AU703457B2 - Method for malting barley and improved malt thus obtained - Google Patents

Abstract

In malting barley, involving (a) a soaking stage, (b) a germination stage, and (c) a kiln-drying stage, the soaking stage includes an acid soaking sub-stage at a pH after stabilisation of 3.5-4.6, at a ratio between the vol. of soak water and the amt. of barley such that the activity of the grain during germination is not modified.

Description

WO 96/36740 PCT/FR96/00713 METHOD FOR MALTING BARLEY AND IMPROVED MALT THUS OBTAINED The subject of the present invention is a method for malting barley, that is to say a method including a steeping step, a germinating step and a kiln drying step, which makes it possible to obtain a low nitrosodimethylamine (NDMA) content.

During the 1980s, brewers took into consideration the presence of NDMA in beer, this substance being believed to be carcinogenic and mutagenic to man. The NDMA present in malt seems to originate from the reaction of secondary and tertiary amines, synthesized by barley during malting, with nitrogen oxide forms (NOx) present in the surrounding air.

Depending on the writers, various types of amines would be the major precursors of NDMA.

T. Wainwright et al., in 1986, following much prior work and in a summarizing article entitled "The Chemistry of Nitrosamine Formation: Relevance to Malting and Brewing" in Journal Inst. Brew., Jan.-Feb.

1986, Vol. 92, p. 61, suggests that hordenine is the major precursor of NDMA and attributes a secondary role to the other amines of dimethylamine (DMA), sarcosine and gramine type.

In 1985, work published by M. M. Mangino R. S. Scanlan and entitled "Nitrosation of the Alkanoids Hordenine and Gramine, Potential Precursors of N-Nitrosodimethylamine in Barley Malt" in Journal Agric. Food Chem., 1985, Vol. 33, p. 705 demonstrates the important role of malt gramine in NDMA formation phenomena because of its very high susceptibility to being nitrosated by NOx agents in the air.

Finally, several writers suggest that the main precursor of NDMA in malt is dimethylamine (DMA), a secondary amine synthesized in the radicles of barley during malting. Work published in 1981 by S. Sakuma et al., in an article entitled "Formation of N- 2 Nitrosodimethylamine during Malt Kilning", published in Reports Res. Lab. Kirin Brewery Co. in 1981, Vol. 24, p. 20, indicates that there exists a direct relationship between the amount of NDMA produced in malt and the amount of DMA in green malt and NOx in the air. More recently, L. J. Yoo, in an article in 1992 entitled "Precursors of Nitrosodimethylamine in Malted Barley. Determination of Dimethylamine" in Journal Agric. Food Chem., Vol. 40, p. 2224, confirms, on the basis of the amounts of amines measured for malt and the production of NDMA after nitrosation, that DMA is the main precursor of NDMA and that its concentration in green malt makes it possible to deduce the amount of NDMA potentially produced on kiln drying. At the same time, in this article and in an article adjoining the latter, by B. Poocharoen et al, entitled "Precursors of Nitrosodimethylamine in Malted Barley. Determination of Hordenine and Gramine" published in Journal Agric. Food Chem., Vol. 40, p. 2220, some of these writers place in great doubt the role of hordenine and of gramine in the production of NDMA in malt, which suggests, according to the most recent data, that the main precursor of NDMA is dimethylamine (DMA) produced in the radicles during germination of the barley.

Currently, the method for reducing the NDMA level commonly used on an industrial scale is based on the addition of sulphur in the kiln drying, and on the employment of burners with a low NO 2 level or alternatively of indirect heating burners, various options explained in an article by W. Flad entitled "Minimizing Nitrosamine Formation During Malt Kilning", which appeared in the November 1989 issue of Brauwelt International.

However, such a method, in addition to the fact that it does not give satisfaction in all cases, in the sense that some industrial batches of malt treated by this method still contain unacceptable doses of NDMA, is also questionable because of the fact that the use S of sulphur leads to the emission of sulphur dioxide 3 during its combustion. The use of sulphur in malting could be brought into question in the medium term because of changes in environmental protection standards.

Now, in the current state of knowledge, there exists no industrially usable method which makes it possible to avoid the use of sulphur and which can result in a low NDMA level without, moreover, exhibiting significant disadvantages with respect to the quality of the malt obtained and/or with respect to the industrial implementation proper and/or with respect to the yield.

Some methods have been provided which make use of an addition of a compound, generally an acid, after the germination and immediately before the kiln drying or alternatively during the kiln drying.

The article by William J. Olson entitled "Control of Malt N-Nitrosodimethylamine in Direct Kilning Without Use of Sulfur Oxides", published in "MBAA Technical Quarterly", Vol. 19, No. 2, 1982 63 to 67), envisages the addition of phosphoric acid and of sugar (dextrose or fructose) before kiln drying, this resulting in NDMA levels which remain of the order of 2 to 3 gg/kg, it being understood that the addition of H 2 P0 3 alone results in malts exhibiting NDMA levels of 20 to 30 gg/kg and the addition of sugar alone results in malts exhibiting NDMA levels of 5 to 8 rg/kg. This method gives variable results, as shown in the article by W. J. W. Lloyd and S. J. Hutchings, entitled "Suppression of NDMA Formation in Malt", which appeared in the report of the 1983 EBC Congress 62). Finally, the addition of phosphoric acid before kiln drying produces foul smells during kiln drying which render the industrial use of the method highly problematic.

