GB2031948A

GB2031948A - Foods, beverages and fodder with a reduced content of histamine

Info

Publication number: GB2031948A
Application number: GB7928972A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1978-09-04
Filing date: 1979-08-20
Publication date: 1980-04-30
Also published as: DK368479A; CH641644A5; PT70133A; NZ191395A; NL7906522A; AU526269B2; DE2933041C2; CA1135110A; JPS5539798A; LU81643A1; HU183030B; AR225293A1; FR2434582B1; IT7925449A0; AT374089B; GB2031948B; ATA554279A; BE878568A; DE2933041A1; AU5028179A

Abstract

A process for producing foodstuffs, beverages and forages with a reduced content of biogen amines and preferably with a reduced content of histamine is described. People with good health are able to degradate histamine which they take up with food by a histaminase. Nevertheless, many people, female and male, are not able to perform a sufficient degradation of the histamine and if these people take up histamine this results in severe troubles, like headache, a development of gastric ulcera, diarrhoea, as well as several kinds of allergic reactions. If histamine is present in forages, like e.g. in silo-fodder, then the histamine passes over into the milk and accordingly also into the diary and products, so that it is desired to have not only foodstuffs but also forages available with low histamine levels or completely histamine free. It now was found out unexpectedly that foodstuffs, beverages and forages having a drastically reduced histamine content or being completely free of histamine can be prepared by adding during any desired step of the preparation of the foodstuff, beverage or forage a microorganism which performs a transamination or desamination of histidine, or an enzyme which results in a transamination or desamination of histidine. According to said process the histidine can e.g. be converted by a primary splitting off of ammonia into the innocuous glutamic acid. The new process is especially important for the production of such foodstuffs, beverages or forages, in which an essential step in the course of the preparation of said products is a conversion performed by a micro-organism like e.g. an alcoholic fermentation or a lactic fermentation. The naturally occurring microorganism strains generally perform a decarboxylation of histidine present in the starting material forming the undesired histamine. The new process makes it possible to produce such foodstuffs, beverages and forages which generally have a high histamine content, like e.g. alcoholic beverages, sour-milk products, pickled cabbage and silo-fodder, nearly or completely free of histamine.

Description

SPECIFICATION A process for producing foodstuffs, beverages and forages with a reduced content of biogen amines In recent years increased efforts were made in order to keep the level of harmful substances in foodstuffs, beverages and forages as low as poss ible, or to produce foodstuffs, beverages and forages completely free of noxious substances, like e.g. pesticides, heavy metals and heavy metal compounds, mycotoxines and other carciogenic agents. Also with regard to forages it is very important to keep them free of any harmful contaminants, because said nox ious substances can be accumulated in the living tissue of the animals, accordingly in the meat, and it is furthermore also possible that an enrichement of said harmful substances occurs in the milk, especially in cow milk and products therefrom.

It is well known in the artthatthe consumption of certain foodstuffs, especially of certain alcoholic beverages, like wine and beer, or of cheese and pickled cabbage can cause headache and migraine, and that furthermore foodstuffs and beverages of said kind can be also harmful for people who suffer from allergic reactions, diarrhoea, gastric ulcera and ulcera of the duodenum.

Investigations performed with beers showed that the above mentioned undesired effects of beer are more frequently encountered after the drinking of top fermented typs of beer than after the drinking of bottom fermented typs of beer. Nevertheless, the severity of the undesired reactions is more dependent from the quantity of beer consumed than from the certain type of beer, like pilsener beer, export beer and soon, consumed. Quite unexpectedly it however was found out that beers having the same content of alcohol, of fusel oil and of constituents of the hop extracts, can however result in quite different severity of the above mentioned undesired reaction.

It however also was found out that beers having a high protein content and content of amino acids are more harmful than beers with low protein content.

Biogenic amines generally are not present in the primary products of agriculture and animal breeding, but are produced in the course of the process of making the final foodstuff or beverage. The most important of said biogen amines is histamine.

It now was found out that several naturally occuring microorganism strains metabolize the histidine present inthe primary products of agriculture or animal breeding by decarboxylating the histidine forming the undesired biogen amine histamine.

Already very low levels of histamine can cause severe physiological reactions in persons sensitive to said substance, like e.g. headache, migraine, sickness and diarrhoea. Furthermore, histamine is an essential factor of allergies and anaphylaxies.

Histamine is a strong base, which forms salts with acids. Histamine is to be found in certain plants, like e.g. spinach and stinging nettle, and it is also present in several animal tissues and human tissues.

Basophilic leucocytes accumulate histamine in inactive form, it however can be liberated in the free form by allergic reactions or by ionizing irradiation or by damage of the tissue or by liberators for histamine.

It is also believed that there is a close relation-ship between the histamine content of the gastric mucous membrane, the ulcus recidive and the gastric secretion. By administering histamine receptor antagonists an essential mitigation of chronic grastric ulcera can be achieved. The same is also true with regard to mitigation of migraine. In order to achieve a migraine prophylaxis an healthy diet having a low histamine level or being histamine free is very important.

Persons having chronic duodenum ulcera accumulate significantly less histamine in the mucous membrane than persons having no gastric difficulties and a healthy duodenum. The healthy persons are able to degradate histamine which they take up by a histaminase. Other persons who do not have said enzyme suffer from headache (Horton Syndrom) which is also called "histaminzephalgis".

