FI75973B - FOERFARANDE FOER ELIMINERING AV MIKROBER I PROCESSVATTEN AV PAPPERSFABRIKER. - Google Patents

Description

75973

This invention relates to the destruction of microbes which cause precipitation, form slime and / or degrade the quality of food paper or board in the process water of paper mills.

Process waters in the paper industry provide very favorable conditions for the growth of bacteria and fungi. Temperature, acidity value, and nutrient concentrations are in many cases almost optimal for many microbes. As a result, paper mills may occasionally experience mucus disturbances caused by microbes growing into piles. This is often accompanied by strong mucus formation. Bacteria or bacterial spores (eg Bacillus) that remain on food paper or board form their own microbiological problem.

Extensive studies in various paper mills that have experienced disturbances in paper production have shown that the most significant causes of disturbance in process waters are filament-forming microbes. The formed filaments are adhered to by mucus-forming bacteria as well as chemical mucus. Harmful microbes are mainly mold and yeast fungi as well as chain-forming bacteria such as Bacillus species.

Mucus disorders have traditionally been controlled with antimicrobial chemicals. In the past, mercury compounds were used, which were abandoned because of their toxicity in the late 1960s, even before their use was banned. There are still forced to use biocides, organic compounds considered to be second class poisons. Each anti-slime chemical has a certain biological half-life in the process water, which varies from a few tens of minutes to several days. Some degradation products may also be bacteriostatic 2,75973. For this reason, the use of chemical pesticides is also ecologically disadvantageous.

As a new opportunity to be used to control microbes in paper mill process water, a natural microbiological slime control method has now been developed. It is based on the utilization of lytic, microbial cell wall degrading enzymes. In this case, the control is directed at the primary disorder, the microbe. Lytic enzymes have now been found to fight just those microbes. Lytic enzymes are labile, rapidly degradable compounds in process waters and are harmless to non-microbes. So a way has now been found to replace existing chemical pesticides with a user- and environment-friendly alternative.

Lytic enzymes are intended to be used primarily to break down clump-forming microbes, as well as to kill food-grade bacteria in process waters in the paper industry. Using lytic enzymes, mucus disorders can also be removed by cleaving the wall structures of microbial cells so that the agglomerates disintegrate.

The advantages of enzymes compared to chemicals are e.g. their non-toxicity to humans and the environment, their specificity and low space requirements. Concentration of the enzyme preparation results in effective concentrations of a few ppm of the enzyme preparation in the process water.

The invention therefore relates to a process in which microbes which cause precipitation and form slime and / or degrade the quality of food-grade paper and board are destroyed either pre- or post-process in the process waters of paper mills by adding lytic enzymes to them.

75973 3

Lytic enzymes produced by microbes or isolated from egg white mainly act on 1,3-bonds in the glucan of the microbial cell walls, so that the cell wall can no longer withstand intracellular pressure and rupture. It is known that lytic enzymes also have protease activity (Separation, Recovery, and Purification in Biotechnology, ACS Symp. Ser. 314 (1986), pp. 9-31: JB Hunter and JA Asenjo, Structural and Simple Models of Enzymatic Lysis and Disruption of Yeast Cells .) Hitherto, it is known to use these enzymes commercially only to release proteins attached to cell walls and for research purposes to release intracellular components.

It is already known to degrade polysaccharide glue using conventional enzymes in the paper industry. There is one commercial preparation, levan hydrolase, (US 3,773,623, US 3,824,184, FI 52,271) which, when attenuated, has an effect only on algal mucus produced by only a few bacteria in the paper industry. In practice, it has proved almost impossible to achieve sufficient potency with this enzyme alone, and further concomitant additions of biocides that kill toxic microbes, especially fungi, have to be added. Two other conventional enzymes for use in paper industry process waters for slime cleavage have also been patented, namely pentosanase-hexosanase (US 4,055,467) and dehydrogenase (US 4,370,199).

There are a few very significant drawbacks to the use of conventional enzymes in the control of microbiological disorders in the paper industry. First, the control targets a secondary phenomenon, mucus that varies from microbial to microcontroller and the enzymes are very specific, leaving mucus that cannot be broken down by the enzyme. Second, the polysaccharide mucus is broken down into smaller components that microbes can use as food. This can also result in the explosive growth and accumulation of dangerous fungi, which disrupts the process.

For example, lytic enzymes are added to the paper mill's process water as an aqueous solution based on preliminary tests performed in the laboratory, for example, 10 ppm for two hours every four hours at certain critical targets in the paper mill. The goal is to keep the microbial density for interfering organisms below a certain critical problem-causing micro-bit density limit.

