PL199644B1 - Application of protein fraction from brewery spent grain and microbiological substrate - Google Patents

Abstract

An application of the protein fraction of brewer's spent grain was discovered. The protein fraction described is a byproduct of the beer brewing process. The microbiological culture medium proposed can be easily employed in the cultivation of Gram-positive soil bacteria of the Actinomycetes series, particularly the species of Streptomyces. Employing the solution of protein fraction extract (PFE) is unusually economical and allows one to obtain bacterial cultures producing secondary metabolites.

Description

Description of the invention

The invention relates to the use of a protein fraction from spent grains as the main component of a new microbial medium. The described protein fraction is a by-product of the beer brewing process. The proposed microbiological medium is perfectly applicable for the cultivation of gram-positive soil bacteria of the Actinomycetes, and especially Streptomyces species. The protein fraction extract (PFE) solution used is extremely economical and allows for obtaining bacterial cultures producing secondary metabolites.

In microbiological practice, in addition to well-defined culture media, many media based on ingredients derived from production waste from the food industry are used. One of the by-products of the beer production process is fermented barley grain. This product is a major disposal problem.

In the beer fermentation process (Fig. 1), unground wort is prepared from dried straw in a mashing process. Water is added to the dried straw and the temperature is slowly increased, followed by enzymatic hydrolysis of carbohydrates and proteins. Then, after the completed hydrolysis, the wort is separated from the sludge in the filtration process, the obtained wort is the subject of further processes leading to the production of beer. The sludge called spent grain SG is a production waste. As a result of grinding the spent grain and fractionation, three fractions are obtained: a coarse fraction (fiber fraction FF, fiber fraction) with a high fiber content, a middle fraction and a fine fraction containing a lot of protein (protein fraction PF, hereinafter referred to as a protein fraction) (Ishiwaki et al. 2000). The hay consists mainly of parts of the pericarp and shells of the barley grain, and contains 28% protein, 17% lipids, and 55% fibers, respectively (Hasson 1993). Brewing husk is commonly used as an additive to ruminant feed and an additive in bakery. The protein fraction is also used as an agent to improve the properties of creams and sauces.

A typical search for industrially useful microorganisms is carried out in the process of testing the properties of soil samples from various ecological niches. Bacteria of the genus Streptomyces are an extremely rich source of biologically active compounds. Only a few media are proposed in the literature for the growth of Streptomyces (Kieser et al. 2000). To improve the selective growth properties of Actinomycetes, it has been proposed to use substances such as L-arginine (El-Nkeeb, Lechevalier 1963), humic acids (Hayakawa, Nonomura 1987) and trimethylprim with nalidixic acid (Hayakawa et al. 1996).

The object of the invention is to propose a method for the disposal of brewing waste, in particular spent grains. Another object of the invention is to propose a new culture medium for microorganisms, in particular a selection medium for bacterial cultures of the genus Actinomycetes and related, which is easily available and ensures a sufficiently high level of secondary metabolite production.

Unexpectedly, the above-described problems have been solved by the present invention. The subject of the invention is the use of a protein fraction from spent grains for the production of a microbiological medium. In a preferred embodiment of the invention, the microbial medium produced is used for the cultivation of gram-positive soil bacteria of the order Actinomycetes. The microbial medium produced may be used according to the invention to cultivate Streptomyces species. Preferably, the medium produced comprises a 2% solution of the extract of the protein fraction (PFE) of spent grains. The produced medium can be used as a medium for industrial cultivation, preferably industrial cultivation of strains of bacteria of the genus Streptomyces, especially under controlled pH conditions and / or as a medium for the isolation of Actinomycetes strains from the environment, preferably new Actinomycetes strains, and / or as a medium for sporulation and / or production of secondary metabolites and / or as a substrate for the search for new biologically active compounds and / or monitoring of environmental changes.

The subject of the invention is also a microbiological medium for the cultivation of gram-positive soil bacteria of the order Actinomycetes, characterized in that it contains a protein fraction from spent grains. Preferably, the medium according to the invention contains a 2% solution of the protein fraction extract (PFE) and / or has a pH of 6 to 8, preferably about 7, in particular 7.2. In a preferred embodiment, the medium of the invention is a solid medium which contains an added agar, preferably in an amount of about 2% w / v. In another preferred embodiment, the medium of the invention is a liquid medium that comprises 20 mM Tris-HCl pH 7.2.

The use of SG fraction as a major component of a new economical microbial medium applicable to the isolation, culture and sporulation of Streptomyces bacteria has been disclosed.

PL 199 644 B1 and related. To fully characterize the described medium, tests were carried out: the growth of various types of bacteria; spore production (sporulation); the production of secondary metabolites; the growth rate of the liquid cultures was also examined.