Also the Bing-headache syndrom and the Harris neuralgis, i.e. pains in the region of the eyes, the forehead and the temporar region, which occur only on one side of the head and generally only in certain periods of the day, frequently at night, and which kind of headache results in tearflow, inflammation of the eyes and the face, as well as swelling of the mucous membrane of the nose and soon, is effected by histamine and can be treated by the administration of antihistaminica.

A desensibilisation and a higher resistency against histamine can be achieved by administering histamine-azoprotein. Generally in the serum there is to be found an antihistamine factor and if said factor is missing, then this is regarded as a symptom of a latent allergy.

The histamine is secreted with faeces and urine.

Histamine enhances the contraction of the smooth muscles and results in a vasomuscular relaxation. In this way the blood pressure is lowered, however the permeability of the capillars increases, so that in the skin pustule and edema can occur. The human serum is able to bound free histamine. Persons which suffer from allergic reactions however have a decreased ability to bound histamine, i.e. bounding is hindered by a so called antipexin.

The level of histamine content in a foodstuff or a beverage, like e.g. wine, which is regarded as being already dangerous is 2 mg histamine per kilogram of foodstuff or beverage (see H. Marquart, "Die Weinwirtschaft", page 127, 1978).

The histamine content of several foodstuffs and beverages, which are on the market now and were prepared according to the usual prior art processes were determined.

Diary products and their histamine content are given in the following table I.

Tablel Histamine content of several diary products Product Histamine content in mgperkgproduct Pasteurized whole milk 0,3 Pasteurized whole milk 0,5 Pasteurized whole milk 0,7 Uperized whole milk 0,8 Uperized whole milk 0,8 Dikmelk 1,2 Yogurt 2,1 Yogurt 1,7 Tilsit cheese, fullfat 50,0 Tilsit cheese, fullfat 60,2 Gouda cheese, fullfat 54,0 Gouda cheese, fullfatfrom Austria 41,0 Gouda cheese, fullfatfrom Netherland 180,0 Camembert, fullfat 35,0 Camembert, fullfat 55,0 Stilton, fullfat, British 158,0 Harz cheese 390,0 Harz farmer cheese 383,0 The European fullfat cheeses have a fat content referred to the dry weight of 40 /O by weight.

It can be seen from the above stated table I that the histamine content of several kinds of whole milk is clearly below the tolerable highest limit according to the publication of H. Marquart, "Die Weinwirtschaft", page 127, 1978. It however is suprising that fresh milk already contains some amounts of histamine. It can be assumed that at least a part of said histamine content of the fresh milk is resulting from histamine which the cows have taken up together with the feed, and especially the silo-fodder, and which histamine passes over into the milk produced.

It furthermore can be seen from the above stated table I that however the histamine content of sour milk products, like yogurt, and of cheeses is far higher than the histamine content of fresh milk. As already mentioned above, several naturally occuring microorganism strains convert histidine, which is present in the milk protein, by decarboxylation into histamine, and therefore the lactic fermentation of the milk and the production of cheese results in an enormous increase of the histamine level. It can be seen from said table that if only 50 g of the Harz farmer cheese are eaten, nearly 20 mg of histamine are taken up, i.e. the 10-fold quantity of the histamine which is tolerable according to the above publication of Marquart for 1 kg of a beverage, like e.g. wine.

Also wines were tested and they also have a rather high histamine content, especially red wine and champagne. Also in this case the histamine is formed during the preparation of the wine or champagne by a decorboxylation of histidine by microorganisms resulting in the formation of histamine. The proteinous material of the musts and the mash are the source for histidine in said products. In the following table 11 the results of the determination of the histamine content of several kinds of wine are stated.

Table Histamine content of several sorts of wine Product Histamine content in my perky Table wine (Mosel) 0,5 Tabel wine (France) 1,6 High quality wine (Mosel) 1,1 Highest quality wine (Kabinett-wine from Rheinhessen) 3,0 Highest quality wine (Kabinett-wine from Rheinhessen) 4,5 Selection wine (Auslese, Rheingau) 1,7 Red wine (Ahr) 5,6 Tokay wine (Hungary) 1,1 Tokay wine, dry (Hungary) 3,2 Red wine (Austria) 7,4 Sparkling wine (Sekt, dry, Germany) 5,1 Champagne (France) 7,8 The tests preformed with beer however showed, as already outlined before, that types of beer which are richer in protein cause more severe troubles than beers with a lower protein content or amino acid content.

The testing of the histamine level of said beers showed that beers richer in protein also have an especially high histamine level, and that even alcohol free beers can show a high content of histamine.

In the following table Ill there are stated the histamine contents of several beers, and furthermore in said table there is given also the number of the tests.

Table lIl Histamine content of several sorts of beer Test No. Type ofbeer Histamine content in mail 1 Old- 4,5 2 Old- 9,8 3 Old- 8,0 4 Old- 6,8 5 Old- 5,0 6 White- 5,3 7 White- 4,9 8 White- 4,0 9 White- 4,7 10 Malt- 6,8 11 alcohol-free 7,5 12 alcohol-free 4,6 13 Export- 3,2 14 Export- 2,9 15 Export- 11,2 16 Export- 7,0 17 Export- 6,6 18 Export- 7,2 19 Pils- 3,5 20 Pils- 0,2 21 Pils- 7,4 22 Pils- 5,8 23 Pils- 5,2 24 Pils- 5,9 25 Pils- 8,7 It now was found out suprisinglythat in foodstuffs, beverages and forages the content of undesired biogen amines and especially the histamine level can be lowered essentially by adding in any step of the preparation of the foodstuff, beverage or forage a microoranism or an enzyme, by which the histadine present in the starting material is converted to innocuous products and not to the undesired histidine.