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

The invention is described in more detail below by means of examples. Example 1

Mold: Aspergillus niger Method:

A mushroom broth (S-S broth) was used as the culture broth: 0.4% yeast extract, 1.0% malt extract, 0.4% D-glucan, pH 7.3. The inoculum was prepared by homogenizing Aspergillus niger grown on an oblique surface with fungal nutrient agar (S-S agar) in sterile water. After inoculation, the mold was grown with shaking to +30 ° C.

The lytic Novozym 234 enzyme was made into a 1% solution. The solution was sterilized by filtration through a bacterial filter (pore size 0.45 μm).

To test the efficacy of Novozym 234 in inhibiting the growth of Aspergillus niger, the following concentrations of enzyme were added to the broth inoculum: 0 ppm (= control), 1 ppm, 5 ppm, 10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm. Growth of Aspergillus niger was monitored by measuring the absorbance of the culture broth at 620 nanometers immediately after inoculation and at 1 hour, 2 hours, 3 hours, 4.5 hours, 6.5 hours, and 9 hours of inoculation with a spectrophotometer.

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Score:

The growth of Aspergillus niger was monitored spectrophotometrically (wavelength 620 nm). The measured absorbance values are shown in Table 1. At concentrations of Novozym 234 of 5 ppm and above, mold growth was clearly slowed. At the concentrations used (<100 ppm), the growth of Aspergillus niger was not completely inhibited but significantly slowed down (see Figure 1).

Table 1.

A 620 values of Aspergillus niger cultures containing different concentrations of Novozym 234 during cultivation.

Novozym 234- concentration 0 ppn 1 ppm 5 ppm 10 ppm 25 ppm 50 ppm 75 ppm 100 ppm

Growing 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, 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 6.5 h 0.16 0.15 0.09 0.04 0.09 0.08 0.02 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 fungus commonly found in the paper industry, which, due to its strong groin growth, causes the formation of agglomerations. It is capable of growing in very variable conditions, which makes it particularly difficult. Table 1 and Figure 1 show how the already 5 ppm Novozym 234 lytic enzyme slows the growth of Aspergillus niger and could be used as a periodic pump feed in a paper mill. An amount of 75 ppm enzyme completely stops growth for 5 hours. 75973 6 supplements have been used in the experiment to illustrate the results, so in reality even smaller amounts of enzyme would be needed in the paper mill process water in the real situation.

Example 2

Yeast fungus: Yeast 1696, isolated from a paper mill Method:

A mushroom broth (S-S broth) was used as the culture broth: 0.4% yeast extract, 1.0% malt extract, 0.4% D-glucose, pH 7.3. Yeast 1696 was inoculated by homogenizing yeast agar grown on fungal nutrient agar (S-S agar) in sterile water. Yeast 1696 was grown in a flask at + 30 ° C.

A 1% solution of Novolym 234 lytic enzyme was prepared and sterilized by filtration (filter pore size 0.45).

In the growth inhibition experiment, Novozym 234 was added to the yeast 1696 broth without detachment at the following concentrations: 0 ppm («control), 1 ppm, 5 ppm, 10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm. The growth of yeast 1696 was monitored by measuring the absorbance of the culture at 620 nanometers immediately after inoculation and 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours and 7 hours after inoculation with a spectrophotometer.

Results: The growth of yeast 1696 isolated from a paper mill was monitored spectrophotometrically. · A values are shown in Table 2.

620

At Novozym 234 concentrations of 25 ppm or greater, the growth of yeast 1696 was significantly slowed. At lower concentrations of Novozym 234, a slowing of yeast growth was also observed, but the effect did not last as long as at higher concentrations (see Figure 2).

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Table 2.

Measured A values of yeast 1696 cultures containing different concentrations of Novozym 234 during cultivation.

620

Novozym 234- concentration 0 ppn 1 ppm 5 ppm 10 ppm 25 ppm 50 ppm 75 ppm 100 ppm

Growing time (= control) 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 2 h 0.08 0.04 0.03 0.03 0.01 0.02 0.01 0.01 3 h 0.09 0.07 0.06 0.06 0.03 0.03 0.03 0.03 4 h 0.11 0.09 0.09 0.09 0.06 0.05 0.05 0.06 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 p

Yeast 1696 caused interruptions in papermaking in a Finnish paper mill by forming slimy piles. In addition to home fungi, yeast fungi have proven to be very difficult due to their chain-forming ability and often intense mucus production. Table 2 and Figure 2 show how as little as 1 ppm of Novozym-234 lytic enzyme slows the growth of the yeast 1696 plant strain. Higher enzyme concentrations emphasize inhibition, and given that the medium in question contains significantly more dissolved nutrients than paper mill process water, it can be assumed that in practice a periodic pump dosing of a few ppm, 2 hours every four hours, would be sufficient to keep yeast stock growth low.

