzer country cheese 383.0

As can be seen from this table, the histamine content of various types of whole milk is well below the tolerance limit specified by Marquardt. It is surprising in itself that fresh milk already has a certain amount of histamine. It can be assumed that this histamine content of the fresh milk is at least partly due to the fact that the cows ingested histamine with the feed, in particular with silage, and this passed into the milk. As can be seen from Table I, however, the histamine content of sour milk products, such as yoghurt, and of cheeses is considerably higher than that of fresh milk. Since microorganisms, as already mentioned above, are able to convert the histidine contained in milk protein into histamine by decarboxylation, this drastic increase in the histamine content occurs during the acidification of the milk and during cheese production. Almost 20 mg of histamine is already consumed when just 50 g of Harzer farm cheese is consumed, i.e. the 10-fold amount that Marquardt regards as the tolerance limit for 1 kg of beverage, such as wine.

Wines also have a fairly high histamine content, especially red wine and champagne.

5

10th

IS

20th

25th

30th

35

40

45

50

55

60

65

641644

4th

In this case too, as will be discussed in more detail below, the histamine is formed by converting histidine from protein substances contained in the must or in the mash into histamine by microorganisms. The results obtained from the wine test are summarized in Table II below.

Table II Histamine content of various wines

Product histamine mg / kg

Table wine (Moselle) 0.5

Table wine (Frankr.) 1.6

Quality wine (Moselle) 1.1

Kabinettwein (Rheinhessen) 3.0

Kabinettwein (Rheinhessen) 4.5

Selection (Rheingau) 1.7

Red wine (Ahr) 5.6

Tokaier (Hungary) 1.1

Tokaians, dry (Hungary) 3.2

Red wine (Austria) 7.4

Sparkling wine, dry (Germany) 5.1

Champagne (France) 7.8

As has already been clarified above, it was found in the beer tests that beers rich in protein are less digestible than those with a low protein or amino acid content. The examination of the histamine content in these beers showed that the protein-rich beers are also particularly rich in histamine and that even non-alcoholic beers can have relatively high histamine contents.

The histamine contents of various beers are compiled in Table III below, and the test numbers are also given in this table.

Table III Histamine content of various beers

No.

Beer type

Histamine mg / 1

1

Old-

4.5

2nd

Old-

9.8

3rd

Old-

8.0

4th

Old-

6.8

5

Old-

5.0

6

White-

5.3

7

White-

4.9

8th

White-

4.0

9

White-

4.7

10th

Malt-

6.8

11

non-alcoholic

7.5

12

non-alcoholic

4.6

13

Export-

3.2

14

Export-

2.9

15

Export-

11.2

16

Export-

7.0

17th

Export-

6.6

18th

Export-

7.2

19th

Pils

3.5

20th

Pils

0.2

21st

Pils

7.4

22

Pils

5.8

23

Pils

5.2

24th

Pils

5.9

25th

Pils

8.7

It has now surprisingly been found that it is possible to produce foods, luxury foods and animal feeds that are low in histamine or histamine-free by killing the substances present on the starting material.

25 existing microorganism flora adds such a microorganism or a corresponding enzyme to the starting material, which degrades existing histidine without the formation of histamine.

The present invention therefore relates to a process for the production of foods, luxury foods and animal feeds which are low in histamine or histamine-free, which is characterized in that the microorganism flora present on the starting material is killed and then during any stage of your step adds a microorganism or a corresponding enzyme to the production of the food, luxury food or animal feed, which degrades the histidine present without the formation of histamine.

In a preferred embodiment of the method according to the invention, a bacterium, preferably from the class of the micrococci, the Pseudomonas species and lactobacilli, a fungus or a yeast, preferably a Saccharomyces species, is added 45 or one obtained from such a microorganism as the microorganism Enzyme added, which degrades histidine without histamine formation by transamination or deamination. In general, the histidine present is broken down to form glutamic acid and / or ketoglutaric acid or salts of these acids.

While the naturally occurring microorganism population mainly contains strains that degrade histidine in an undesirable manner to form histamine, it has now surprisingly been found 55 that a small number of strains, histidine, can also occur in the naturally occurring microorganism population degrades without the formation of histamine, and such deposited pure cultures 60 can also be obtained from microorganisms at the collection points for microorganisms which degrade histidine without the formation of histamine.

The following reaction scheme is used to explain the undesired histidine degradation, which gives 65 histamine via decarboxylation, and also a desired histidine degradation, which delivers the harmless by-products glutamic acid and / or ketoglutaric acid via deamination:

5

Histidine

641644

N-

II

- CH "-

CH - ÇOOH NH,

-CO.

• CH

II

CH C - CH ~ - CH0 - NH,

\, /

N '

H

histamine

- NH.

Glutamic acid.