The object of the present invention accordingly is a process for producing foodstuffs, beverages, and forages with a reduced content of biogen amines, wherein during any desired step of the preparation of the foodstuff, beverage or forage there is added a microorganism which performs a transamination or desamination of histidine, or there is added an enzyme which results in a transamination or desamination of histidine.

When the inventive process is performed then the microorganism added can e.g. be a bacterium, preferably one of the class of micrococci, the species of the pseudomonas and of the lactobacilli, or it also can be a fungus or a yeast, preferably a species of saccharomyces, and the used strain of microorganisms must result in a transamination or desamination of histidine. Instead of the microorganism itself there can also be added an enzyme which was isolated from said classes of microorganism which results in a transamination or desamination of histidine.

According to a preferred embodiment of the inventive process a foodstuff, a beverage or a forage is prepared by adding in the course of the preparation a microorganism or an enzyme, which degradates the histidine which is present in the foodstuff, the beverage or the forage, forming glutamic acid or ketoglutaric acid or a salt of said acids. It is so possible to produce foodstuffs, beverages and forages which have a drastically reduced content of biogen amines, preferably histamine, or which are practically completely free of such biogen amines.

The inventive process is especially important when a foodstuff, a beverage or a forage shall be produced according to a process, in which an essential step is a conversion performed by a microorganism. Examples for such conversions performed by a microorganism are e.g. an alcoholic fermentation like e.g. the alcoholic fermentation performed during the process of the preparation of a wine or a beer, or a lactic fermentation or also the conversions occuring during the production of a cheese. The lactic fermentation is of importance not only with regard to the preparation of sour milk products, like sour milk, yogurt or curd, but also in the course of the production of a pickled vegetable, like e.g. pickled cabbage or a silo-fodder, a lactic fermentation has to be performed.

If the inventive process is applied to a process for the preparation of a foodstuff, a beverage or a forage in which an essential step is a conversion performed by a microorganism, then the following two embodiments are especially advantageous.

According to the one preferred embodiment of the inventive process, the microbes present in the starting material are killed and thereafter a conversion performed by the microorganism is made by inocculating the starting material with a pure culture of the corresponding microorganism which on the the one hand performs the desired conversion, and on the other hand results in a transamination or desamination of the histidine present in the starting material.

If for example according to said preferred embodiment of the invention, a lactic fermentation shall be performed, then from the corresponding technical cultures of lactobacillus helveticus or lactobacillus bulgaricus or lactobacillus acidophilus there has to be isolated a single cell culture, and said culture has to be cultivated in a nutrient medium to which pantothenic acid, niacin, riboflavin and calcium had been added.The so resulting pure cultures of the above mentioned microorganisms have an optimum fermentation temperature in the range of 40 - 44"C, and the maximum temperatures at which a fermentation can be performed with said microorganisms is in the range of 50 - 52"C. The above mentioned pure cultures of lactobacillus helveticus, lactobacillus bulgaricus and lactobacillus acidophilus respectively result in a homofermentative fermentation of glu cose and maltose converting said sugars into d(-) lactic acid and no carbon-dioxide is formed during said conversion. The pure cultures of said microor ganisms furthermore also desaminate several amino acids, like e.g. histidine, arginine, citrulline, ormithine, proline and hydroxyproline liberating ammonia.

Many of the microorganism strains, like e.g. lactobacillus strains which are deposited with culture collections are able to perform the desired desamination of histidine liberating ammonia. In as far as the pure cultures of said deposited strains perform said desamination they can be used as microorganism strains for performing the inventive process.

One example for such a deposited microorganism strain is M. Rogosa, which is deposited with the German culture collection "Deutsche Sammlung von Mikroorganismen (DSM)", Griesebachstrasse 8, 34 Göttingen, DT. with the deposition number DSM 20074. Said microorganism strain is also deposited with the American type culture collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, US, with the deposition number ATCC 9649.

In the same way as outlined above for the cu Itivation of lactobacillus helveticus, lactobacillus bulgaricus and lactobacillus acidophilus, there can be also cultivated other lactobacillus strains to result in the desired pure cultures, and according to said process also a single cell culture of lactobacillus delbrücki was prepared, which desaminates histidine liberating ammonia.

If a lactic fermentation is performed with the single cell cultures of the lactobacillus strains then in addition to the fermentative production of lactic acid also from histidine present in the starting material there is first splitt off ammonia, and the histidine converted to glutamic acid. Accordingly, no undesired conversion of the histidine into histarnine by splitting off carbon-dioxide from the histidine occurs.

If fresh milk is sterilized and thereafter inocculated with the above described single cell cultures of lactobacillus helveticus or lactobacillus bulgaricus or lactobacillus acidophilis, and the lactic fermentation performed at a temperature in the range of 40 - 45"C, then there results a sour milk or yogurt which is practically free of histamine or which respectively has a histamine content which is not higher than the histamine content of the fresh milk used for the preparation of the sour milk. Generally it is advantageous to optimize the growth conditions of the microorganisms during a lactic fermentation by adding to the milk used as starting material pantothenic acid, niacin and riboflavin.A further essential mineral ingredient, which pure cultures of lactobacillus helveticus, lactobacillus bulgaricus and lactobacillus acidophilus need for a rapid growth is calcium.