Another method for acidification immediately before kiln drying is described in the article by W. A. Hardwick et al. entitled "N-Nitrosodimethylamine in Malt Beverages Anticipatory Action by the Brewing 4 Industry", published in "Regulatory Toxicology and Pharmacology 2" 38 to 66), Academic Press Inc., 1982. It consists of an acidification of the surface of the grain at a pH of the order of 2.5 54), so as to decrease the amount of NDMA. The phenomenon involved consists in "freezing" the hordenine, one of the potential precursors of NDMA, which is, under these conditions, in its protonated form incapable or virtually incapable of giving rise to formation of NDMA in the presence of NOx. This technique does not appear to have given rise to industrial applications, for the reasons set out in the article by P. A. Brookes, entitled "The Effects of Nitrosodimethylamine Palliatives on Malt Properties" and published in the Journal of Inst. Brew., 1982, Vol. 88, p. 256-260, namely significant problems of handling and of safety which would be presented by use on an industrial scale.

Another known means for decreasing the amount of hordenine is to slow down germination.

Such a slowing down can be obtained, according to the prior art, by addition of ammonium persulphate or of NO 2 after the steeping and before the germination or alternatively by acidification in the steeping making it possible to limit the growth of the germ or alternatively by the choice of conditions (moisture content, temperature, germination time) making it possible to limit the growth of the germ.

The addition of ammonium persulphate or of bromate during malting is envisaged in the article by T. Wainwright and D. D. O'Farrell entitled "Ammonium Persulphate in Malting: Control of NDMA and Increased Yield of Malt Extract" which appeared in the Journal of Inst. Brew., May-June 1986, Vol. 92, p. 232-238. This method, which has not been used on an industrial scale, requires the addition of gibberellic acid.

The use of acidification of the steeping water for decreasing the growth of the embryo is known from the three following articles by T. Wainwright:

A

j i UJ I .4" 5 "Nitrosodimethylamine: Formation and Palliative Measures", which appeared in Journal Inst.

Brew., 1981, Vol. 87, p. 264-265; "N-Nitrosodimethylamine Precursors in Malt", p.71 80 (cited above); "Nitrosamines in Malt and Beer", which appeared in Journal Inst. Brew. (1986), Vol. 92, p. 73 to It results from these articles that any action, in particular an appropriate acidification during steeping, which limits the growth of the embryo reduces the amount of hordenine, a potential precursor of NDMA mainly formed in this tissue, by a factor of approximately 10 and consequently the amount of NDMA produced in the finished malt.

However, the most recent of these three articles ("Nitrosamines in Malt and Beer") shows 76, Vol. 1, 3 and 4) that all the treatments which can be envisaged for decreasing the growth of the embryo have negative effects on the quality of the malt and cannot be used directly on an industrial scale.

It should be noted, by way of information, that methods, not used industrially, employing inactivation of the embryo have been suggested by the following two articles: G. H. Palmer et al., "Combined Acidulation and Gibberellic Acid Treatment in the Accelerated Malting of Abraded Barley", published in J. Inst.

Brew., Vol. 78 (1972), p. 81-83; D. Crabb and G. M. Palmer, "The Production and Brewing Value of Malt made without Embryo Growth", which appeared in ASBC Proceedings, 1972, p. 36 to 38.

These articles do not relate to the problem of NDMA, which would only be because of their date of publication prior to the identification of the problems due to NDMA. Their aim is to kill the embryo in order for the latter not to develop radicles and plumules which would result in a loss of extract. The method involves the addition of gibberellic acid. It is /NT F f ~g -6necessary to start from abraded barley because of the fact that the pericarp of germinated barley is impermeable to gibberellic acid.

The first article gives, as example, the addition to abraded barley of 0.006N

H

2 S0 4 in the first steeping and 0.01N

H

2 S0 4 in the second steeping, as well as of gibberellic acid (0.5 to 0.1 ppm) at the end of each steeping. This has the effect of inactivating the embryo, of reducing the size of the radicles (by more than of increasing the amount of soluble proteins, of stimulating the aleurone layer and of increasing the amount of extract because of the absence of growth of the embryo and of the radicles.

The second article relates to destroying the abraded barley embryo using 0.02% of acid (unspecified), with addition of gibberellic acid making it possible to supply that which would have been produced during normal germination.

These methods, which require a step of abrasion of the barley and addition of gibberellic acid and which additionally result in an increase in the amount of soluble proteins, have not given rise to industrial uses either.

French Patent FR-1,360,637 (Stauffer Chemical Company) relates to a malting method which provides a malt with an attractive and more vivid colour with a decrease in the development of the radicles during the germination. It makes use of an acidic steeping at the rate, typically, of one litre of steeping water per 100 grams of barley, the initial pH of the steeping varying between 1.7 and 2.4. Such ratios by volume of the steeping water to the amount of barley have the consequence that the pH of the steeping water for a long time remains the same as the initial pH. As the barley remains for a very long time at a low pH this results in a slowing down in the germination, which leads to the disadvantages already mentioned.

,i 7 It results from the above that no method is currently known which can be used on an industrial scale, without addition of sulphur during kiln drying, which is without more or less significant disadvantages and which would make it possible to obtain malt with a low NDMA level.

