The present invention concerns a procedure for isolating from cereal a microbe inhibitor and/or a combination of microbe inhibitors. The invention fur¬ ther concerns a microbe inhibitor and/or a combination of microbe inhibitors. Moreover, the invention concerns the use of a microbe inhibitor and/or a combination of microbe inhibitors.

Microbes are in this context in particular understood to be bacteria and various fungi, such as yeast fungi, moulds, decay fungi, trichophytid fungi, etc., and in general all microbes known in themselves which cause damage in economic activity and in health care.

Microbial growth and contamination cause major damage in various fields of industry, business and production. Bacteria mostly cause damage in foodstuff industry, particularly in meat processing industry, and through contamination generally in health care. Yeast and mould fungi cause damage particularly in foodstuff industry, notably in bakery industry, dairy industry, jam and sweets industry, and in production of prepared foods, in fodder industry, timber and board industry, paper and pulp industry, etc. Decay fungi cause damage particularly in wood conversion industry and in paper and pulp industry.

Attempts have been made to fight microbes by greatly different procedures and methods, depending on the branch of industry and kind of product concerned. It has been common practice to apply in microbe fight¬ ing e.g. methods of treatment which destroy microbes, such as heat treatment, various irradiation methods, particular chemicals which destroy microbes, etc. At¬ tempts to fight microbes have further been made with the aid of general sanitation, i.e., endeavours have

been made to keep the product apart from the microbes responsible for contamination with the aid of protec¬ tive packaging, protective gas, cleaning or any other procedure. Furthermore, one has endeavoured to inhibit the growth of harmful microbes by means of specific microbe inhibitors, particularly chemicals and microbe inhibitors isolated from nature or from artificial sub¬ strates.

In general, the chemicals employed in de- stroying microbes and/or inhibiting their growth are comparatively dangerous, i.e., the chemicals are in most instances toxic or even dangerous to the environ¬ ment and to persons coming into contact with them; in this connection may for instance be mentioned chemicals used in timber treatment and the serious environmental problems which they cause. In general, there is at present a need to find and develop new agents inhibi¬ ting microbial growth, microbe inhibitors which could be used particularly in foodstuff industry in such manner that they would cause lower toxicity than before to foodstuffs, and in other fields as well.

The object of the present invention is to eliminate the drawbacks mentioned in the foregoing. Specifically, the object of the invention is to dis- close a novel microbe inhibitor which is derived from natural substances and which is usable, better than before, particularly in connection with foodstuffs and with other products and fields as well. The object of the invention is specifically to disclose a microbe inhibitor and a combination of microbe inhibitors of lowest possible toxicity, causing the least possible side effects and on the whole superior to those of prior art in its application and other properties, and better fit to use, suitable to be used in a wide range of various fields and various products, particularly in connection with foodstuffs.

Furthermore, the present invention is advan-

tageous as to the cost involved, thereby differing from any other inhibitor produced e.g. by fermenting. Price is nowadays one of the salient factors restricting the use of inhibitors. The invention is based on extensive cereal studies that have been carried out and from which un¬ expectedly the fact has emerged that when cereal meal is suspended in water and from the water a microbe inhibitor fraction is separated which has molecular weight less than 30,000, suitably 7,000 to 30,000, advantageously 10,000 to 25,000, a microbe inhibitor fraction inhibiting microbial growth in versatile and efficient manner is obtained which has low toxicity and which is appropriate to be used in the greatest diver- sity of fields. The cereal to be used in the procedure of the invention is, for instance, barley, wheat, rye, oats, rice and/or corn; a particularly advantageous starting material is barley.

In the procedure of the invention the inherent defensive system of plants is utilized. It is known in the art that in plants there occur compounds control¬ ling microbial growth, microbe inhibitors. The plants are using these microbe inhibitors in order to fight plant diseases or other pests which threaten them. The novelty in the invention is particularly that microbe inhibitors have now for the first time ever been obser¬ ved in aqueous suspensions made from meal of said ce¬ reals, that is, being present dissolved in water. The invention is based on this observation and on isolation and separation of the microbe inhibitors from said process waters, employing separation procedures mainly known in themselves in the art.

The microbe inhibitors may be used as they are, e.g. in the form of the aqueous suspensions ob- tained, e.g. in the bakery industry by admixing the aqueous suspension to the dough, usually in the form of products which are dispensed in liquid form or sprayed.

in the form of a product to be admixed in solid form (prepared e.g. by means of inspissation) to the desired material, e.g. to a foodstuff or to another material, or admixed in the form of any further processed product or mixture whatsoever, such as a carrier substance.