However fresh milk contains already sufficient calcium, so that a further addition of calcium is not necessary when a sour milk is prepared according to said process.

From the so resulting sour milk if desired also a curd, which is free of histamine, can be prepared, and said curd can be sold as such or it can be used for the preparation of special kinds of cheese, like e.g. "Mainzer Handkase" or "Olmützer Quargeln" or also Harz-cheese.

The single cell cultures of lactobacillus delbrücki cultivated according to the process outlined above were used to prepare pickled cabbage, pickled cucumbers and silo-fodder having a reduced histamine level or being free of histamine.

Pickled cabbage is produced by cutting white cabbage and adding the desired herbs and spices just in the same way as in the conventional processes for preparing pickled cabbage. Then however the resulting material is not fermented with the naturally occuring lactobacillus strains present in the starting material but the naturally occuring microbes are killed by submitting the product to a heat treatment. The so sterilized material is then inocculated with such a strain of lactobacillus delbrucki which converts histidine to glutamic acid, and thereafter the lactic fermentation and the further treatment of the product is performed analogous to the prior art processes for the preparation of pickled cabbage.

Just in the same way also pickled cucumbers are made by first sterilizing the starting material and thereafter inocculating with the strain of lactobacil lus.

As outlined above, the inventive process is also applicable for the preparation of fermented forages, i.e. the so called silo-fodder. In this case the green fodder used as starting material like e.g. grass, leguminous plants like clover and species of medicago, green maize and similar fodder are first submitted to a heat treatment in order to kill all the microbes present in the starting material, and thereafter the material is inocculated with the above mentioned single cell pure culture of the lactobacillus delbrücki. The inocculated material is then fermented in the usual way in the watertight tanks used for the usual preparation of fermented fodder, i.e. in the fodder silos.In the same way silo-fodder can be also prepared using as starting material partially dried up green fodder, and in this case there is first added a sugar containing material like molasses or beet chips to the partially dried starting material, and then said mixture is first submitted to the heat treatment in order to kill all the microbes present and then the inocculation with the above stated single cell pure culture of the lactobacillus delbrncki is performed. The so prepared histamine free silo-fodder or silo-fodder having a very low histamine level when fed to cows does not introduce into their organism histamine which is transferred by the organism into the milk. The silo-fodder prepared according to the inventive process accordingly makes it possible to produce practically histaminefree fresh milk.

If the conversion performed by the microorganism is an alcoholic fermentation, then the preparation of an alcoholic fermentation product, which is free of histamine, can be achieved by sterilizing the used starting material and performing the alcoholic fermentation with such a strain of saccharomyces which performs the desired alcoholic fermentation and furthermore a transamination or desamination of histidine present in the starting material.

If according to said embodiment of the invention a wine is prepared, then the used must is first steril ized and the so resulting product then inocculated with the above mentioned single cell pure culture of saccharomyces. In the course of the alcoholic fer mentation then any histidine present in the starting material is transaminated or desaminated, and accordingly not converted to the undesirable histamine.Some of the above mentioned single cell pure cultures of saccharomyces however are not best suited for the performance of the alcoholic fermentation then therefore it might be more advantageous to perform the production of wine according to the second preferred embodiment of the invention described later, i.e. by first submitting the histidine present in the starting material to a transamination or desamination using an other microorganism strain than a saccharomyces strain and performing thereafter the resulting product which contains no more histidine to the alcoholic fermentation with saccharomyces.

Also the secondary fermentation in the process for making champagne is generally not performed using a special single cell culture of yeast which in addition to the alcoholic fermentation also performs a transamination or desamination of histidine, but also in this case generally a pure culture of a lactobacillus is added in order to perform the transamination or desamination of histidine. Also said process is explained in detail lateron.

It can be seen from table I that some sorts of cheese are specially rich in histamine. For the production of many sorts of cheeses, like e.g. a special kind of hard cheese or soft cheese, generally not one single strain of a microorganism is necessary but frequently the special kind of cheese is prepared by the action of several different microorganisms. If accordingly a cheese free of histamine or having a low histamine content is prepared according to the inventive process, then it is necessary to cultivate from all the microorganism strains, which are neces saryforthe preparation of the special kind of cheese desired, such single cell cultures which result in a transamination or desamination of histidine.

Sometimes this might be rather difficult.

The Roquefort-cheese is produced by a fermentation of a milk-coagulate, which is prepared by adding rennet to the milk of sheep. A certain blue strain of mould then converts said milk-coagulate into the Roquefort-cheese. According to the traditional process for making Roquefort-cheese the inocculation of the milk-coagulate is achieved by adding to it mouldy bread. From the blue mould of the Roquefort-cheese there now was cultivated such a single cell culture which results in a desamination of histidine. Roquefort-cheese now was prepared according to the inventive process by starting from the milk-coagulate, which was produced according to the prior art technique of producing a milk coagulate for Roquefort-cheese, and to said coagulate then there was added the pure culture of the blue mould.The further production of the cheese and the ageing of the product was performed according to the prior art process. The resulting product was a Roquefort-cheese which had an extreme low content of histamine which however as to its taste and its smell was rather similar to Roquefort-cheese prepared according to the traditional procedure.