HOOC - CH2 - CH2 - CH - COOH

NO

HOOC - CH0 - CH, - C - COOH

2 2 il

Ketoglutaric acid

Due to the fact that in the naturally occurring microorganism population there are mainly strains which degrade histidine to form histamine, the process according to the invention is particularly important wherever a conversion caused by microorganisms takes place in the course of the production of a food, luxury food or animal feed, because it is now possible with the aid of the process according to the invention to obtain the products formed in these production processes histamine-free by using such a pure culture of a microorganism which breaks down histidine without the formation of histamine.

If the method according to the invention is used in the production of a foodstuff, luxury food or feed, in which an essential step is a conversion caused by microorganisms, then two particularly advantageous embodiments of the method according to the invention are possible.

According to one preferred embodiment of the method according to the invention, after killing the microbial flora of the starting material, the conversion is carried out with such a pure culture of a microorganism strain which also breaks down any histidine present without the formation of histamine. The conversion carried out with the pure culture of the microorganism strain in the starting material can be, for example, an alcoholic fermentation or a lactic acid fermentation.

If, according to this preferred embodiment of the process according to the invention, an alcoholic beverage, for example beer, wine or spirits, or an alcohol-free secondary product obtained from a product produced by alcoholic fermentation, for example alcohol-free beer or table vinegar, is produced, then the germ flora of the starting material is destroyed the alcoholic fermentation is carried out by inoculation with such a pure culture of a yeast strain, in particular a Saccharomyces species, which, in addition to the alcoholic fermentation, degrades histidine contained in the starting material without the formation of histamine.

If, according to this one preferred embodiment of the process according to the invention, a food, luxury food or feed is produced in which an essential step of the production process is a lactic acid fermentation, for example producing a sour milk product, a yoghurt, a curd cheese, a sauerkraut or a silage ,

then, after killing the microbial flora of the starting material, lactic acid fermentation takes place by inoculation with such a pure microorganism culture, preferably lactobacillus pure culture, which breaks down histidine contained in the starting material without the formation of histamine. Examples of corresponding pure cultures are those of Lactobacillus helveticus or Lactobacillus bulgaricus or Lactobacil-55 lus acidophilus, and particularly preferably of Lactobacillus delbrückii.

The embodiment of the method according to the invention described above can also be used in cheese production. In this case, after the bacterial flora 60 of the quark serving as the starting material or the milk coagulate produced by adding rennet has been killed, the cheese is produced by inoculation with at least one pure microorganism culture which breaks down histidine contained in the starting material without the formation of histamine. 65 According to the other preferred embodiment of the method according to the invention, a food, stimulant or animal feed is produced by a production process in which an essential one

641 644

6

The step is a conversion caused by microorganisms, for example alcoholic fermentation or lactic acid fermentation, in that after killing the microorganism flora of the starting material, histidine contained in the starting material is broken down with the aid of a corresponding microorganism or an enzyme obtained therefrom without the formation of histamine, so that in the case of subsequent, by the microorganism caused essential conversion of the starting material in this practically no histidine is present or histidine formed during the conversion is immediately degraded by the first microorganism or the enzyme without the formation of histamine, so that there is no histidine present by the second Microorganism can be converted to histamine via a histidine decarboxylation.

If, according to this second preferred embodiment of the method according to the invention, an alcoholic beverage, for example beer, wine or spirits, or a non-alcoholic secondary product obtained from a product produced by alcoholic fermentation, for example an alcohol-free beer or table vinegar, is produced, then after the germ flora is killed Such a pure culture of a microorganism or an enzyme is added to the starting material, which degrades the histidine contained in the starting material without the formation of histamine, and after the histidine degradation has taken place, the alcoholic fermentation is carried out by inoculation with a yeast culture, which can optionally also be a yeast strain, which histidine is present Would decarboxylate histamine. In this case, the microorganism used for the degradation of histidine is preferably a pure culture of a Lactobacillus, in particular a pure culture of Lactobacillus delbrückii, which forms lactic acid and breaks down the histidine formed by proteolysis without the formation of histamine, whereupon after the histidine degradation has taken place, the alcoholic fermentation is expediently inoculated with an inoculation Pure yeast culture is carried out, which at least partially degrades ammonia and glutamic acid and the lactic acid formed by the lactobacillus.

In order to determine whether a pure microorganism culture, for example a microorganism strain obtained from a collection point, degrades histidine in the desired manner or not, all one has to do is cultivate this microorganism strain in a suitable nutrient medium and add histidine to it. As soon as the microorganism strain has grown in this nutrient medium for a time, the histamine content is then determined in the nutrient medium. This was done using the fluorometric method described in the P.A. Shore et al. in J. pharmacol. exp. Ther. 127, 183 (1979). This method has high sensitivity and is specific for histamine. A condensation of o-phthalaldehyde with any histamine present is carried out, the reaction product of the aldehyde and the histamine having a strong, constant fluorescence after excitation with radiation of 360 nm. The fluorescence is determined at 150 nm. An advantage of this method for the qualitative and quantitative determination of histidine is that it is very specific, the presence of closely related compounds such as n-alkyl histamine, acetyl histamine, serotin and similar compounds not interfering with this detection method.