The object of the invention is to solve this problem and the invention thus relates to a method which can be used on an industrial scale and which makes it possible in particular to obtain a malt with a low NDMA level.

The basic idea of the invention is that it is possible to control the acidification during the steeping, so that the germination is only very slightly modified or not modified at all, while acting on the mechanism which results, after kiln drying, in a low amount of NDMA, without affecting the properties of the malt or complicating the industrial implementation or decreasing the yield.

The method according to the invention is thus characterized in that the steeping step includes at least one acidic steeping substep which is carried out at a pH which, after stabilization, is between 3.5 and 4.6 at ambient temperature, the ratio of the volume of the steeping water to the amount of barley being chosen so that the activity of the grain during germination is not substantially modified, everything else being equal. This makes it possible to take into account the buffer effect of the barley.

With these pH values, the germination of the barley is unaffected or only very slightly affected whereas, for example, a pH which, after stabilization, would be equal to 3 would result in a significant modification in the germination and thus in the quality of the malt. The malt obtained according to the invention exhibits, after kiln drying, a low NDMA level, for example lower than 2 Fg/kg and preferably lower than 1 pg/kg.

C51/1

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l< L o -8- According to a preferred embodiment, the said stabilized pH is between 3.8 and 4.6, the preferred value being 4.5. This is because, according to the invention, the results obtained are virtually the same, for example, for pH values of 3.5 and 4.5 and it is therefore advantageous to operate at the higher value, which corresponds to the addition of the lower amount of acid to the same volume of steeping water.

The acid can be hydrochloric acid and/or alternatively phosphoric acid.

The said ratio of the volume of the steeping water to the amount of barley is advantageously between 0.8 1 and 1.2 1 per kg of barley and preferably equal to substantially 1 1 of steeping water per kg of barley.

At least one steeping substep has a duration advantageously of between 4 and 6 hours.

In all cases, a decrease in the soluble proteins is observed (decrease in the Kolbach index) but, in the case of phosphoric acid, an additional advantage is a significant fall inthe viscosity of the wort, which is due essentially to the decrease in the level of soluble P-glucans in the malt. It should be noted that, as phosphoric acid is employed under cold conditions during the steeping, the problem of smells and of difficulties in handling which has been mentioned above in the case of the addition of phosphoric acid during kiln drying is no longer posed.

Acid can be added to the steeping water during each of the steeping substeps.

According to a preferred alternative form, the steeping step includes three substeps and acid is added to the steeping water during the first and second of these substeps.

The invention also relates to a malt obtained with substantial stimulation of the germination and which is characterized in that it exhibits an extract of between 81% and 82% of dry matter a level of soluble proteins of between 4 and a viscosity of %r J 1) 9 between 1.4 and 1.5 mPa-s, a modification homogeneity 72%, an NDMA level 10- 9 and optionally a level of soluble P-glucans 200 mg/l.

Other characteristics and advantages of the invention will become more apparent on reading the following description, in combination with the appended drawing in which Figure 1 takes up again the two major hypotheses for the formation of NDMA in malt published in the literature, in this case the formation of NDMA from DMA produced during the germination and from DMA formed by successive nitrosation of hordenine and of nitrosohordenine during the kiln drying.

Figure 2 gives a comparative illustration of the change in the pH during steeping, according to Patent FR-1,360,637 and according to the invention.

Figure 3 gives a comparative illustration of the filterability of the test and control malts according to Tests 8 and 9 of Tests II.

The results of Tests I below are represented in Tables 1 and 2.

The results of Tests II below are represented in Tables 3 and 4. The influence on the quality of the malt of the substantial stimulation in germination after acidic steeping is illustrated in Table 4.

The results of Tests III below are represented in Table The invention has been made use of in several series of tests carried out under micromalting conditions or alternatively on an industrial scale.

TESTS I: HC1/H 3 P0 4

COMPARISON

Barley varieties: Three brewing varieties were tested on the micromalting scale. All are spring varieties (Alexis, Vodka and Nomad).

Micromalting procedure: The steeping: Barley stored at 13 0 C is subjected to 3 successive steepings, interrupted by 2 periods of aeration of the grain.

0~' 10 1st steeping 6 hours 130C 1st aeration 14 hours 25 0

C

2nd steeping 5 hours 14°C 2nd aeration 13 hours 200C 3rd steeping 2 hours 160C For the acidic steeping tests, hydrochloric acid (HC1) and phosphoric acid (H 3

PO

4 are added to the first 2 steepings: HC1 Test, pH 4.5 after stabilization HC1 Test, pH 3.5 after stabilization

H

3 P0 4 Test, pH 4.5 after stabilization

H

3 P0 4 Test, pH 3.5 after stabilization The steeping is carried out with a proportion of approximately 1 litre of steeping water per kilogram of barley.

The germination: This step, which last 96 hours, can be subdivided into three separate stages: 1st phase: 14 hours 200C 2nd phase: 2 hours 180C 3rd phase: 80 hours 160C The kiln drying: The germinated barley is dried under micromalting conditions by a gradual increase in the temperature.

1st phase: 3 hours 62°C 2nd phase: 2 hours 650C 3rd phase: 2 hours 68°C 4th phase: 2 hours 73°C 5th phase: 1 hour 78°C 6th phase: 2 hours 800C 7th phase: 6 hours 83°C After kiln drying and before storing the malt, the radicles are separated from the grain by mechanical impacts.