According to the second preferred embodiment of the inventive process for producing a foodstuff, a beverage or a forage, according to a process in which an essential step of the preparation is a conversion performed by a microorganism there is used for said conversion not a special strain of said microorganism but a naturally occuring strain of it, which can also result in the undesired decarboxylation of histidine to form histamine. According to said second preferred embodiment of the invention, however before the conversion with the microorganism or at the same time the conversion with the microorganism occurs, there is performed a degradation of the histidine present in the starting material forming innocuous by-products, preferably a desamination or transamination of the histidine present, or the histidine formed.When thereafter the fermentation is performed with a naturally occuring microorganism there is either no more histidine present or histidine formed during the fermentation is immediately converted into innocuous by-products, so that a naturally occuring microorganism strain has no histidine available which can be decarboxylated by it to form the undesired histamine.

According to said second preferred embodiment of the inventive process accordingly a fooodstuff, a beverage or a forage is prepared according to a process wherein an essential step of said process is a conversion performed by a microorganism, like e.g.

an alcoholic fermentation or a lactic fermentation, and in said process before the stated conversion, or together with the stated conversion performed by the microorganism, the histidine present in the starting material orthe histidine formed in situ is desaminated ortransaminated by a second microorganism or desaminated ortransaminated by a corresponding enzyme and so removed from the material. Therefore, when thereafter or simultaneously the essential conversion performed by the first microorganism occurs or proceeds, in the starting material practically no histidine is present which could be decarboxylated by the microorganism performing the essential conversion into the undesired histamine.

Accordingly, if the the product to be prepared is an alcoholic beverage then it is possible according to said second preferred performance of the inventive process to produce a final product free of histamine by killing before the alcoholic fermentation the microbes present in the starting material introducing thereafter a microorganism or an enzyme, which performs a transamination or desamination of the histidine present in the starting material and as soon as all the originally present histidine was converted into innocuous by-products the alcoholic fermentation is induced by inocculating the material with a yeast culture. The used yeast culture can be in this case also such a culture which as such would perform a decarboxylation of the histidineto form histamine. At the moment the yeast is added however in the material to be fermented no longer any histidine is present, and also histidine which is in situ generated by dyeing cells of the yeast immediately converted into innocuous secondary products by the first microorganism which is still present or the enzyme which was added before. Therefore, the yeast culture performing the alcoholic fermentation does not find any essential amounts of histidine present, which could be converted to the undesired histamine.

The last mentioned embodiment of the invention can be e.g. used for the production of a beer. According to the prior art procedures for making a beer, already in those working steps which are performed before the alcoholic fermentation is induced there are present microorganisms which can result in a conversion of histidine into the undesired histamine.

Histidine is present in the protein component of the cereals, e.g. in the protein of the barley, in essential quantities. According to the book "Nährwert- Tabellen" of Souci, Fachmann & Kraut, Wiss. Verlagsgesellschaft, Stuttgart, 1977, barley flower contains 1,4 g to 2,5 g of histidine per kilogram of barley flower. Barley from which the glumes were removed has a protein content of 97 - 113 g per kilogram, and this about corresponds to 1,8 g of histidine per kilogram of barley. During the preparation of the malt a proteolysis of the proteins of the cereals occurs in the wortmasher and in the brewing pan. At this time already microorganisms, like e.g. saccharomyces diastaticus, lactobacillus fermentu m, lactobacillus pastorianus and pediococcus cerevisiae are present, which can result in a decarboxylation of the histidine to form histamine.The microorganisms present are of course not especially cultivated pure single cell cultures, but the microorganism strains present in the natural environment and said strains, as already explained before, generally metabolize histidine by decarboxylating it which results in the undesired histamine.

According to the prior art production of beer, the decarboxylation of the histidine which results in the undesired histamine performed by the above stated microorganisms is not stopped until the end of the saccharification process, and thereafter stopped by the thermal inactivation. In the course of the primary alcoholic fermentation and during the ripening microbes present in the fermentation tanks and among them also several strains of saccharomyces cerevisiae metabolize still present histidine by decarboxylating it, so that in the beer essential amounts of histamine are formed.

It now was surprisingly found out that beer, which is free of histamine can be prepared according to the inventive process by performing already before the alcoholic fermentation a thermal inactivation of the microorganisms of the malt and a thermal inactivation of the microorganisms present in the mash.

Proceeding like this no histamine is formed in the starting material before the alcoholic fermentation of the beer is induced by the inocculation with yeast.

The alcohlic fermentation can be made according to both preferred embodiments of the invention, i.e.

either by using a pure culture of saccharomyces cerevisiae which besides the alcoholic fermentation also results in a desamination of the histidine (this process will be explained lateron in the example), or by removing the histidine of the starting material by the action of a microorganism, which results in a desamination ortransamination of the histidine, or by the addition of a corresponding enzyme. This procedure will be described now.

The mash is first sterilized by a heat treatment, preferably by heating it for a period of 30 - 40 minutes to a temperature in the range of 62 - 65"C.