In the present case, the determination of the histamine is carried out in culture media or in process products, both products which have been produced by the process according to the invention and products which have been produced by a process for comparison purposes, by first adding to the organic material, in which the histamine content is to be determined, perchloric acid is added. The mixture is then made alkaline and extracted with n-butanol. The organic layer is separated and from this the histamine is shaken out with an acidic aqueous medium. In this acidic aqueous medium, it is then condensed with the o-phthalaldehyde, and then the excitation of the fluorescence and the measurement of the fluorescence at 150 nm are carried out. It was found that the fluorescence of this acidic solution persisted for at least 5 hours is stable and the higher the histamine content of the sample, the higher the intensity of the fluorescence. Histamine concentrations in the aqueous acidic medium which are in the range from 0.005 to 0.5 g / ml can be determined according to this i5 method. The histamine content of the sample can be read off directly using calibration curves. In order to test the reliability of this method, measured, very small amounts of histamine were added to various products, for example sauerkraut, wine, beer and milk products, and it was found that 95-100% by weight of the added histamine was added this test procedure could be determined.

25 If, for example, a lactic acid fermentation is to be carried out by the process according to the invention, then individual cell cultures can be isolated from appropriate technical cultures of Lactobacillus helveticus or Lactobacillus bulgaricus or Lactobacillus acidophilus and these 30 in a suitable nutrient solution with the addition of pantothenic acid, niacin, riboflavin and calcium multiply. The actual pure cultures of the aforementioned microorganisms obtained in this way have an optimum fermentation temperature in the range of 40-44 ° C., the maximum temperatures at which fermentation with these microorganisms is possible being in the range of 50-52 ° C. The above-mentioned pure cultures of Lactobacillus helveticus or Lactobacillus bulgaricus or Lactobacillus acidophilus ferment glucose and maltose homofermentatively with the formation of d (-) lactic acid, but without the formation of carbon dioxide. These microorganisms in question deaminate histidine and some other amino acids such as arginine, citrul-lin, ornithine, proline and hydroxyproline with the release of ammonia.

45 In this context, it should be expressly pointed out that a large number of microorganism strains, for example lactobacillus strains, which are deposited at conventional collection points, are able to carry out the desired deamination of histidine with the release of ammonia. If pure cultures of these deposited strains have the property mentioned, they are suitable for carrying out the process according to the invention.

In the same way as described above for the Lactobacil-55 lus helveticus and the other Lactobacillus strains mentioned, a "single cell culture" of the Lactobacillus delbrückii was also prepared.

If lactic acid fermentation is now carried out with the single cell cultures of the lactobacillus thus obtained, 60 in addition to the formation of the lactic acid, histidine contained in the starting material is also converted into glutamic acid after primary ammonia elimination, and there is no undesired conversion of histidine with carbon dioxide elimination, which leads to the undesirable histamine.

If a sterilized sweet milk with the single cell cultures of Lactobacillus helveticus or Lactobacillus bulgaricus or Lactobacillus acido-

7

641 644

Inoculated by philus and the lactic acid fermentation carried out at a temperature in the range of 40-45 ° C, then you get a sour milk that is practically completely free of histamine or whose histamine content is not higher than that of the fresh milk used for the production of sour milk. In general, it is advantageous to add pantothenic acid, niacin and riboflavin to the milk used as the starting material in order to optimize the growth conditions during lactic acid fermentation. Calcium is an essential mineral component that the pure cultures of Lactobacillus helveticus or Lactobacillus bulgaricus or Lactobacillus acidophilus need for their growth. However, fresh milk already contains sufficient amounts of calcium, so that the addition of calcium is unnecessary in the sour milk production described here.

If desired, a histamine-free curd can also be produced from the sour milk obtained in this way, and this can either be sold as such or it can be used for the production of certain types of cheese, for example for the production of Mainz hand cheese or Olomouc quark, or else of resin cheese.

The single-cell culture of Lactobacillus delbrückii obtained by the method described above was used to produce sauerkraut, sour cucumbers and silage with a reduced histamine content or free of histamine at all.

In the production of sauerkraut, the correspondingly chopped white cabbage is seasoned as usual and then not fermented by the action of the lactic acid bacteria on it, but by thermal treatment the existing microbial flora is destroyed, and then the artificial inoculation with the histidine to convert the lactobacillus to glutamic acid del brückii strain, and the inoculated material is processed into sauerkraut in the usual way.

In the same way, also in the production of sour cucumbers, the starting material is first sterilized and subsequently inoculated.