In the procedure of the invention the cereal is ground by any grinding method known in itself in the art, e.g. dry milling or wet milling, described for instance in the patent application EP 267637. Further, the meal thus obtained is suspended in water e.g. in proportion 1:10 to 10:1, advantageously 2:1 to 5:1. The suspension thus obtained may be treated with an enzyme, e.g. cellulase, as disclosed in the cited, earlier application print, e.g. in association with the conven- tional starch process. From the solution are separated the starch and fibrous substances by any process known in itself in the art, e.g. by straining, filtration, centrifuging and/or any other separating method known in the art. The microbe inhibitor fraction is separated from the solution thus obtained by centrifuging, ultra¬ filtration, gel filtration, evaporating and/or any other separating method known in the art.

The microbe inhibitor fraction may be concen¬ trated by conventional methods, e.g. ultracentrifuging, ultrafiltration, gel filtration and/or evaporation, or in the way described in the embodiment examples follo¬ wing below. The microbe inhibitor fraction may be con¬ centrated and dried e.g. to powder form, and it may then also present itself e.g. as a lyophilized, finely divided powder. It may equally be bound to a carrier, e.g. to starch or equivalent. Furthermore, the microbe inhibitor fraction may present itself as a concentra¬ ted, free-flowing liquid (e.g. 0 to lOfold or even higher concentration), dry matter content e.g. 0.1 to 50 to 100%. In the concentration process the microbe inhibitor fraction may for instance be ultrafiltered and, if desired, evaporated in vacuum (temperature

below 45°C) .

The microbe inhibitor fraction of the inven¬ tion has been found to tolerate well temperatures up to 50°C; the temperature dependence is shown in Fig. 3; the inhibitor fraction tolerates several hours at tem¬ peratures below 50°C (tried up to 5 hrs). The inhibiti¬ on acticity of the microbe inhibitor measured using Trichoderma as a test organism was after 3 months at room temperature slightly lowered (to get 100 % capaci- ty, one needed 30 % more inhibitor), in refrigerator temperature the activity remained unchanged.

The microbe inhibitor fraction may be further fractionated, e.g. by molecular weight or specific ac¬ tivity, employing procedures known in themselves from molecular fractionating, e.g. ultracentrifuging, ul¬ trafiltration, gel filtration, etc.

The separated and fractionated microbe inhib¬ itors may be used as fractions, i.e., as microbe in¬ hibitors having certain given activities, or as a com- bination constituted by a plurality of inhibitors, in accordance with the specific use and other require¬ ments, e.g. as a single fraction to inhibit a single microorganism or as a combination which has been found to be the most efficient for retarding or inhibiting the growth of bacteria and fungi in general.

In the foodstuff industry, the microbe inhib¬ itor and/or combination of microbe inhibitors produced as taught by the invention may in particular be used to prevent spoiling of bakery products, to prevent spoi- ling of meat and meat products, prepared food products and/or fast food, to prevent spoiling of jams, berry and food products and/or sweets, and/or to prevent spoiling of products of the dairy industry. Furthermo¬ re, the microbe inhibitor and/or combination of microbe inhibitors prepared as taught by the invention is ap¬ propriate to be used to prevent spoiling of various salads and dressings. The microbe inhibitor and/or

combination of microbe inhibitors according to the invention can also be used together with any known inhibitor, for example with nicine or some chemical inhibitor such as sorbic acid. When used in connection with foodstuffs, the microbe inhibitor fraction of the invention has been found to prolong the storage, shop and consumption times of foodstuffs, to retard the spoiling of finished foods and to retard mould infestation of bread. More- over, the microbe inhibitor fraction improves the structure of the bread by increasing the formation of carbon dioxide in the dough about 10 to 20%; it may be dispensed into the bread dough because it will not inhibit baking yeast, as for instance sorbic acid does. In addition, the microbe inhibitor fraction functions also at neutral pH, pH adjustment is not necessary. The microbe inhibitor fraction can be processed in such a way that it gives grain flavour or colour to the prod¬ uct or that no extra flavour or colour is found. Addi- tionally, the acidity of the inhibitor fraction can be adjusted appropriate for the field of application, without any changes in inhibition capacity (tested by a standard method, at pH-range 3.5 - 5.0) The microbe inhibitor fraction of the invention used in connection with foodstuffs may replace some of the presently used chemical preservative agents, such as sorbic acid, propionates and benzoates.

In the paper, pulp and wood conversion indus¬ try, the microbe inhibitor and/or combination of mi- crobe inhibitors prepared as taught by the invention is appropriate to be used to prevent spoiling of paper stock, particularly to prevent slime forming and to eliminate process problems caused by slime as well as quality defects of the paper. In the sawmill industry, the microbe inhibitor and/or combination of microbe inhibitors prepared as taught by the invention can be used generally to prevent decay of timber, e.g. to

counteract brown rot fungi, white rot fungi and/or soft rot fungi, and to prevent likewise mould attack of timber and/or mould-induced discoloration. In the board industry and elsewhere in the wood conversion industry and in connection with handling of timber raw material, the microbe inhibitor and/or combination of microbe inhibitors prepared as taught by the invention can likewise be used to counteract decay and/or mould at¬ tack of timber and defects caused by them. In the fodder industry, the microbe inhibitor and/or combination of microbe inhibitors prepared as taught by the invention is appropriate for use to counteract spoiling induced by fungi or bacteria and/or mould attack of fodder raw materials and/or finished fodders.