Thereafter the mash is cooled to a temperature of 45"C, and at said temperature the pure culture of lactobacillus delbrücki, which was prepared as explained before, is inocculated. After said inocculation of the lactobacillus delbrncki into the mash there occurs a violent homofermentative conversion of glucose and maltose forming lactic acid, and furthermore at the same time from all the histidine present primarily ammonia is splitt off and it is metabolized resulting in glutamic acid. During this step periodically samples are taken in order to monitor the increase in the concentration of lactic acid, and at the same time occuring conversion of histidine into glutamic acid.

At the end of said acidification step nearly all the histidine which had been originally present in the mash is converted into L-glutamic acid, and thereafterthe main fermentation is performed, i.e. the alcoholic fermentation of the beer by adding to it yeast, i.e. saccharomyces cerevisiae. In this case the alcoholic fermentation is preferably performed by inocculating such a yeast pure culture, which metabolizes also the ammonia which had been formed before in the course of the desamination of the histidine, as well as also the formed glutamic acid and furthermore also at least a part of the lactic acid which had been produced by the lactobacillus delbrücki. Accordingly, preferably the alcoholic fermentation is performed using such a strain of saccharomyces cerevisiae which is able to remove the side-products which had been before produced by the lactobacillus delbrücki. On the other hand however the lactobacillus delbrücki is still present when the alcoholic fermentation is performed, and therefore any histidine which is formed in situ by a proteolysis of yeast cells is again removed by the lactobacillus delbrücki according to the desamination reaction. Therefore, it is possible to produce according to said process a beer which has an extremely low histamine content, or is completely free of histamine.

The above stated process can be also applied to the production of an alcohol-free beer, and therefore it is also possible to produce alcohol-free beers which are free of histamine, or have an extremely low content of histamine.

The above explained conversion of histidine into ammonia and glutamic acid can be also performed in the course of the preparation of other alcoholic products, like wine, spirits and so on, and accordingly corresponding histamine-free alcoholic products can be produced. From said alcoholic products then there can be prepared histamine free secondary products, like e.g. a histamine-free vinegar.

If a histamine-free wine is prepared according to the embodiment of the invention explained above, then in the same way as in the process for preparing the beer first a thermal sterilisation of the musts, or if a red wine shall be prepared optionally also a thermal sterilisation of the mash is performed, in order to kill all microbes present in the starting material.

Thereafter, either the must or the mash is submitted to the alcoholic fermentation by inocculating it with a single cell culture of saccharomyces cerevisiae, which does not decarboxylate histidine, or from said sterilized starting material before the alcoholic fermentation the histidine is first removed by inocculating the single cell pure culture of the lactobacillus delbrücki prepared according to the process outlined before, or by inocculating an other lactobacillus strain, which results in a desamination of the histidine present.

If said first mentioned embodiment of the inventive process is performed, then after the inocculation of the single cell culture of the lactobacillus delbrucki the must or the mash is maintained at the optimum fermentation temperature for said microorganisms which is about in the range of 40 - 45"C until the histidine present in the must or the mash had been converted into ammonia and glutamic acid. Also in this case the lactobacillus delbrucki or other lactobacillus cultures used result in a lactic fermentation of carbohydrates of the musts or the mash resulting in lactic acid.As soon as the histidine presend had been metabolized by the lactobacillus the main fermentation, i.e. the alcoholic fermentation, is performed by inocculating a pure culture of yeast Also in this case preferably such a strain of saccharomyces cerevisiae is inocculated which metabolizes in the course of the alcoholic fermentation also the ammonia, the glutamic acid and the lactic acid, which were formed by the preceeding conversion with the lactobacillus as by-products.

The above outlined process can also advantageously be used for the preparation of distilled alcoholic beverages, i.e. spirits. If a clean destillation of the raw material is performed generally no essential amounts of histamine enter into the destilled material during the destillation. Nevertheless, it was found out that spirits at present on the market, like e.g. cognac, whisky and similar spirits often contain great amounts of histamine. Also in this case either the alcoholic fermentation can be performed selecting a special yeast pure culture or before the alcoholic fermentation a degradation of the histamine present can be performed using the pure culture of lactobacillus delbrücki or an other pure culture of a lactobacillus, which converts all the histidine present in the starting material into innocuous secondary products.

According to a further embodiment of the inventive process the formation of histamine can be also prevented in the course of such a conversion performed with microorganisms in which generally in the starting material no essential amounts of histidine are present, in which however histidine is formed by a proteolysis of dead cells of the microorganism making the essential conversion in the process for making the foodstuff, beverage or forage.

Atypical example for a process in which the starting material is free of histidine, in which however histidine is formed in situ is the preparation of a sparkling wine. The sparkling wine is prepared by adding to a wine, which resulted from the primary fermentation, a mixture of succrose and yeast and thereafter the secondary fermentation is performed in a closed vessel, like e.g. a pressure tank or a closed bottle. During said secondary fermentation the added succrose is fermented by the living yeast cells to result in alcohol and carbondioxide, which remains dissolved in the raw sparkling wine. An undesired process occuring in the production of sparkling wine is that the proteins of the yeast, respectively the histidine of said proteins is decarboxylated by the living yeast cells to result in histamine.It can be seen from the table II that because of said degradation of the proteins champagne has a far higher content of histamine than the average value of a white wine.

If the inventive process is applied for the preparation of a sparkling wine, which is nearly or completely free of histidine then it generally is especially advantageous to perform said process according to the second preferred embodiment of the inventive process. Accordingly, to the wine which resulted afterthe primary alcoholic fermentation of the musts there is added a mixture of the pure culture of a lactobacillus strain, which results in a desamination of the histidine, together with succrose and yeast.