Kiln drying on an industrial scale: A portion of the green malt from the variety Alexis was collected under micromalting conditions and dried on an industrial scale without burning sulphur

LU)

LL

11

(SO

2 so as to promote the formation of NDMA from precursors present in the green malt.

The green malt is dried by means of direct heating of the type with a low NOx content on two circular trays.

The first step is carried out on the upper kiln tray for a time of 20 hours. The initial temperature of 550C is brought to 68-700C during this period, which has the effect of bringing the moisture content of the grain from 42% to The second step is carried out on the lower tray. This step is composed of 3 steps. A gradual rise in the temperature over 12 hours to 75 0 C, a heating surge to 80 0 C over 4 hours and then a step of cooling the grain by ventilation over 4 hours. During this second step, the moisture content of the grain falls from 10 to 4%.

Analysis of the malts: Conventional parameters: The main criteria for quality of the malt (extract, viscosity, Kolbach index, diastatic power, pH, soluble P-glucans) were measured by methods described in Analytica-EBC. Malts dried on the micromalting scale were analysed for this reason.

Quantitative determination of Nitrosodimethylamines (NDMA): The NDMA levels were measured for malt dried in industrial kilns and after separation of the radicles.

The NDMAs are obtained by extraction of 15 g of malt in 100 ml of distilled water at 500C for one hour with moderate agitation. After extraction by dichloromethane on a tube of "Chemelut" type, the extract is concentrated to 0.5 ml dichloromethane (DCM) and 10 l1 are injected into the GC/TEA ("Thermal Energy Analyser") system.

The NDMA content is calculated with respect to a calibration carried out on a malt sample containing increasing amounts of NDMA.

12 In the analyses, nitrosodipropylamine (NDPA) is added as internal standard.

RESULTS

NDMA level in the malt (Table 1): Whatever the acid employed (HCl or H 3 P0 4 and the acidity, the NDMA levels produced during the kiln drying of the green malts (Alexis) are lower than those of the untreated control: the NDMA values for finished malt are lower by a factor of more than 2.

Soluble proteins (Table 2): The level of soluble proteins (Kolbach index) in the tests is lower than the values of the control, whatever the barley variety under consideration, the pH and the acid employed.

Level of soluble P-glucans (Table 2): The acidification of the steeping water results in a decrease in the soluble P-glucans in the malt.

This phenomenon is particularly significant during the use of H 3 P0 4 which makes possible a reduction of 15 to 50% in the soluble P-glucans, depending on the variety.

Viscosity (Table 2): The fall in viscosity is significant in all cases using phosphoric acid.

pH of the wort (Table 2): The acidification in the malting induces a slight fall in pH (0.2-0.1 pH point) of the wort manufactured from the malts.

Diastatic power (Table 2): The diastatic power is influenced by the type of acid and the acidity without it being possible to lay down general rules, because this phenomenon is variable from one variety to another.

The other criteria for quality of the malt which have been analysed remain unchanged, taken as a whole.

CONCLUSION

This study shows that the decrease in NDMA production in the malt by acidification of the steeping water is not limited to the employment of hydrochloric 13 acid (HC1) The use of phosphoric acid (H 3

PO

4 in the steeping makes possible an equivalent decrease in the NDMA level produced in the finished malt.

Moreover, a stronger acidification (stabilized pH of 3.5) does not introduce advantages with respect to that which consists in adjusting the steeping water to a stabilized pH of On considering the other quality factors of the malt, our study demonstrates the positive effect of the employment of H 3 PO4 or of HC1 in the steeping on the overall quality of the malt. First of all, a decrease in the level of soluble proteins in the malt (Kolbach index) will be noticed.

Secondly, the decrease in the soluble P-glucans and in the viscosity of the malt is significant in these tests.

The pH values of the wort and the diastatic power values are also influenced by the acidification of the steeping water.

The above observations make it possible to envisage pushing the germination further, so as to obtain a level of soluble proteins (Kolbach index) identical to the control. This should result in an increase in the malt extract making possible higher yield values in the brewhouse. In this case, the other parameters of the malt would remain unchanged.

TABLE 1 Samples NDMA level in the malt 10-9 Control 1.8 Tests HC1, pH 4.5 0.7 HC1, pH 3.5 0.8