As already mentioned, the method according to the invention is of great importance in the production of fermentation feed, so-called silage feed. In this case, the green fodder used as the starting material, for example grass, alfalfa, green corn and the like, is first sterilized by heat treatment and then artificially inoculated with the pure cell culture of Lactobacillus delbrückii mentioned above and in the usual manner in the watertight containers provided for this purpose. the usual feed silos, fermented. In the same way, silage is made from wilted silage, after adding sugar-containing substances, e.g. molasses or beet pulp, to the wilted silage, this mixture is first made germ-free and then artificially inoculated with the pure cell culture of Lactobacillus delbrückii mentioned. The histamine-free or low-histamine silo feed produced in this way is extremely desirable because when it is fed to cows, histamine ingested from the feed is prevented from passing into the milk. When using such a silage, it is possible to produce fresh milk which is practically histamine-free.

If the conversion caused by microorganisms is an alcoholic fermentation, then the formation of a histamine-free alcoholic fermentation product can be achieved, as already mentioned, by first sterilizing the starting material and then carrying out a corresponding fermentation with such a Saccharomyces strain, which transaminates or deamination of histidine contained in the starting material.

It is possible to produce a wine in this way by first sterilizing the must and then inoculating with a single cell pure culture from Saccharomyces. As a result, histidine present in the starting material is transaminated or deaminated during alcoholic fermentation and is not converted into harmful histamine. However, since the single-cell pure cultures of Saccharomyces mentioned are often not optimally suited for carrying out alcoholic fermentation, the procedures described below for wine production are those in which histidine contained in the starting material is first removed by other microorganisms and only then alcoholic fermentation is generally more advantageous. Secondary fermentation in champagne production is generally not carried out with a specially selected yeast strain, but rather by adding pure Lactobacillus cultures, as will also be explained in more detail below.

As can be seen from Table I, some cheeses are particularly rich in histamine. The difficulty with cheese production according to the method according to the invention is that in many cheeses not a single microorganism is responsible for the formation of the special hard or soft cheese, but that several different microorganisms can participate in the production of a special type of cheese. If one now wants to produce a low-histamine cheese according to the method according to the invention, then individual cell cultures which lead to transamination or deamination of histidine must be grown out of all the microorganism strains responsible for the production of the special type of cheese.

The Roquefort cheese is known to be made by the action of a certain blue mold on break, which was obtained from sheep's milk by precipitation of rennet. While in traditional Roquefort production an infection of the break with the blue mold is caused by the addition of moldy bread, a single cell culture was now grown from the blue mold of the Roquefort cheese, which led to a deamination of the histidine. The curd was then inoculated with this pure blue culture, and this made it possible to produce Roquefort cheese that contained only very small amounts of histamine, but after maturing it was very similar in taste to the traditionally made Roquefort cheese .

As already mentioned, if you want to produce a food, luxury food or feed in which an essential step in the production process is a conversion caused by microorganisms, it is not absolutely necessary to use such an organism strain for this conversion which does not lead to any decarboxylation of the histidine leads to the formation of the harmful histamine, but it is also possible to convert histidine contained in the starting material into harmless products beforehand, so that in the subsequent action of the microorganism there is no longer any histidine which can be converted to histamine by decarboxylation. If, for example, the product to be manufactured is an alcoholic beverage, then a practically histamine-free end product can also be obtained by killing the microbial flora of the starting material before alcoholic fermentation, then adding a microorganism or an enzyme that transaminates or deaminates the product in Aus5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

641644

histidines contained in the starting material, and then, as soon as the histidine present has been broken down into harmless conversion products, the alcoholic fermentation is carried out by inoculation with a yeast culture. In this case, the yeast strain used can also be one which would in itself lead to a decarboxylation of histidine with formation of histamine, but since at this point in the material to be fermented there is no longer any histidine at all, no undesired conversion of the histidine can occur Histamine.

This last-explained embodiment of the method according to the invention is also very advantageous in beer production, because even in those work steps which take place before alcoholic fermentation, a conversion of histidine to histamine caused by microorganisms can take place. Histidine is contained in cereal protein, for example barley protein, in considerable quantities. According to Souci, specialist u. Kraut, nutritional value tables (Wiss. Verlagsgesellschaft, Stuttgart 1977), the histidine content of barley flour is 1.4 to 2.5 g per kg of barley flour, and the histidine content of defoliated barley with a protein content of 97 to 113 g per kg corresponds to about 1. 8 g-per kilogram. During the malting process, proteolysis of the grain protein takes place in the mash tun and in the brewing pan. In addition, microorganisms such as Saccharomyces diastaticus, Lactobacillus fermentum, Lactobacillus pastorianus and Pedio-coccus cerevisiae are already present at this point in time, which can lead to decarboxylation of the histidine to form histamine. In this case, the microorganisms in question are, of course, not specifically cultivated pure single-cell cultures, but rather the strains present in the natural environment, which, as already explained above, generally lead to this undesired decarboxylation of the histidine .

This microbially induced decarboxylation of the histidine with the formation of harmful histamine can only be interrupted by thermal inactivation at the end of the saccharification process, but not during primary fermentation or during maturation. Microbes that enter the fermentation tanks and cause histidine decarboxylation, which numerous breeds of Saccharomyces cerevisiae are capable of, produce considerable amounts of histamine.