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Example 3

Bacteria: Bacillus subtills, ATCC 23059 Method:

As a culture broth, tryptone-hi 1 extract extract containing 0.5% tryptone, 0.25% hH extract and 0.5 * sodium chloride, pH 9.2, was used. A fresh bacterial culture grown in the corresponding broth was used as a broth. In the experiment, growth was performed by shaking at + 37 ° C.

As a lytic enzyme, centrifuged and filtered Cytophaga sp. NCIB 9497 breeding client. (The pore size of the filter was 0.45 μm). Yeast extract containing 2.0% yeast extract, pH 1, was used as the broth of the enzyme producer.

7.0. Incubation took place at + 37 ° C and lasted 16 hours a.

In the growth inhibition assay, filtered Cytophaga sp. NCIB 9497 culture broth 16% by volume upon inoculation of the bacterial culture medium to be tested. An equivalent amount of h11 extract (2.0% hH extract) was added to the control. Growth was monitored by measuring the degradable ATP content in the culture (adenosine n1 tri phosphate) immediately after inoculation and 2 hours, 4 hours and 23 hours after inoculation by bioluminescence. the more live, active microbes.

Score:

The growth of Bacillus Subtil 1 s ATCC 23059 was monitored by measuring the change in ATP content of the culture. The measured ATP-p1 distances are shown in Table 3. Cytophaga sp. Addition of NCIB 9497 to Bacillus subtlHs ATCC 23059 significantly slowed bacterial growth and significantly prolonged the 1ag lie.

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Table 3

Filtered Cytophaga sp. Containing lytic enzymes. Effect of NCIB 9497 broth on the growth of Bacillus subtllls ATCC 23059. In the table, ATP concentrations are reported in nanograms / 100 μl.

Sample Oh 2h 4h 23 h Incubated Incubated Incubated Incubated B. subtllls ATCC 23059 (control) 22.7 29.2 444 120 000 B. subtllls ATCC 23059 + enzyme solution 4.34 3.16 3.54 64 600

Conclusions:

Bacillus have proven to be problematic in the paper industry, both because of their ability to form chains and, therefore, because of their ability to form agglomerates, which remain in the final product, which can cause major problems, especially in food grade paper.

The growth of this Bacillus subti11s strain of ATCC 23059 is completely stopped by the enzyme solution produced by Cytophaga sp for up to 4 hours, which results in a practical pump lift in the paper industry which is completely sufficient for 1n.

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Example 4

Bacteria: Bacillus sp. Strain of a Finnish paper mill The method was performed as in Example 3.

Results:

The growth of Bacillus $ p in the Finnish paper mill was monitored by measuring the change in the ATP content of the crop. The measured ATP concentrations are shown in Table 4. Cytophaga sp. Addition of NCIB 9497 medium to Bacillus sp. Slowed bacterial growth by clearly prolonging the 1ag phase.

Table 4

Filtered Cytophaga sp. Containing lytic enzymes. Effect of NCIB 9497 broth on the growth of the Bacillus sp. Strain of a Finnish paper mill. In the table, ATP concentrations are reported in ng / 100 μl.

Sample Oh 2h 4 h 23 h 1nkubo1tu 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 experiment is able to confirm the functionality of the enzyme produced by Cytophaga sp also in a "wild" strain of Bacillus sp. Isolated from a Finnish paper mill. Also in this case, the power remains excellent for more than 4 hours, which is a sufficient result from a practical point of view.

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Example 5

Bacteria: Bacillus sp. Strain from a Swedish paper mill The method was performed as in Example 3.

Score:

The growth of Bacillus sp in the Swedish paper mill was monitored by measuring the change in the ATP content of the crop. The measured ATP concentrations are shown in Table 5. The filtered Cytophaga sp. Addition of NCIB 9497 broth Bacillus sp. clearly slowed bacterial growth and prolonged lag.

Table 5

Lytic enzyme-containing filtered Cytophaga sp. Effect of NCIB 9497 broth on the growth of the Bacillus sp. Strain in a Swedish paper mill. In the table, ATP concentrations are reported in ng / 100 μl.

Sample Oh 2 h 4 h 23 h

Incubated incubated incubated

Bacillus sp.

(= control11) 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 Cytophaga sp. Complete inhibition is maintained for at least 4 hours, during which time control without enzyme inhibition is more than 30-fold greater than its growth intensity.

Claims (3)

12 75973 A method for killing microbes which cause precipitation, form mucus and / or impair the quality of food paper or board in the process water of paper mills, characterized in that a lytic enzyme having qucanase and protease activity which degrades microbial cell walls is added to the process water of the paper mill. Process according to Claim 1, characterized in that a lytic enzyme is added to the process water of the paper mill, preferably to a concentration of at least 1 ppm. 3. Use of precipitating, mucolytic and / or microbicidal lytic enzymes with glucanase and protease activity that degrade microbial cell walls in paper mill process water.