SE469313B - PROCEDURE FOR ELIMINATION OF MICROBES IN THE PROCESS WATER FROM PAPER FACTORIES USING LYTICAL ENZYMES, WHICH HAVE A GLUCANAS AND PROTEAS ACTIVITY - Google Patents

Description

ï-yva: IN 469 313 2 It is now a new opportunity for microbial control in process the water in paper mills has developed a natural microbiological sludge control agent. It is based on the use of lytic enzymes, which degrade the cell walls of microbes. Combat the target then targets the primary disorder, the microbe. MAN have now found lytic enzymes, which fight precisely these micro- ber. The lytic enzymes are labile compounds, which are broken down quickly in the process water and are harmless to non-microbes.

So a way has now been found to replace the current chemical pesticides with a consumer- and environmentally friendly alternative tiv.

The intention is to use lytic enzymes primarily for decomposition of microbes, which form clusters, and to destroy bacteria, which spoil the food qualities of paper industrial process water. By using lytic enzymes can to eliminate mucus disorders, by cleaving the microbial cells wall constructions so that the accumulations are broken down.

The advantage of the enzymes compared to chemicals is i.a. their poisonous freedom for man and the environment, their specificity and low space requirements. By concentrating the enzyme preparation you get effective levels already with a few ppm enzyme preparations in the process the water.

The invention thus relates to a method and a use according to the features set out in claims 1 and 3.

Lytic enzymes, produced by microbes or isolated from egg whites, acts mainly on 1,3-bonds in the glucan in the walls of the microbial cells, the cell wall no longer holding the pressure in the cell and bursts. So far it is known to use these enzymes commercially only to release egg whites, stuck to the cell walls and for research purposes to release the internal parts of the cell. 5-fï'äí 3 469 313 It is previously known to degrade polysaccharide mucus using common enzymes in the paper industry. There is a commercial cellular preparation, levan hydrolase, (US 3,773,623, US 3,824,184, SF 52 271) whose disadvantage is its effect only on levanslem, which only a few bacteria produce in the paper industry. IN In practice, it has proved almost impossible to obtain one sufficient effect with only this enzyme and one is forced at the same time adding toxic microbes, mainly fungi, lethal biocides. Also two other common enzymes for use in the paper industry's process water for cleavage of slime has patented, namely pentosanase-hexosanase (US 4,055,467) and dehydrogenase (US 4,370,199).

When using common enzymes to eliminate microbiome logical disturbances in the paper industry occur some real major disadvantages. Firstly, the fight is aimed at one secondary phenomenon, mucus, which varies from one microbe to another other and the enzymes are very specific, so that it remains mucus, which the enzyme is unable to break down. Secondly, it is split the polysaccharide mucus in smaller components, which the microbes can use as nutrition. This can result in an explosive increase and accumulation of the dangerous fungi, which causes disruptions in the process.

To the process water at a paper mill is added, for example in the form of an aqueous solution of lytic enzymes on the basis of preliminary near laboratory samples, for example 10 ppm for two hours with four-hour intervals at critical points in paper mills ken. The aim is to maintain the microbial density of the disruptive organisms. below a limit, which is critical of the problematic microbial density.

The lytic enzyme preparation may be in solid, water-soluble form or in liquid form, formulated in water according to normal practice.

Vt fl ni 469 313 The invention is described in more detail below by way of example.

Example 1 Mold: Aspergillus niger Method: As a culture fluid, fungal nutrient fluid (S-S fluid) was used: 0.4% yeast extract, 1.0% malt extract, 0.4% D-glucan, pH 7.3. The transition product was made by homogenizing with fungal nutrient agar (S-S agar) with obliquely grown Aspergillus Niger in sterile water. After the transfer, mold was grown in agitation at a temperature of + 30 ° C.

From the lytic Novozym 234 enzyme, a 1% solution was prepared. ning. The solution was sterilized by filtering it with bacteria. rief filter (pore size 0.45 um).

When testing the effect of Novozym 234 in inhibiting Asperger's gillus niger's growth was added to the culture fluid in connection with the transfer of the following levels of enzyme: 0 ppm (= control), 1 ppm, 5 ppm, 10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm. Asperus nigers growth was observed by measuring the absorbance of the culture fluid in 620 nanometers immediately after inoculation and 1 hour, 2 hours, 3 hours, 4.5 hours, 6.5 hours and 9 hours after spectrophot metric transfer.

Results: The growth of Aspergillus niger was observed spectrophotometrically (wavelength 620 nm). The measured absorbance values are shown in Table 1.

At levels of Novozym 234 of 5 ppm and more, the mold growth increased clearly slower. At the levels used (100 ppm) the the growth of Aspergillus niger was not completely, but it was retarded noticeable (cf. figure l). lqvaa 469 313 Table l.

A 620 values for Aspergillus niger cultivar strains containing different levels of Novozym 234 during cultivation.