It has now surprisingly turned out that a histamine-free beer can be produced by

that prior to alcoholic fermentation, thermal inactivation of the germ flora of the malt and thermal inactivation of the germ flora of the mash is carried out, and then the histidine contained in this starting material is broken down by describing the mash described above in connection with lactic acid fermentation Single cell pure culture of Lactobacillus delbrückii is added.

The sterilization of the mash before the addition of the pure culture of Lactobacillus delbrückii is expediently achieved by heating it to a temperature in the range from 62 to 65 ° C. for 30 to 40 minutes. The mash is then cooled to 45 ° C. and inoculated with the pure culture of the Lactobacillus delbrückii at this temperature. In the mash treated in this way, the Lactobacillus delbrückii vigorously converts glucose and maltose to form lactic acid, and at the same time the histidine present is converted into glutamic acid after primary ammonia elimination. During this step, the progression of lactic acid formation and

8th

constantly controls the parallel conversion of the histidine into glutamic acid.

As soon as the histidine present has largely been converted into the 1-glut-s amic acid after the completion and acidification of the mash, the main fermentation is carried out, i.e. the alcoholic fermentation of the beer by adding yeast, i.e. by adding Saccharomyces cerevisiae, by. This alcoholic fermentation is expediently carried out by inoculation with a pure yeast culture which partially metabolizes both the ammonia formed during the histidine degradation and the glutamic acid formed from the histidine and also the lactic acid formed by the Lactobacillus delbrückii. The alcoholic fermentation should therefore be carried out with a strain of Saccharomyces cerevisiae 15 which optimally degrades the by-products previously formed by the Lactobacillus delbrückii. In the described manner it is possible to produce a histamine-free beer. If one applies this process to the production of an alcohol-free beer, 20 then this can also be obtained histamine-free.

The preceding conversion of histidine into ammonia and glutamic acid is also carried out with great advantage in the production of other alcoholic products, for example wine and spirits, and then one also obtains histamine-free alcoholic products or secondary products produced from them, for example histamine-free vinegar.

In the production of histamine-free wine according to this last-described embodiment of the method according to the invention, analogously to the production of beer, the must is first thermally sterilized or, if necessary, the mash in the production of red wine, in order to kill unwanted primary germs. The must or mash is then inoculated with a small amount of the pure cell culture of Lactobacillus delbrückii or other suitable lactobacilli, and the material is then left at the optimal fermentation temperature of these microorganisms in the range from 40-45 ° C until the histidine contained in the must or in the mash is converted to 40 ammonia and glutamic acid. In this case, too, the Lactobacillus delbrückii or the other added Lactobacillus species simultaneously produce lactic acid by fermenting carbohydrates. As soon as the desired histidine degradation has taken place, the main fermentation is carried out, i.e. the inoculation with a pure yeast culture, in which case a Saccharomyces cerevisiae strain is also used, which optimally metabolizes not only alcoholic fermentation but also the ammonia formed, glut-50 amino acid and lactic acid as far as possible.

The procedure mentioned above can also be used to advantage in the production of spirits, that is to say distilled alcoholic beverages. Although in the case of a cleanly carried out distillation generally no substantial amounts of histamine are carried into the distillate during the distillation, it has nevertheless been found that commercially available spirits, such as cognac and the like, often contain large amounts of histamine. In this case too, the existing histidine can be converted into harmless by-products either by selecting 6o a special yeast strain or, generally more advantageously, before alcoholic fermentation by treatment with a pure culture of Lactobacillus delbrückii or another Lactobacillus delbrückii pure culture will.

According to a further embodiment of the method according to the invention, the formation of histamine in conversions caused by microorganisms can also

9

641644

be prevented where normally no substantial amounts of histidine are present in the starting material, but then histidine is formed from dead cells of the microorganism, the action of which causes the conversion in the food, feed or foodstuffs.

A typical process during which histidine is formed is sparkling wine. In the production of sparkling wine, a mixture of sucrose and yeast is known to be added to a wine obtained by primary fermentation, and the secondary fermentation is then carried out in a closed vessel, for example a pressure tank or a closed bottle. With the help of the yeast, the sucrose is fermented to alcohol, and the resulting carbon dioxide remains dissolved in the raw sparkling wine. An undesirable process that takes place in the production of sparkling wine is that the egg white originating from the yeast or the histidine derived from this protein is then decarboxylated to histamine by the surviving yeast cells. As can be seen from Table II given above, champagne has a much higher histamine content than the average values customary for wine due to this protein breakdown with the formation of histamine.

If one uses the method according to the invention for producing a histidine-free sparkling wine, then a mixture of one of the lactobacillus pure cultures described above, which lead to deamination of the histidine, with sucrose and the yeast is added to the wine obtained by primary fermentation. In the secondary fermentation taking place in the pressure vessel, the histidine originating from the yeast is then deaminated by the lactobacillus present when it is formed. As a result, the living yeast cells present no longer have any histidine available which can be decarboxylated to form harmful histamine. With this procedure it is possible to produce a practically histamine-free sparkling wine.