H

3 P0 4 pH 4.5 0.7

H

3 P0 4 pH 3.5 0.8 ci< 14 TABLE 2 Variety Parameters Units Control HC1 HC1 H 3 P0 4

H

3 P0 4 pH 4.5 pH 3.5 pH 4.5 pH Alexis Moisture content 4.5 4.4 4.3 4.2 4.2 Extract DM 81.1 81.1 81.6 81.4 81.6 Total proteins DM 11.7 12 11.9 12.3 11.7 Sol. proteins 4.6 4.1 4 4.3 4.2 Kolbach index 39.3 34.2 33.6 35 35.9 Viscosity mPa-s 1.63 nd nd 1.49 1.53 Sol. P-glucans mg/l 357 358 333 249 nd pH 5.89 5.69 5.82 5.75 5.81 Diast. Power WK 308 297 286 350 286 Nomad Moisture content 4.4 4 4.4 4 4.1 Extract DM 82.5 82.5 82.4 83 82.7 Total proteins DM 9.8 9.9 10 9.8 9.9 Sol. proteins 3.9 3.6 3.6 3.7 3.8 Kolbach index 39.8 36.4 36 37.8 38.4 Viscosity mPa-s 1.69 nd nd 1.61 1.65 Sol. 0-glucans mg/1 366 378 328 318 nd pH 6.01 5.8 5.9 5.88 5.9 Diast. Power WK 140 96 97 96 82 Vodka Moisture content 4.5 4.4 4.8 4.2 4.3 Extract DM 81.4 81.6 81.8 81.8 81.9 Total proteins DM 10.4 10.6 10.5 11 10.6 Sol. proteins 4.4 3.6 3.7 3.6 3.9 Kolbach index 42.3 34 35.2 32.7 36.8 Viscosity mPa's 1.61 1.61 1.55 1.51 1.59 Sol. P-glucans mg/l 405 380 230 192 nd pH 5.96 5.85 5.89 5.86 5.85 Diast. Power WK 286 200 176 261 197 nd: not determined TESTS II Barley varieties: Four brewing varieties were tested (Tests 1 to on an industrial scale (200 tonnes of malt). Two are spring varieties (Prisma and Alexis); two are winter varieties (Clarine and Plaisant).

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'L>

15 Malting procedure: The steeping: Barley stored at 130C is subjected to 3 successive steepings, interrupted by periods of aeration of the grain.

250 m 3 of water moderated to 120C are added to 240 tonnes of barley. After a period of 6 hours, the first steeping water is discharged and the grain is aerated for 11 hours with air entering at a temperature of 20'C. In this stage, the moisture content of the grains is between 26 and 28%.

A second steeping is carried out with the same volume of water for 3 hours, followed by a period of aeration of 8 hours at 160C. At this stage, the moisture content of the grain is 35-37%.

The final steeping corresponds to a period under water of 2 hours, followed by an aeration of 3 to 4 hours at 160C.

In the final stage of the steeping, the grains have reached a moisture content of the order of 40 to 42%.

For the tests (acidic steeping), hydrochloric acid is pumped from a storage tank and mixed with the water supplying the steeping vessel.

The germination: This step is carried out for 5 days in circular boxes stacked in a vertical installation of "Malting Tower" type and the grains are turned over every 8 hours, so as to homogenize the germination.

At the beginning of the germination, the steeped barley is sprinkled, so as to reach a moisture content of the grain of 43%. The temperature of the air entering is controlled at 150C.

The kiln drying: The germinated barley (green malt) is conveyed from the germinating box to the kiln where the green malt is dried by means of direct heating of the type Swith a low NO, content on two circular trays.

16 The first step is carried out on an upper kiln tray for a time of 20 hours. The initial temperature of is brought to 68-70'C during this period, which has the effect of bringing the moisture content of the grain from 42% to The second step is carried out on the lower tray. This step is composed of 3 steps. A gradual rise in the temperature over 12 hours to 75 0 C, a heating surge to 80°C over 4 hours and then a step of cooling the grain by ventilation over 4 hours. During this second step, the moisture content of the grain falls from 10 to 4%.

In the case of the controls carried out in parallel to each test, 75 kg of sulphur are burnt during the kiln drying.

After kiln drying and before storing the malt, the radicles are separated from the grain by mechanical impacts. Quantitative determination of the NDMAs is carried out after separation of the radicles.

Quantitative determination of the Nitrosodimethylamines: The NDMAs are obtained by extraction of 15 g of malt in 100 ml of distilled water -at 50'C for one hour with moderate agitation. After extraction by dichloromethane on a tube of "Chemelut" type, the extract is concentrated to 0.5 ml DCM and 10 p1 are injected into the GC/TEA ("Thermal Energy Analyser") system.

The NDMA content is calculated with respect to a calibration carried out on a malt sample containing increasing amounts of NDMA.

In the analyses, NDPA is added as internal standard.

RESULTS:

The observations made on the micromalting scale (Tests I) are validated on an industrial scale (200 tonnes): the application of acid to the steeping water makes it possible to obtain malt with a low 1 nitrosamine content and with a quality identical to 4 j I :'.ii 17 that of the control malt treated with sulphur (see Table 3).

This has been verified on 5 industrial tests with different barley qualities (winter/spring).

The application of different doses to different steepings shows that the employment of acid in the first two steepings is sufficient. In terms of volume, it may be considered that approximately 500 litres of acid added to the steeping water make it possible to achieve the objective.

At these volumes of acid, and with approximately 1 litre of steeping water per kilogram of barley, the pH values of the steeping waters after homogenization and pH equilibrium are between 3.8 and 4.1 in the first steeping and between 3.8 and 4.6 in the second steeping.

A test carried out with a low acid dose (Test 4 in Table 3) shows the need for a minimum amount of acid in the steeping in the absence of sulphur in the kiln drying. With such a dose of acid, the pH values at equilibrium in the first and in the second steepings are 5.1 and 4.8 respectively.

In parallel with the "nitrosamines" criterion, the other quality criteria of the malt were observed.

The values of some of them differ between the test and the control (see Table 3).

Firstly, the amount of sulphur dioxides is very low in the malts of the tests with respect to the control malt. This, of course, is related to the absence of application of sulphur in kiln drying in the case of the tests.