In the field of health care, the microbe in¬ hibitor and/or combination of microbe inhibitors pre¬ pared as taught by the invention is appropriate to be used to fight, in general, particularly fungi, such as trichophytid fungi and bacteria, and fungal and bac¬ terial contaminations.

The invention is described in detail in the following with the aid of embodiment examples, refer¬ ring to the attached drawings, wherein Fig. 1 presents an industrial procedure according to the invention for preparation of microbe inhibitors from cereal, in diagram form,

Fig. 2 illustrates the effect of pH on the inhibition capacity elicited by a microbe inhibitor fraction pro- duced by the procedure depicted in Fig. 1,

Fig. 3 illustrates the effect of temperature on the inhibition elicited by the same fraction, Fig. 4 presents the degree of inhibition elicited by gel-filtrated microbe inhibitor fractions. In Fig. 1, the cereal, e.g. barley, wheat, rye, oats, rice and/or corn, advantageously barley 1, is supplied into a hammer mill 2 and ground to meal 3.

The husks 4 of the cereal are removed from the hammer mill. The meal 3 is suspended in water 5 and thereto is admixed enzyme 6, the suspension 7 thus obtained is strained through a sieve 8. From the coarser fibre fraction thus obtained the water is removed in a de- watering step 9, and the product thus obtained is dried and pelleted in a pelleting step 10. The finely divided suspension from the sieve 8 is conducted to a separati¬ on concentrator 11, from which the process water 5 is returned to circulation and the concentrated matter is conducted to separation-fractionation 12; the more concentrated matter is conducted from here to a starch separating process 13,14,15, and the supernatant is conducted to the microbe inhibitor separating step 16. Generally, the microbe inhibitor fraction consists of a water-soluble protein fraction which travels generally e.g. in the above-described starch process along with the process waters. Endeavours are in the separating process to separate the microbe inhibitor fraction from said fraction, using separating methods known in them¬ selves in the art, e.g. methods based on molecular weight.

The microbe inhibitor fraction, molecular weight 7,000 to 30,000, advantageously 10,000 to 25,000, is separated e.g. by the aid of ultracentifug- ing, ultrafiltration, gel filtration and/or any other separating procedure known in itself in the art. The microbe inhibitor fraction may be further fractionated e.g. on the basis of molecular weight, using ultra centrifuging, ultrafiltration, gel filtration and/or any other separating method known in itself in the art. If desired, the microbe inhibitor and/or combination of microbe inhibitors may be further concentrated, using ultracentifuging, ultrafiltration, gel filtration and- /or any other concentrating procedure known in itself in the art.

In one of the research applications of the

process according to the figure 1, the microbe inhibi¬ tor was prepared by using separation, evaporation and sterile filtration.

Lyophilized microbe inhibitor was also pre- pared by using process according to the figure 1. The supernatant was centrifuged, sterile filtrated and lyophilized. The lyophilized microbe inhibitor was suspended in water to its original volume and the inhi¬ bition activity was measured using the standardmethod. The inhibition activity of the lyophilized inhibitor was the same as the supernatant ^• s.

The effect of pH and temperature on the ac¬ tivity of the microbe inhibitor prepared by the process according to figure 1 was tested. The effect of pH on the inhibition was tested at pH-range 3-9 using standard method in Trizma- base/HCl or Tris HC1/KOH buffers. The possible growth retardation caused by pH or buffer was taken into con¬ sideration. Test organisms were Penicillium C3 bread mold, Zygosaccharomyces cidri and Bacillus stearother- mophilus. The results are shown in figure 2.

The effect of temperature on microbe inhibi¬ tor capacity was tested. The inhibition fraction was kept 1 to 5 hours at the test temperatures before the measurement of inhibition capacity. The results are shown in figure 3.