The ferment complex responsible for this conversion was isolated from the microorganism pure cultures which lead to transamination or deamination of the histidine. For this purpose, the cells were mechanically destroyed, the liquid containing the fermentation complex was isolated and lyophilized. It was found that 10 "3 g of the ferment material thus obtained are able to degrade 1 g of histidine. The ferment complex thus obtained was used in the same manner as described in detail above for the pure culture of Lactobacillus delbrückii in order to degrade histidine into harmless products in a starting material, giving results analogous to those obtained using the Lactobacillus delbrückii pure culture.

Histidine is in and of itself an essential amino acid, i.e. an amino acid that humans have to ingest with food. If the foodstuffs produced by the process according to the invention are protein-rich, such as sour milk products or cheese, the selection of a special microorganism strain does not produce a completely histidine-free foodstuff, but only prevents the microorganism strain from undesired decarboxylation of histidine with the formation of histamine carries out. If histidine is broken down by the microorganism strain, it is, as explained in detail earlier, converted, for example, into harmless glutamic acid.

If, however, the food produced by the process according to the invention is not an essential supplier of proteins, as is the case, for example, with alcoholic beverages, there are of course no concerns whatsoever to completely remove small amounts of histidine contained in these products, because that is for those In any case, nutritional protein substances have to be supplied to the human organism by other sources, such as meat, eggs and milk products.

The present invention further provides foods, luxury foods or animal feeds which are low in histamine or histamine-free and are produced by the process according to the invention.

The invention will now be explained in more detail by means of examples.

example 1

Production of a low or histamine-free beer

When the process according to the invention is carried out, the beer is produced essentially according to the brewing processes customary hitherto. In this connection, reference is made, for example, to the section on beer brewery in the “Textbook on Chemical Technology” by OST-RASSOW, edition 1965, pages 1394-1405, published by Johann Ambrosius Barth, Leipzig.

The essential work steps in carrying out a brewing process will now be explained and highlighted at the end, as a result of which the method according to the invention differs from the previously customary method.

1st step - malt preparation:

The main raw material for beer, namely barley, does not initially contain any fermentable extract. When the barley germinates in the malting plant, enzymes are formed that convert the grain content into soluble form during mashing. This creates soluble sugar from the insoluble starch, which is fermentable. In addition, other enzymes formed during germination convert high-molecular substances to low-molecular ones. By drying the germ material, namely the kiln, ultimately color and aroma substances are formed for the beer.

Step 2 - Brewing:

In the production of the "wort", the soluble parts of the malt, approximately 75% of the grain content, are allowed to dissolve in water through the action of the enzymes formed in the malting plant. After the wort has been filtered from the undissolved parts, the wort is boiled with the addition of hops and the desired original wort percentages are adjusted by evaporating more or less water.

3rd step - alcoholic fermentation:

After the hops are filtered off from the hot wort, they are cooled to the fermentation temperature as quickly as possible using cooling equipment. The fermentation temperature is 5-7 ° C when producing bottom-fermented beer and 14-16: C when producing top-fermented beer. The brewer's yeast is added and the alcoholic fermentation is carried out at the specified temperatures, usually for 8-10 days. The beer obtained in this way is then subjected to secondary fermentation in the fermentation vessels, finally the yeast is removed by filtration and the beer is filled into bottles or barrels and stored.

This conventional brewing process is modified as follows in order to produce a low-histamine or histamine-free beer using the method according to the invention.

It has been shown that the surface of the barley to be converted into malt is already covered with many microorganisms5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

641 644

10th

men is contaminated. This means that not only the desired conversion of insoluble starch into soluble sugars takes place during the mashing process in the presence of water, but also quite large amounts of the histidines present in the barley proteins are decarboxylated to histamine by these microorganisms. It was also shown that the microorganisms that break down the histidine to histamine are sensitive to heat.

When the brewing process is carried out according to the method according to the invention, the undesirable microorganisms located on the surface of the barley are therefore killed by carrying out a short-term treatment with boiling water, i.e. a so-called blanching process, or by immersing the barley in water which has a temperature of 75 -100 ° C. The water is discarded and the barley is washed several times with aseptic water at a temperature of 50 ° C.

Then, in the same way as in the conventional brewing process, the germinated barley is kilned and, just as in the conventional working process, the malt is crushed and mixed with warm, but germ-free water and the wort is boiled with the hops.

After the hops have been removed, the wort must be cooled under absolutely sterile conditions when the process according to the invention is carried out, using aseptic coolers. This step is essential to prevent the wort from being re-infected with microorganisms in the environment during the cooling process. The wort is then alcoholically fermented by adding such a single-cell pure culture to yeast, namely to Saccharomyces cerevisiae, which breaks down histidine without the formation of histamine. This pure culture of Saccharomyces cerevisiae was the one deposited with the German Collection for Microorganisms, Grisebachstrasse 8, D-3400 Göttingen, under the number DSM 70449 and can be freely obtained there at any time.