Secondly, the level of the P-glucanase activities is always greater than or equal to 400 IRVU in the case of the tests, whereas it is of the order of 300 IRVU in the case of some controls. These figures give the impression that the acid "dopes" the production of hydrolases in the case where conventional Smalting conditions would not make it possible for the Sgrain to fully express its potential.

18 At the same time, a decrease in the Kolbach index is witnessed in the case of the tests, which is reflected by a lower amount of soluble proteins.

Finally, in some cases, a decrease in the viscosity of the malts of the tests is witnessed.

The other quality criteria of the malt remain unchanged, taken as a whole.

Because of the confirmation during these industrial tests of a fall in the level of soluble proteins in the tests, it was desired to verify, in the context of Tests II, if it was possible to restore the level of soluble proteins by substantially stimulating the germination in the test.

For this reason, two options were experimented with (Tests 6 to 9, listed in Table Option 1 (Tests 6 and 7) consists in adding gibberellic acid mg/tonne) to the test at the beginning of germination. Option 2 (Tests 8 and 9) consists in lengthening, by a period of 5 hours, the final phase under air of steeping in the test. In the latter case (Option the characteristics of the malt were observed at 4 and 5 days of germination. In both options, the amount of acid added corresponds to that used for Test 3 (400 and 150 litres). The results are reported in Table 4.

The results of these additional tests confirm the idea mentioned beforehand, namely a significant increase in the malt extract during the reestablishment of the value of the soluble proteins by a substantial stimulation of the germination phase. This increase is between 0.4 and 1.3 extract points, depending on the test under consideration.

A more significant decrease in the viscosity than that observed in the preceding tests is also found. This difference in viscosity is related to a better degradation of the constituents of the cell walls by the expression of high cytolytic activities.

For example, the levels of soluble -glucans in the test malts are reduced by the expression, in these 19 malts, of higher endocellulase and glucanase activities than in the control malts.

This characteristic has a significant influence on the quality of filtration of the wort in the mash tun described by Grandclerc J. et al., 1988, in "Simplification de la m~thode de filtration du brassin Tepral, description et mithode" ["Simplification of the method of filtration of the Tepral mash, description and method"], published in the French review Bios, No. 19, p. 88-92. When a 50/50 mixture of the Iranis test malts of Tests 8 and 9 is compared with respect to a 50/50 mixture of the control malts of Tests 8 and 9, a marked difference in filtration behaviour is observed. During the first 300 seconds of the test of Figure 3 (Phase the filtration is gravitational and, after T 300 s, the filtration is carried out under a pressure of 1 bar. Figure 3 illustrates this fact by showing a marked improvement in the rate of filtration in the case of the mixture of the tests, in particular in Phase II, with respect to the mixture of the control malts.

At the same time, a better modification homogeneity of the malt is observed in all cases.

Finally, the performances of malt quality related to this type of malting method are illustrated very significantly in the observation of the malts produced in 4 days of germination (Test The adoption of acidic steeping makes it possible to produce, after 4 days of germination, a malt with a quality identical to that of the malt produced in 5 days under standard malting conditions.

Moreover, the physiological changes related to the acidic steeping make it possible to limit the respiration of the grain, which leads to improving the malting yield by approximately 1 point

CONCLUSION

The object of the study is achieved: on an Sindustrial scale, the employment of acid in the steeping makes it possible to limit the production of Pl [LI 20 nitrosamines in the malting in the absence of sulphur in the kiln drying.

The malts produced under these conditions are in accordance in terms of quality with, in all cases, a lower amount of sulphur dioxide and a constant and high level of -glucanase activity.

In certain cases, these malts are also characterized by a lower Kolbach index and a lower viscosity.

In tests of re-establishment of the level of soluble proteins by substantially stimulating the germination, it has been found, with respect to the control, that, at the same time as controlling the NDMAs, the adoption of such conditions made it possible to significantly increase the malt extract value and to significantly decrease the soluble -glucans in and the viscosity of the corresponding wort. These observations, at levels of soluble proteins which are identical to those which are measured on the control, demonstrate that the malting process claimed makes it possible not only to control the level of NDMA produced but also to manufacture malt of better quality overall.

INFLUENCE OF THE RATIO OF THE VOLUME OF STEEPING WATER TO THE VOLUME OF BARLEY TREATED: The variation in the pH curve during the first 6 hours of steeping is illustrated in Figure 2. The "Stauffer A" and "Stauffer B" tests correspond to the extreme conditions of Patent FR-1,360,637 2, lefthand column), namely a steeping water containing 0.1% of acid for Test A and 1% of acid for Test B, and, in both cases, 10 volumes of water per 1 volume of barley 1 litre of steeping water per 100 g of barley).

The test according to the invention corresponds to a steeping water containing 0.2% of acid, i.e. 400 1 of acid per 200 tonnes of water, for a test corresponding to 200 tonnes of grain.