To measure the inhibition caused by the microbe inhibitor, a standardmethod presented in the example 1, was used. Other possible methods to be used are the so-called cup plate assay and continuos growth observation spectrofotometrically by Bioscreen -device (Labsysterns, Helsinki, Finland)

EXAMPLE 1, standard method for measuring the inhibition The appropriate medium and microbe culture,

150 μl altogether, and inhibitor and water, to the final volume of 165 μl, are added to the microtiter

plates. Instead of inhibitor, there is water in the control wells. Three parallel tests of each sample are made. The growth is observed spectrophotometrically at appropriate intervals at 690 nm by Biomeck 1000 (Beck- man Instruments Inc. Fullerton, USA). The concentration of the microbe culture (mold spores or bacteria/ yeast) is so chosen that the absorbance in the beginning of the measurement is 0.02 - 0.3. The inhibition rate is counted by comparing the result with the control test. The incubation temperature is the growth temperature of the chosen micro-organism. In the examples presenting the properties of microbe inhibitor, Trichoderma reesei mold has been used as a standard microorganism unless otherwise stated. The microbe inhibitor fraction prepared as taught by the invention was in tests found to inhibit the following microbes (+++ strong inhibition, ++ dis¬ tinct inhibition, + weak inhibition, - no inhibition, that is less than 20%):

Microbes spoiling bakery products

- Yeasts

Zygosaccharomyces bailii ++ - Moulds

Aspergillus niger ⁺ ( ⁺⁺ )

Cladosporium ++

Trichosporon ++

Bread mould C Penicillium sp +++

Bread mould 1 Penicillium sp ++

Bread mould 2 Penicillium sp +/-

Bread mould 4B Rhizopus sp +

Microbes spoiling meat products

- Bacteria

Yersinia enterocolitica +++ Salmonella (Gram, facult. anaer.) enteritidis ++

Escherichia coli +/- Bacillus cereus ++ thermophilus +++

- Yeasts Debaryomyces hansenii ++

Rhodotorula glutinis +++

Trichosporon cutaneum ++

- Moulds Alternaria ++

Aspergillus niger ++

Cladosporium ++

Eurotium ++ Rhizopus ++

Microbes spoiling paper pulp stock

Alternaria spp. ++ Cladosporium spp. ++

Trichoderma spp. +++

Aspergillus niger +

Penicillium cyclopium ++

Microbes causing decay of timber

- Brown rots

Antrodia sp, Poria sp +++

Piptoporus betulinus ++

- Dry rot

Merulius +++

Soft rots

Trichoderma + Alternaria ++

Microbes causing mould attack on timber

Cladosporium ++

Generally, the microbe inhibitor fraction was found to inhibit strongly or distinctly the following microbes:

Merulius tremellous +++

Trichoderma reesei +++

Zygosaccharomyces cidri +++ Rhodotorula glutinis +++

Poria placenta +++

Trichophyton mentagrophytes +++

Zygosaccharomyces rouxii ++

Z. bailii ++ Z. microellips. ++

Saccharomycodes ludwigii ++

Trichosporon cutaneum ++

Eurotium herbariorum ++

Trichothecium roseum ++ Cladosporium resinae ++

Aspergillus awamori ++

A. ficuum ++

A. kawachii ++

A. oryzae ++ Rhizopus oryzae ++

Piptoporus betulinus ++

Acinetobacter ++

Staphylococcus aureus +++

Fusarium culmorum ++ F. graminearum ++

Leuconostoc mesenteroides ++

Pseudomonas putida ++

EXAMPLE 2, gel filtration of the inhibitor fraction

In the present study, a 13 ml sample was taken from the supernatant of the protein separating process in the process of Fig. 1, the sample being diluted and gel-filtrated using Shepadex G 75. In the gel filtrati¬ on 70 fractions were collected, which were subjected to inhibition testing. Compared to the standard proteins (bovine serum albumine and cytochrome C) the fractions with more than 80 % inhibition capacity are within the range of 10000 to 25000 daltons. The results are pre¬ sented in Fig. 4 (inhibition 1 = 100%).

EXAMPLE 3, breadmakinq

In the present study a dough was made, mixing proportions for 80 g dough 49 g flour, 30 ml water/salt solution (24 g/1), 0.9 g yeast. The inhibitor was added so that the water quantity decreased correspondingly (the inhibitor had dry matter content 5 to 7%), 8 ml of inhibitor solution were added, better results were achieved with a quantity of 20 ml. The inhibitor frac¬ tion was taken from a process as depicted in Fig. 1, supernatant 17, it was centrifuged, the supernatant formed and dialyzed for one day against water.

The dough was made up and baked and the bread was cooled down. The cooled bread was cut into slices, the slices were sprayed with a bread mould mixture and enclosed in bags (Minigrip) . The mould attack on the bread slices was followed for about 8 days.

The results are shown in table 1.

TABLE 1, inhibition of bread moulding

Treatment Result of comparison (organoleptic)

Inh. of inv. Good

Control Moulded

Sorbate Fair

Propionate Fair, unpleasent flavour

(16 mg/80 g)

Table 1, inhibition of bread moulding

When a microbe inhibitor according to the invention was used, the structure of the bread turned out better in the baking test, owing to the ample car¬ bon dioxide that was formed.

The embodiment examples are intended to il¬ lustrate the invention, without delimiting it in any way whatsoever.