The pure culture of the lactobacillus strain was prepared according to the process described before.

After said mixture is added to the wine, the secondary fermentation is performed in the closed vassel and the histidine which is formed by a proteolysis of yeast cells is immediately desaminated by the pure strain of the lactobacillus. Therefore, the still living yeast cells have no more any histidine available which could be decarboxylated by the yeast to form the undesired histamine. After the secondary fermentation the further treatment of the raw sparkling wine is performed according to well known prior art processes, i.e. the lactobacillus is removed from the raw sparkling wine together with the yeast either according to the traditional champagne method by the so called "disgorging" or by submitting the raw sparkling wine to a counter pressure filtration.Several methods for the preparation of sparkling wine are e.g. in detail disclosed in the USA-patent ........

USA patent application 925,669, filed July 18, 1978 of A. Lembke.

From pure cultures of microorganisms which result in a transamination or desamination of the histidine there was isolated the ferment complex which is responsible for said transamination or desamination. This isolation was effected by mechanically disrupting the cells and isolating a liquid material containing said desired ferment complex. Said liquid was submitted to a lyophilization. It was found out that 10-39 of the resulting dry enzyme material are able to degradate 1 g of histidine. Said dry material containing the enzyme complex was used in the processes described before instead of the living pure cultures of lactobacillus, i.e. lactobacillus delbrncki, in order to convert histidine present in the starting material or histidine formed in situ during an alcoholic fermentation into innocuous by-products.

It was found out that the results achieved with the dry enzyme containing material were analogous to those gained by the use of the living pure culture of lactobacillus.

Histidine is as such an essential amino acid, i.e. an amino acid which the human being has to take up with the diet. In as far as a product prepared according to the inventive process is rich in proteins, like sour milk products or cheese, the microorganism strains used according to the inventive process for the performance of the lactic fermentation and for the preparation of the cheese respectively will not result in that the so prepared foodstuff is completely free of histidine. The special microorganism strains only prevent that the microorganism metabolizes histidine by decarboxylating it and forming the undesired histamine. Only some of the histidine present in said starting materials with high protein content will be metabolized by the microorganism so that innocuous by-products, like glutaminic acid, are formed.

If however the foodstuff or beverage prepared according to the inventive process is no essential source of protein, like e.g. an alcoholic beverage, then it is not at all critical to remove from said products any histidine present completely, because the histidine necessary for the human organism in any case must be supplied by other foodstuffs having a high protein content, like meat, eggs, milk products and soon.

The invention now will be illustrated by an example.

EXAMPLE Preparation of a nearly histamin-free beer When beer is produced according to the prior art processes, then three essential working steps have to be performed. We e.g. refer to the explanation of the beer production given in the "Lehrbuch der chemischen Technologie" of OST-RASSOW, Edition 1965, pages 1394-1405, Johann Ambrosius Barth, Publishing Company, Leipzig.

1. Preparation ofthe malt: The most important raw material for the preparation of beer, i.e. barley, unless submitted to a special treatment, does not contain fermentable extractable materials. The barley is submitted to germination in the malt house, and during said process enzyms are formed, which convert the water insoluble starch of the barley into soluble sugar. Further enzyms are generated during the germination which convert also further materials of high molecular weight into substances with low molecular weight. Thereafter the germinated barley is dried, and thereby dyestuffs, flavours and fragrances are developed.

2. The brewing: The malt is crushed and treated with warm water (mashing). Substances which are already water sol uble are dissolved and further amounts of starch are deg;adated enzymatically into soluble maltose. After the filtration of the wort the hop is added and the wort is boiled and more or less water can be vapo rated during the boiling. The hop wort is separated from the hop.

3. The alcoholic fermentation: After the hop is removed from the hotwort, said wort has to be cooled within a very short period of time to the fermentation temperature. The fermentation temperature is in the range of 5 - 7"C if a bottom fermented beer is produced, and in the range of 14 - 1 6 C if a top fermented beer is brewed. The brewer's yeast is added and the alcoholic fermentation performed at the stated temperature generally for 8 10 days. The so resulting beer then is submitted to a post-fermentation in storage cellars and finally the yeast is removed by filtration and the beer filled into bottles or barrels.

Said prior art brewing process is modified as follows if according to inventive process a beer free of histamine is prepared: It was surprisingly found out that the surface of the barley converted into the malt is already contaminated with many microorganisms. This results in that during the mashing in the presence of water not only the desired conversion of the insoluble starch into soluble sugars occurs but also the rather large quantities of histidine present in the proteins of the barley are decarboxylated by said microorganisms to form the undesired histamine. Furthermore, it was found out that the microorganisms which metabolize the histidine yielding the undesired histamine are sensitive to heat.

According to the inventive process therefore the undesired microbes are removed from the surface of the germed barley by a short treatment with boiling water (blanching) or by immersing it into water having a temperature of 75 - 100"C. The water is dis- carded and the barley washed further several times with water of a temperature of 50"C.

Thereafter, according to the prior art processes the malt is crushed and the mashing process and also the boiling of the wort after the hop had been added is performed in the same way as in the prior art process.