PFE microbiological substrate prepared from production waste in the beer brewing process has features important for applications in biotechnology. It can be used as a cheap, economical medium for industrial cultivation, a medium for the isolation of Actinomycetes strains from the environment, as a medium for sporulation and production of secondary metabolites. An important feature of the PFE medium is the selective stimulation of the growth of Actinomycetes bacteria, this feature allows the identification of previously unknown strains isolated from the environment. PFE medium is a cheap and easy to prepare product obtained from waste, therefore, using controlled pH conditions, it can be used on a large scale in the industrial cultivation of strains of bacteria of the genus Streptomyces. It is possible to use this medium to search for new biologically active compounds and to monitor environmental changes.

The presented figures supplement the content of this description. Fig. 1 shows a diagram of the brewing process and the production of production waste. Figure 2 shows the observed spore production of the Streptomyces coelicolor strain on PFE solid medium a) after 43 hours; b) after 72 hours. Figure 3 shows the observed production of undecylprodigiosin by the Streptomyces coelicolor strain on PFE solid medium: a) after 43 hours; b) after 72 hours. Figure 4 shows the production of actinorhodin observed by the Streptomyces coelicolor strain on PFE solid medium: a) after 43 hours; b) after 72 hours. Figure 5 shows the growth curves of Streptomyces coelicolor and the amount of colored metabolites produced in liquid culture: a) growth on minimal SMM medium; b) growth on PFE substrate.

The following abbreviations are used in this description: BSG (brewers' spent grain) means brewing spent grain, FF (fiber fraction) means fiber fraction, PF (protein fraction) means fraction protein fraction extract, PFE stands for protein fraction extract, SMM stands for supplemented minimal medium.

P r z k ł a d 1

Preparation of the PFE (Protein Fraction Extract) substrate

The medium was prepared as follows: Two grams of dry protein fraction (PF) from the spent grains was added to 100 ml of distilled water and autoclaved for 30 min at 121 ° C. The cooled solution was filtered through Whatman # 1 paper. Then, after removal of the solids, the solution was adjusted to pH 7.2 with 1 M NaOH and re-autoclaved for 25 min. The solid medium contained addition of agar (2% w / v). The liquid medium contained 20 mM Tris-HCl pH 7.2.

P r z k ł a d 2

The use of PFE substrate for the cultivation of various microorganisms

In order to obtain the characteristics of the PFE substrate, its suitability for the cultivation of various microorganisms, especially actinomycetes, was investigated.

Materials

The protein fraction (PF) and the fiber fraction (FF) of the spent grain were from Dr. Bo Lofquist, BoMill AB, Sweden. Sixteen Actinomycetes strains and six other strains were grown in PFE medium. Strain Streptomyces coelicolor A3 (2) M145 (SCP1 ^_ SCP2 ^_ ) (Kieser et al., 2000), was obtained from H. Kieser (Norwich, UK). The following strains were derived from the Polish Collection of Microorganisms (PCM) were used in the experiments: Nocardia asteroides PCM 2254 ^T, Nocardia otitidiscaviarum PCM 2143, Rhodococcus equi PCM 559 ^T, Rhodococcus erythropolis PCM 2150, Rhodococcus rhodochrous PCM 909, Gordonia bronchialis PCM 2167 ^T dietzi maris PCM 2292, Streptomyces griseus PCM 2448, Streptomyces albus PCM 2319, Streptomyces flavoscleroticus PCM 2303, Streptomyces exfoliatus PCM 2367, Nocardiopsis dassonvillei PCM 2492, Nocardiopsis alborubida PCM 2490, Citrobacter sub4646 PCM 2346. salamae cheese. Erlangen PCM 2533, Shigella flexneri PCM 1793, Pseudomonas aeruginosa PCM 2058, Staphylococcus aureus PCM 2101.

Methods

Determination of Streptomyces coelicolor growth curves

S. coelicolor A3 (2) was grown in two media: Supplemented minimal medium (SMM) (Kieser et al. 2000, with modifications by Dr. E. Takano submitted personally) and PFE medium. In both cases, the cultures were carried out in 250 ml beakers with air springs. Beakers and springs

Were coated with silicone. 50 ml of medium were inoculated with 5x10 ⁶ spores. The cultures were carried out at 28 ° C, in a shaker at 220 rpm / min for 48 hours.

The antibiotic concentrations were checked as follows: the actinorhodin concentration was tested in the medium, 0.75 ml of the medium was added to 0.75 ml of 2 M KOH, mixed and centrifuged after 30 minutes (10 min, 20,000 g). The absorbance at 633 nm was then measured and the actinorhodin concentration was calculated using the molar factor ε-25320. To determine the concentration of undecylprodigiosin, 10 ml of a culture was taken and centrifuged (3000 g, 10 min), then the cells were extracted with 1 ml of methanol for 24 hours with gentle mixing. The extract was acidified with 1 ml of 0.5 M HCl, centrifuged and the absorbance of the supernatant was measured at 530 nm, the concentration of undecylprodigiosin was calculated using the molar ratio ε = 100500.