The pH values found during the steepings are given in Figure 2 and correspond to the table below: 4 yC A f 1 C 21 T(h) Stauffer A Stauffer B Test Control 0.25 2.3 1.3 2.67 7.4 2.28 1.3 3.15 7.45 0.75 2.35 1.31 3.4 7.45 1 2.37 1.23 3.6 2.39 1.23 3.72 7.4 2 2.4 1.24 3.8 2.4 1.25 3.85 7.45 3 2.4 1.23 3.88 7.45 2.42 1.23 3.92 4 2.41 1.25 3.95 7.45 2.42 1.26 3.96 7.4 2.44 1.22 3.98 7.4 6 2.45 1.22 4.05 7.4 In the Stauffer A and Stauffer B tests, it is the water to which acid has been added which imposes its pH. During the first six hours of steeping, the pH remains below 2.5 and 1.3 respectively. In the Tepral test, the starting pH is 2.67 but it becomes 3.5 after approximately one hour. After six hours, it reaches the final value of 4.05. A significant buffer effect due to the barley is thus found with the volume of water/weight of the grain ratio chosen in the test, which makes it possible to prevent the grain from being maintained for an excessively long time at an excessively low pH. The physiology of the activity of the grain is thus prevented from being disturbed during the germination. The pH varies little during most of the steeping substep.

22 Table 3: Effect of the acidic steeping on the overall quality of the malt

I

I

Parameters Test 1 Test 2 Test 3 Test 4 Test Prisma Alexis Alexis Clarine Plaisant Vol. HC1 Vl V2 V3 Analysis of the malt Extract Kolbach Sol. prot.

Hartong 45

D.P.

Viscosity

NDMA

SO

2 Sol. 0-glucans P-Glucanase Unit T C T C T C litre 250 0 350 0 400 0 litre 250 0 170 0 150 0 litre 250 0 0 0 0 0 T C T C I I DM 81.4 81.3 37.8 40.2 4.2 4.3 38.0 33.0 WK 220 200 mPa s 1.46 1.46 ppb(10 9 0.5 0.5 ppm(10' 6 7.0 27.0 mg/l 44 85 IRVU 480 300 82.5 83.1 34.7 38.6 3.4 3.6 36.5 34.0 200 190 1.59 1.65 0.5 0.8 6.0 10.0 80 45 400 330 82.1 82.9 37.2 38.3 3.5 3.6 35.0 38.0 250 250 1.51 1.58 0.9 0.5 3.0 22.0 140 160 420 400 80.3 79.6 30.3 30.9 3.3 3.4 28.5 26.0 180 190 1.58 1.60 1.7 0.8 3.0 7.0 493 550 400 300 80.2 79.8 37.2 43.4 4.1 4.9 34.0 41.0 330 350 1.51 1.52 1.4 1.2 2.0 32.0 130 140 440 430 T: Test, C: Control Table 4: Effect on the quality of the malt of the substantial stimulation in germination after acidic steeping Parameters Test 6 Test 7 Prisma Prisma Malting conditions Gib 30 Gib 30 Test 8 Iranis 4 days Test 9 Iranis 5 days Unit T C T C T C T C Analysis of the malt Extract DM 80.9 80.4 80.8 80.4 80.4 79.9 81.9 80.6 Homogeneity 72 65 75 65 84 57.0 84 77 Sol. prot. 4.6 4.4 4.3 4.4 4.9 4.9 5.0 Hartong 45 38.1 35.8 36.1 35.8 43.2 42.2 46.1 42.9 D.P. WK 270 260 230 260 300 290 290 310 Viscosity mPa.s 1.50 1.59 1.49 1.59 1.46 1.52 1.46 1.50 NDMA ppb(10- 9 1.6 0.9 1.2 0.9 0.2 0.2 0.1 0.2 Sol. 0-glucans mg/l 195 240 190 240 100 180 70 130 P-Glucanase U/kg nd nd nd nd 357 286 411 355 Endocellulase mU/g nd nd nd nd 499 332 591 415 T: Test, C: Control 23 TESTS III:MECHANISM OF ACTION OF THE ACID ON THE NDMA

LEVEL

Barley varieties: Samples of germinated barley were withdrawn at the end of the germination phase and before kiln drying (green malts) in the case of the industrial test Test 3 of Table 3 (Alexis) and in the case of the micromaltings Vodka and Nomad of Table 2. In each of the cases, both the control green malt without treatment and the malt resulting from the acidic steeping were collected.

Malting procedures: The malting conditions for these samples are summarized in the description of Tests I and II described above.

Analysis of the green malts: Several potential precursors of NDMA were analysed from the green malts: hordenine and gramine according to a method published in 1981 by P.T. Slack and T. Wainwright in an article entitled "Hordenine is the Precursor of NDMA in Malt" in Journal Inst. Brew., Vol. 87, p. 260.

sarcosine is extracted under similar conditions to hordenine, esterified with fluorodinitrobenzene (FDNB) and analysed by HPLC.

dimethylamine (DMA) was extracted with methanol, derivatized with fluorodinitrobenzene

(FDNB)

and analysed by HPLC in accordance with the method published in August 1991 in Verein Deutscher Ingenieure and entitled "Determination of Aliphatic Amines by

HPLC".

The NDMA content of the green malts was determined according to the method described in Test I.

Moreover, a visual study of the green malts was carried out so as to estimate the effect of the acid on the growth of the radicles and of the plumules, these tissues being the major participants in the production S of the precursor amines of NDMA.

c~jj 24

RESULTS:

The concentrations of secondary

(DMA,

sarcosine) and tertiary (hordenine, gramine) amines measured in the green malts collected before kiln drying are expressed in parts per billion ppb with respect to dry weight and are represented in Table The figures show that gramine and sarcosine are in the green malt in very small amounts and that only hordenine and dimethylamine are present in measurable amounts. In the case of the industrial malt, the concentrations of these two amines are 2 to 10 times greater for hordenine and DMA respectively with respect to the mean of the two micromalts analysed.