Novozvm 234- EEEF 0 ppm 1 pp 5 ppm 10 ppm 25 ppm 50 ppm 75 ppm 100 ppm latency time (= control). 0 h 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 h- 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2 h 0.05 0.05 0.00 * 0.00 0.01 0.00 0.00 0.00 3 h 0.10 0.09 0.03 0.01 0.01 0.03 0.01 0.00 0.00 4.5 h 0.15 0.14 0.06 0.02 0.05 0.04 0.00 0.00 0.00 6.5 h 0.16 0.15 0.09 0.04 0.09 0.08 0.02 0.03 0.03 9 h 0.25 0.26 0.18 0.16 0.13 0.13 0.12 0.12 Conclusions: Aspergillus niger is a typical general in the paper industry. recurrent mold, which with its strong thread growth causes kar accumulations. It also grows in very changing conditions, and is therefore particularly troublesome. From Table 1 and Figure 1 shows how already a 5 ppm Novozym 234 -lytic enzyme retards the growth of Aspergillus niger mold and it could be used with periodic pump dosing in a paper mill. An enzyme- amount of 75 ppm stops growth completely for 5 hours. At pro- MHN: IN 469 315 supplementary preparations have been used to illustrate the results, and in reality even less enzyme in the process water of a paper mill in a real situation tion.

Example 2 Yeast: Yeast 1696, isolated in a paper mill Method: As a cultivation liquid, fungal nutrient liquid (S-S liquid) was used: 0.4% plant extract, 1.0% malt extract, 0.4% D-glucose, pH 7.3. Of yeast In 1696, a transition product was prepared by homogenizing a yeast fungus surface grown with plant nutrient agar (s-S agar) in rilt water. The yeast of 1696 was grown in agitation at a temperature of 30o3C.

From lytic Novozym-234 enzyme a 1% solution was prepared, which was sterilized by filtration (the pore size of the filter was 0.45 μm).

In growth inhibition tests, Novozym 234 was added to yeast in 1696 liquid in connection with the transfer with the following concentrations: 0 ppm (= control), 1 pp, 5 pp, 10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm. The growth of the yeast in 1696 was observed by measuring the strain absorbance at 620 nanometers immediately after transfer and 1 hour, 2 hours, 3 hours, 3 hours, 4 hours, 5 hours, 6 hours and 7 hours after the spectrophotometric transmission.

Results: The cultivar of yeast 1696 isolated at a paper mill observed spectrophotometrically: the A620 values are shown in Table 2. zym 234 content is 25 ppm or greater, the yeast's 1696 is retarded growth significantly. At lower Novozym 234 levels were observed also a slowdown in yeast growth, but the effect was sufficient not then as long as at higher concentrations (cf. Figure 2). i-f fl zí f 469 313 7 Table 2 A -values of Yeast 1696 strains containing different levels of Novozyme 234 during culture.

Novozym 234- halt 0 ppm 1 ppm 5 ppm 10 ppm 25 ppm 50 ppm 75 ppm 100 ggxn Growth time '(= k0tr0ll) 0 h 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 h 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2 h 0.08 0.04 0.03 0.03 0.03 0.01 0.02 0.01 0.01 0.01 3 h 0.09 0.07 0.06 0.06 0.03 0.03 0.03 0.03 0.03 4 h 0.11 _ 0.09 0.09 0.09 0.06 0.05 0.05 0.05 0.06 5 "h _ 0.21 0.18 0.18 0.18 0.12 0.11 0.11 0.12 6 h 0.29 0.27 0.27 0.26 0.20 0.16 0.16 0.17 7 h 0.45 0.45 0.45 0.45 0.34 0.27 0.30 0.31 Conclusions: Yeast 1696 caused interruption in paper production in a Finnish paper mill by forming slimy clusters. except molds, yeasts have proved to be very troublesome due to of its chain-forming ability and often sarcastic mucus formation. Chart 2 and Figure 2 show how already 1 ppm Novozym-234-lytic enzyme tarder the growth of yeast 1696 strain at the factory.

At higher enzyme levels, the inhibition is accentuated and then taken into account that the culture broth in question contains significantly more take nutrients other than the paper mill's process water, it can be assumed, that in practice periodic pump dosing would suffice a few ppm, for 2 hours at four hour intervals, to keep the yeast strain growth at a sufficiently low level. m Myvaa l 469 315 Example 3 Bacteria: Bacillus subtilis, ATCC 23059 Method: Tryptone yeast extract, in which it is used, is used as the culture broth there were 0.5% tryptone, 0.25% yeast extract and 0.5% sodium chloride, pH 9.2. A fresh bacterial culture was used as the transfer product grown in the corresponding nutrient fluid. In the test, it happened occasionally . . O while stirring at a temperature of +37 ° C.

Centrifuged and filtered Cytophaga was used as the lytic enzyme culture fluid Cytophaga sp. NCIB 9497. (The pore size of the filter was 0, 45 um). As a culture broth for the enzyme producer, yeast extract was used. liquid, which contained 2.0% yeast extract, pH 7.0. The incubations took place at + 37OC and lasted 16 hours.