Example 2

Production of a histamine-free wine

The grapes were pressed in the usual way and the must was degummed. However, the must was then pasteurized immediately by subjecting it to heat treatment at a temperature of 62-68 ° C. for about 30 minutes or by subjecting the must to a thin film of heat treatment at a temperature of 71-74 ° for only 1 minute C submitted.

The pasteurized must was then immediately cooled to a temperature of 15-20 ° C and inoculated with a pure single-cell culture of Lactobacillus delbrückii, which breaks down histidine without the formation of histamine. A suitable strain for this is the strain called M. Ro-gosa, which has the accession number DSM 20074 in the German Collection of Microorganisms (DSM), Grisebachstrasse 8, D-3400 Göttingen. This strain of microorganisms is also deposited at the American Collection Agency, American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 - USA, and has the number ATCC 9649 there and can be obtained freely from both depositaries.

Lactic acid fermentation of the inoculated must was carried out for 24-48 hours at a temperature of 15-20 ° C. During this lactic acid fermentation, all of the histidine present in the must was removed by the Lactobacillus delbrückii, and a portion of the sugar components of the must was also broken down by this Lactobacillus delbrückii to form lactic acid.

The must was then inoculated with yeast and the alcoholic fermentation carried out in the usual way. During this alcoholic fermentation, part of the lactic acid produced by the Lactobacillus delbrückii was broken down by the yeast, but part of the lactic acid remained in the wine, thereby improving the taste of the wine obtained.

In this case, the yeast that was used to carry out the alcoholic fermentation was not a single-cell pure culture, but a commonly occurring yeast that would degrade histidine to histamine.

At the end of the alcoholic fermentation, the wine was subjected to the usual treatments, i.e. cooled, filtered and bottled.

The wine was tested for its histamine content and no histamine was found at all. The taste, smell and color of the wine were excellent.

For comparison purposes, in a second batch the fresh must was pasteurized and cooled in the same way as explained above, but then there was no inoculation with the individual cell culture of Lactobacillus delbrückii, but the sterilized must was immediately inoculated with the same natural yeast as in the case of first working process. Alcoholic fermentation, cooling, filtering and bottling were carried out in the same way as in the process described above.

In this case, the color and the smell of the wine were again excellent, but the taste was less rounded and harmonious than that of the wine produced by the process according to the invention. In this case the wine showed a histamine content of 2.5 mg / kg, i.e. a histamine content, which is roughly the same order of magnitude as the usual white wine available on the market.

Example 3

Production of histamine-free sour cucumbers

50 kg of freshly harvested sour cucumbers, namely the non-mature small cucumbers, as are usually used for this purpose, were placed in a sealable container for the production of sour cucumbers. In this container, the cucumbers were subjected to steam treatment for 1 to 1 minute in order to kill any microorganisms present on the surface of the cucumbers.

Salted water was boiled and the usual spices, such as sliced onions, dill and the like, were added and the boil continued for 2 minutes to ensure that all germs present had been killed. The aqueous solution was then cooled to about 45 ° C and poured into the containers holding the cucumbers while maintaining the aseptic conditions. Inoculation was then carried out with the pure single cell culture of Lactobacillus delbrückii described in Example 2, which breaks down histidine without the formation of histamine. Lactic acid fermentation was allowed to proceed for 2 weeks at a temperature of 35-45 DC.

The acidic cucumbers were then filled into containers and sealed, while maintaining the sterile conditions.

When the product was tested for its histamine content, no histamine was found, from which it can be seen that the histamine content of the product is clearly below 0.1 mg / kg.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

11

641 644

For comparison purposes, another batch of freshly harvested cucumbers was treated in the same way, but the treatment of the product with the steam was not carried out, and after the addition of the aqueous solution there was no inoculation of the product, but the lactic acid fermentation became natural with the help of the same occurring lactic acid bacteria that were present on the starting materials.

Lactic acid fermentation was carried out at the same temperature conditions and for the same time as in the method described above and the product was filled into containers again.

In this case the product showed a histamine content of 9.8 mg / kg.

Example 4

Preparation of a histamine-free sauerkraut Freshly harvested white cabbage was cut, salted, and the usual spices such as juniper berries were added, and then steamed for 1 minute to kill any microorganisms present on the raw material. Then the starting product was left to stand until its temperature had dropped below 45 ° C. The single cell culture was then inoculated with Lactobacillus delbrückii, which degrades histidine without the formation of histamine and which was also used according to Example 2. Lactic acid fermentation was carried out for 2 hours at a temperature in the range of 35-45 ° C. No histamine was found in the end product obtained in this way.