If attention is turned to the hordenine concentrations in the green malts, it is noticed that the amount of hordenine produced during the germination is lower by 50% to 100% in the acidic test sample with respect to the control in the case of the micromalts and is produced at an identical level in the control sample and the acidic test sample in the case of the industrial malt (Alexis).

In the case of DMA, the values for micromalts and industrial malt are more in agreement with one another in comparison with the preceding case. In all cases, the concentration of DMA in the acidic steeping test sample is lower by a factor of the order of 4 with respect to the control. In the case of the Nomad test, the values measured show that the amount of DMA in the acidic steeping test green malt is 10 times lower than the amount measured for the control.

The employment of acid in the steeping has no significant effect on the amount of NDMA formed during the germination or on the size of the radicles and of the plumules produced during the germination of the grain.

-7 1 Table 5: Concentration of NDMA precursors in the green malt, expressed in ppb (10 9 by dry weight Amines Vodka Nomad Alexis (micromalt) (micromalt) (industrial test) Control Acidic Control Acidic Control Acidic test test test Sarcosine <10 <10 <10 <10 <10 DMA 124 34 493 46 3400 800 Hordenine 36,000 18,000 35,000 27,000 71,000 75,000 Gramine <10 <10 <10 <10 <10 NDMA <0.3 0.5 0.3 0.3 0.3 0.2 26

CONCLUSION:

This study demonstrates the role of the acid used in the steeping on the amount of NDMA precursors present in the grain at the end of germination.

The values of amines measured in this study show that the addition of acid to the steeping significantly and reproducibly decreases the amount of DMA present in the green malt.

A lower availability in the green malt of this secondary amine, regarded as one of the main precursors of NDMA, thus very logically results in a reduction in the formation of NDMA during the nitrosation induced by NOxs during kiln drying, which makes it possible to dispense with sulphur during this final malting step while maintaining the required NDMA standards.

The influence of the acid on the level of hordenine in the green malt is less significant and reproducible from one test to another, which suggests that the reduction in the NDMA level by acidification of the steeping water is essentially related to the decrease in DMA in the green malt by this malting process.

This mechanism of action of the acid is distinguished from the mechanism related to the decrease in growth of the tissues (embryo, radicles) and the amount of hordenine cited by Wainwright et al.

in "Nitrosamines in Malt and Beer" in Journal Inst.

Brew., 1986, Vol. 92, p. 76; the addition of acid to the steeping carried out according to our conditions has no significant effect on the size of the radicles and of the plumules developed during the malting or on the amount of hordenine produced.

At the same time, additional malting tests intended to re-establish the slightly lower level of proteins of the tests make it possible to conclude that the acidification of the grain in the steeping leads to physiological events which result in malt being obtained which is superior in quality to the control.

The adoption of acid in steeping, in conjunction with a 27 substantial stimulation of the grain in germination, makes it possible, on the one hand, to produce malts with a high extract and low viscosity while controlling the level of soluble proteins produced and, on the other hand, the adoption of these malting conditions also make it possible to manufacture, in 4 days of germination, malts with a quality identical to that of the malts obtained in 5 days of germination under standard conditions.

Finally, the adoption of this method and the physiological phenomena which it engenders results in an increase in yield in the malting.

C

Claims (10)

1. 2. Method according to Claim 1, characterized in that the said stabilized pH is between 3.8 and 4.6.
2. 3. Method according to Claim 2, characterized in that it includes two acidic steeping substeps and in that the said stabilized pH is between 3.8 and 4.1 during the first substep and between 3.8 and 4.6 during the second substep.
3. 4. Method according to one of Claims 1 to 3, characterized in that at least one acidic steeping substep is carried out with a ratio of 0.8 to 1.2 litres of steeping water per kg of barley. Method according to Claim 4, characterized in that the said ratio is substantially 1 litre of steeping water per kg of barley.
4. 6. Method according to one of the preceding claims, characterized in that at least one steeping substep has a duration of between 4 and 6 hours.
5. 7. Method according to one of the preceding claims, characterized in that the acid is added to the steeping water during each of the said steeping substeps.
6. 8. Method according to one of Claims 1 to 6, characterized in that the steeping step includes three substeps and in that acid is added to the steeping water during the first and second of these substeps.
7. 9. Method according to one of the preceding claims, characterized in that the said acidification is carried out with hydrochloric acid. e 29 Method according to one of Claims 1 to 8, characterized in that the said acidification is carried out with phosphoric acid.
8. 11. Method according to one of the preceding claims, characterized in that the germination step is stimulated with respect to a standard industrial germination, so as to re-establish the value of the level of soluble proteins at a level substantially equal to that obtained from the same barley variety obtained without acidic steeping and subjected to the said standard germination.
9. 12. Malt, characterized in that it exhibits: an extract of between 81 and 82% of dry matter; a level of soluble proteins of between 4 and a viscosity of between 1.4 and 1.5 mPa-s, a modification homogeneity 72%; an NDMA level 10 9
10. 13. Malt according to Claim 12, characterized in that it exhibits a level of soluble -glucans of less than 200 mg/l. C C L F 4