A further essential step of the inventive process is the cooling of the wort after the hop had been removed. Said cooling has to be made with absolutely clean sterilized open coolers or closed coolers, in order to prevent that during the cooling a reinfection of the wort with undesired microorganisms occurs, which microorganisms could result in a decarboxylation of the still present histidine to form histamine) Also when the wort is brought to the desired extract content by adding water, an absolutely sterile water has to be used.

Thereafter, the innocculation of the wort with the yeast is made by using a single cell culture of saccharomyces cerevisiae which strain does not carboxylate histidine forming histamin.

The special pure culture of saccharomyces cerevisiae was cultivated according to similar processes as outlined before for the cultivation of lactobacillus strains.

Claims

1. A process for producing foodstuffs, beverages and forages with a reduced content of biogen amines, wherein during any desired step of the pre paration of the foodstuff, beverage or forage there is added a microorganism which performs a transami nation or desamination of histidine, or there is added an enzyme which results in a transamination or desamination of histidine.

2. Process according to patent claim 1, wherein the microorganism added is a bacterium, preferably one of the class of micrococci, the species of the pseudomonas and of the lactobacilli, a fungus our a yeast, preferably a species of saccharomyces, or that there is added an enzyme which was isolated from said classes of microorganism which results in a transamination or desamination of histidine.

3. Process according to patent claim 1 or 2, wherein a foodstuff, a beverage or a forage is prepared, which has a drastically reduced content of said biogenic amines or which is completely free of biogenic amines, preferably of histamine, wherein there is added a microorganism or an enzyme which is able to degradate the histidine which is present in the foodstuff, the beverage or the forage, forming glutamic acid or ketoglutaric acid or a salt of said acids.

4. Process according to one of the patent claims 1 - 3, wherein a foodstuff, a beverage or a forage is prepared, in which process an essential step of the preparation is a conversion performed by a microorganism, e.g. an alcoholic fermentation or a lactic fermentation, wherein said conversion is performed using a microorganism strain, which results in a transamination ordesamination of the histidine present in the foodstuff, the beverage or the forage.

5. Process according to one of the patent claims 1 - 4 for preparing a foodstuff, a beverage or a forage, wherein an essential step of said process is a conversion performed by a microorganism like e.g.

an alcoholic fermentation our a lactic fermentation, in which process before said conversion performed by the microorganism there is first removed from the starting material the histidine contained therein and /orthe histidine which is produced as byproduct during the conversion performed with the microorganism by the application of a further microorganism which results in a desamination ortransamination of the histidine, or by an enzyme which results in a desamination ortransamination of the histidine, so that in the course of the essential conversion of the starting material performed by the first microorganism the starting material is practically free of histidine or histidine which is formed as byproduct by the conversion of the first microorganism is again removed by the second microorganism or the used enzyme, so that there is no longer present histidine which can be converted to histamine by the microorganism performing the conversion of the starting material.

6. Process according to patent claim 4, characterized in that an alcoholic beverages like e.g. beer, wine or spirits are prepared, or that a secondary product which is free of alcohol is prepared from an intermediate product resulting from an alcoholic fermentation, like e.g. a beer which is free of alcohol or a vinegar, in which process the microorganisms present in the starting material are killed before the performance of the alcoholic fermentation and thereafter the alcoholic fermentation is performed by innoculating the material with such strain of yeast, preferably a pure culture of a saccharomyces species, which in addition to the alcoholic fermentation also results in a transamination or desamination of the histidine present in the starting material.

7. Process according to patent claim 5, characterized in that an alcoholic beverage, preferably beer, wines or spirits, or a secondary product which is free of alcohol is prepared from a product made by an alcoholic fermentation, like e.g. a beer free of alcohol or a vinegar is prepared, in which process before the performance of the alcoholic fermentation the microorganisms present in the starting material are killed, and thereafter a microorganism or an enzyme is added, which results in a transamination or desamination of the histidine present in the starting material, and that after the histidine is degradated the alcoholic fermentation is performed by the inocculation with a yeast culture, which yeast culture can be also a strain of yeast which would decarboxylate any present histidine to form the undesired histamine.

8. Process according to patent claim 7, in which the microorganism used for the degradation of the histidine is a pure culture of a lactobacillus, preferably a pure culture of lactobacillus delbrücki which performs a lactic fermentation, and furthermore converts the histidine formed by a proteolysis into glutamic acid or ketoglutaric acid, in which process after the degradation of the histidine the alcoholic fermentation is performed by an inocculation with a pure culture of a yeast, which at least partially metabolizes ammonia and glutamic acid and furthermore also the lactic acid produced by the lactobacillus.

9. Process according to patent claim 4, wherein a foodstuff, a beverage or a forage is prepared applying a process in which an essential step is a lactic fermentation, e.g. a sour milk product, a yogurt, a curd, a pickled cabbage or a silo-fodder is prepared, in which process in the used starting material the microorganisms present are killed and the lactic fermentation is performed by inocculating with a pure culture of lactobacillus which converts histidine present in the starting material forming glutamic acid, e.g. by using as corresponding pure culture of lactobacillus delbrncki.

10. Process according to patent claim 4 or 5, in which the prepared foodstuff is a cheese, in which process as starting material there is used curd or a milk-coagulate produced by the addition of rennet and the preparation of the cheese is performed by inocculating said starting material with at least one pure culture of a microorganism which results in a transamination or desamination of the histidine which is present in the starting material.

11. Foodstuff, beverage or forage, which has a reduced content of biogenic amines or is free of biogenic amines, especially histamine.