Example 5

Preparation of a histamine-free yogurt Fresh milk was pasteurized and cooled to a temperature in the range of 35-45 ° C. A single cell pure culture of such a strain was then inoculated with Lactobacillus yoghourt, which degrades histidine without the formation of histamine. In addition, the starting material was inoculated with a single-cell pure culture of such an i5 strain of Streptococcus thermophilus, which also degrades histidine without the formation of histamine.

The milk inoculated with the two single cell pure cultures was left at a temperature of 35-45 ° C. for 12 hours.

20 The yoghurt thus produced was then filled into the containers and cooled.

When the end product was tested for histamine, no histamine was found and the product had an excellent taste.

s

Claims (11)

641 644 1. A process for the production of foods, luxury foods and feeds which are low in histamine or histamine-free, characterized in that the microorganism flora present on the starting material is killed and then a microorganism or. During any stage of the production of the food, luxury foods or feed a corresponding enzyme is added which breaks down the histidine present without the formation of histamine. 2. The method according to claim 1, characterized in that the added microorganism is a bacterium, preferably from the class of micrococci, the Pseudomonas species and lactobacilli, a fungus or a yeast, preferably a Saccharomyces species, or that one of such Microorganism obtained enzyme adds, which degrades histidine without the formation of histamine by transamination or deamination. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that a microorganism or an enzyme is added which breaks down the histidine present to form glutamic acid or ketoglutaric acid or corresponding salts. 4. The method according to any one of claims 1 to 3, characterized in that one produces a food, luxury food or feed, an essential step of the manufacturing process is a microorganism-induced conversion, such as alcoholic fermentation or lactic acid fermentation, by after the killing the microbial flora of the starting material carries out the conversion with such a pure culture of a microorganism strain which also breaks down any histidine present without the formation of histamine. 5. The method according to any one of claims 1 to 3, characterized in that one produces a food, luxury food or feed, an essential step of the manufacturing process is a microorganism-induced conversion, for example alcoholic fermentation or lactic acid fermentation, by following the The killing of the microorganism flora of the starting material in the starting material degrades histidine with the aid of an appropriate microorganism or an enzyme obtained therefrom without the formation of histamine, so that during the subsequent essential conversion of the starting material caused by the microorganism, practically no histidine is present in the latter or during this Conversion of histidine formed by the first microorganism or the enzyme is immediately degraded without the formation of histamine, so that there is no histidine which can be converted to H by the second microorganism via a histidine decarboxylation istamin can be converted. 6. The method according to claim 4, characterized in that one produces an alcoholic beverage, for example beer, wine or spirits, or an alcohol-free secondary product obtained from a product produced by alcoholic fermentation, for example alcohol-free beer or vinegar, by killing the germ flora of the starting material carries out the alcoholic fermentation by inoculation with such a pure culture of a yeast strain, in particular a Saccharomyces species, which, in addition to the alcoholic fermentation, degrades the histidine contained in the starting material without the formation of histamine. 7. The method according to claim 5, characterized in that one produces an alcoholic beverage, for example beer, wine or spirits, or a non-alcoholic secondary product obtained from a product produced by alcoholic fermentation, for example an alcohol-free beer or a table vinegar, by the Killing the microbial flora of the starting material adds such a pure culture of a microorganism or an enzyme that breaks down histidine contained in the starting material without the formation of histamine and, after histidine degradation has taken place, carries out the alcoholic fermentation by inoculation with a yeast culture, which may also be a yeast strain, the histidine present would decarboxylate to histamine. 8. The method according to claim 7, characterized in that the microorganism used for the degradation of histidine is a pure culture of a Lactobacillus, in particular a pure culture of Lactobacillus delbrückii, which forms lactic acid, and the histidine formed by proteolysis is broken down without the formation of histamine, whereupon one then takes place after Histidine degradation carries out the alcoholic fermentation by inoculation with a pure yeast culture which at least partially degrades ammonia and glutamic acid and the lactic acid formed by the lactobacillus. 9. The method according to claim 4, characterized in that one produces a food, luxury food or feed, in which an essential step of the manufacturing process is a lactic acid fermentation, for example a sour milk product, a yoghurt, a curd cheese, a sauerkraut or a silage is produced by after killing the microbial flora of the starting material, the lactic acid fermentation is carried out by inoculation with such a pure microorganism culture, preferably Lactobacillus pure culture, which degrades the histidine contained in the starting material without the formation of histamine, for example with a corresponding pure culture of Lactobacillus helveticus or Lactobacillactobacillus acid or Lactobacillus bulgarusus or Lactobacillus and especially preferred of Lactobacillus delbrückii. 10. The method according to claim 4, characterized in that the food produced is a cheese, wherein after the killing of the germ flora of the quark serving as the starting material or the milk coagulate produced by adding rennet, the cheese production is carried out by inoculation with at least one microorganism pure culture, which is carried out in Starting material containing histidine degrades without the formation of histamine. 11. Foodstuffs, luxury foods or animal feeds produced by the process according to claim 1, which are low in histamine or histamine-free.