In the plant inhibition test, 16 volumes of ~ ~% filtered culture medium were added. liquid Cytophaga sp. NCIB 9497 in the culture fluid of the bacterium as would be tested in connection with the transfer. During the inspection, a corresponding amount of yeast extract liquid (2.0% yeast extract).

Growth was observed by measuring the ATP content as decomposed in the strain (adenosine triphosphate) immediately after transfer and 2 hours, 4 hours and 23 hours after the transfer with biolum device. The degraded amount of ATP is directly proportional against the growth intensity of the microbes - the more degraded ADP, the more more living active microbes.

Results: The culture of Bacillus subtilis ATCC 23059 was observed by measure changes in the level of ATP in the strain. The measured ATP the levels are shown in Table 3. The addition of the culture fluid Cytphaga sp. NCIB 9497 containing lytic enzymes for the strain Bacillus subtilis ATCC 23059 markedly retarded bacterial growth and clearly extended the team stage. \ -fl "~ | ' 469 313 Table 3 The effect of filtered culture broth Cytophaga sp NCIB 9497 holding lytic enzymes on the growth of Bacillus subtilis ATTC 23059. In the table, the AT levels in units of nanograms / 100 pl.

Sample 0h 2h 4h 23h incubated incubated incubated incubated B.subtilis ATCC 23059 (= control) 22.7 29.2 444 120 000 B.subtilis ATCC 23059 + enzyme solution 4.34 3.16 3.54 64 600 Conclusions: Bacillus species have been shown to be problematic in paper industry both because of its chain-forming ability and thus emergence of accumulations, and their ability to form resistant spores, which remain in the final product, which in particular in per of food quality can cause major problems.

The growth of the strain ATCC 23059 Bacillus subtilis in question can be made to stop completely for over four hours with an enzyme solution produced by Cytophaga sp, a result which constitutes a completely sufficient inhibition time in view of pump dosages in practice in the paper industry.

Example 4 Bacteria: Bacillus sp strain at a Finnish paper mill The method was performed as in Example 3. \. 'p¿; IN 469 313 10 Results: The cultivation of Bacillus sp at the Finnish paper mill was measured by measuring changes in the ATP content in the strain. The measured ATP levels are shown in Table 4. Addition of culture broth NCIB 9497 to the Bacillus sp strain retarded the bacterium grown by clearly extending the team stage.

Table 4 Effect of filtered culture broth Cytophaga sp. NCIB 9497 containing lytic enzymes on the growth of the strain Bacillus sp at a Finnish paper mill. The table indicates the ATP levels in units nq / 100 ul.

Sample O h 2h 4h 23 h incubated incubated incubated incubated Bacillus sp. (= control) 16.3 25.1 293 39 200 Bacillus sp. + enzyme solution 3.32 9.08 2.84 59 800 Conclusions: This test can confirm that an enzyme produced by Cytopha- ga sp also affects a "wild" strain Bacillus sp isolated on a Finnish paper mill. Even in this case, the effect is maintained in over 4 hours, a result that is sufficient in practice.

Example 5 Bacteria: A strain Bacillus sp. at a Swedish paper mill.

The method was performed as in Example 3. \ qvz; 469 313 ll Results: The cultivation of Bacillus sp at the Swedish paper mill was measured by measuring changes in the ATP content in the culture. The measured ATP levels are shown in Table 5. Addition of filtered culture fluid Cytophaga sp. NCIB 9497 to the Bacillus SP plant clearly delayed the growth of the bacterium and prolonged the 1 g stage.

Table 5 The effect of filtered culture broth Cytophaga sp. NCIB content the lytic enzymes on the growth of the Bacillus sp. strain on it Swedish paper mill. The table indicates the ATP levels in units ter ng / 100 pl.

Sample O h 2 h 4 h 23 h incubated incubated incubated incubated Bacillus sp. (= kontrQll) 14.7 27.1 468 81 400 Bacillus sp. + enzyme solution 2.79 4.72 2.64 67,000 Conclusions: The lytic enzyme produced by Cytphaga sp has a broad spectrum, as it also proved in this test to have a long-lasting effect on Bacillus the lice sp. strain that caused the problem and was isolated from it Swedish paper mill. Complete inhibition is maintained unless at least 4 hours, during which the control without enzyme addition has more than 30-fold in relation to the growth intensity.

Claims (3)

469 [Wii 12 l šgígâtkrav A process for the elimination of microbes, which causes precipitates, forms mucus and / or degrades the quality of food paper or board in the process water of paper mills, characterized in that lytic enzyme having a glucanase and protease activity is added to the paper mill process water. degrades the cell walls of microbes. A method according to claim 1, characterized in that lytic enzyme to a content of advantageously at least 1 ppm is added to the process water of the paper mill. Use of lytic enzymes, which have a glucanase and protease activity that degrades the cell walls of microbes, to eliminate microbes that cause precipitation, mucus formation and / or degrade the quality of food paper or board in the process water of paper mills. m