Assay for the production of secondary metabolites by S. coelicolor

Two sets of plates were prepared: 2% PFE and standard BHI medium (Difco). S. coelicolor spores were plated on the plates in amounts ranging from 1x10 ¹ to 1x10 ⁸ . The production of colored secondary metabolites was observed for seven consecutive days. Growth was performed at 28 ° C. Production of metabolites was scored on a 0-3 scale based on observation.

Preparation of soil samples

Soil samples were collected in and around the city of Lund. One gram of each soil sample was dried with warm air at 40 ° C for 24 hours. Then the sample was transferred to a sterile test tube and mixed intensively for 5 min with 3 ml of sterile distilled water. PFE of each solution was seeded onto solid PFE, and incubated at 37 ° C for 7 days. Bacterial growth was studied by microscopic observation, naked eye observation, dye production, and spore production.

Collection strain tests

The collection strains were grown on BHI and PFE media at 37 ° C for 48 hours. Gram-negative bacteria were also grown in MacConkey solid medium at 37 ° C for 24 hours.

Results

Substrates prepared on the basis of: spent grain SG; spent grain fiber fraction (FF); protein fraction (PF) (Table 1).

T a b e l a 1

Growth of microorganisms from three different soil samples on substrates prepared with different fractions of spent grain.

The substrate contains Soil sample Category of microorganisms (%) The strain sporal Strains non-sporulating Mushrooms SG 2% - 6% AND thirty 70 0 K. thirty 70 0 M. twenty 40 40 FF 2% - 6% AND 10 thirty 60 K. 25 40 35 M. 25 15 60 PF 2% - 4% AND 80 twenty 0 K. 90 10 0 M. 90 5 5

Samples taken from three different locations were labeled A, K and M, SG - brewers' spent grains, PF - protein fraction, FF - fiber fraction. The number of colonies of the individual microorganisms found on the plates was expressed as a percentage.

The substrate prepared from the spent grain promoted the growth of bacteria, the substrate made of the fiber fraction favored the growth of the fungi. The fiber fraction mainly contains cellulose, and fungi are known cellulase producers. The medium prepared from the protein fraction turned out to be selective for Streptomyces and other Actinomycetales bacteria. With the exception of the two Nocardiopsis strains: N. dassonvillei and N. alborubida strains which are Nocardia-like strains belonging to the order ActinomycePL, all tested strains were grown on solid PFE medium. Streptomyces strains growing on PFE medium produced spores at the same time as when grown on rich organic BHI medium. We observed weak growth of selected strains of other tested gram positive and gram negative bacteria on PFE medium (Table 2).

T a b e l a 2

Cultures of strains growing on 2% (w / v) PFE medium

Actinomycetes and related strains Other strains Nocardia asteroides PCM 2254 ^T Nocardia brasiliensis Nocardia otitidiscayiarum PCM 2143 Gram-negative: Citrobacter freundii PCM 2346 Salmonella enterica subsp. salamae cheese. Erlangen PCM 2533 Rhodococcus equi PCM 559 ^T Rhodococcus erythropolis PCM 2150 Rhodococcus rhodochrous PCM 909 Shigella flexneri PCM 1793 Pseudomonas aeruginosa PCM 2058 Gordonia bronchialis PCM 2167 ^T I will add: Staphylococcus aureus PCM 2101 Dietzia maris PCM 2292 Streptomyces griseus PCM 2448 Streptomyces albus PCM 2319 Streptomyces flavoscleroticus PCM 2303 Streptomyces exfoliatus PCM 2367 Coryneform clinical isolate CI

The growth of non-Actinomycetes organisms was weaker on PFE solid medium than on BHI solid medium. Since the land and similar ecological niches are dominated by Streptomyces strains and related organisms, the PFE substrate has proved to be good for the selection of strains producing new unknown biologically active substances. The study of further growth characteristics on PFE medium was carried out with the use of a model strain for the genus Streptomyces, Streptomyces coelicolor A3 (2). The S. coelicolor strain has been the subject of much research and is a well characterized strain of Streptomyces (Kieser et al. 2000). For comparison, the tests used rich organic BHI medium and a liquid minimal SMM medium.

One of the major problems with culturing Streptomyces strains under industrial conditions is obtaining reproducible growth conditions leading to sporulation. The composition of the medium has a key influence on the course of growth and sporulation of Streptomyces. We sowed the spores of S. coelicolor A3 (2) in various concentrations on the PFE and BHI media. On the PFE medium, the colonies produced spores initially white, later stained with a polyketide dye gray (Figs. 2a and 2b). Streptomyces did not produce spores on BHI medium.

The production of two colored polyketide compounds, actinorhodine and undecylprodigiosin (Ishizuka et al. 1992), which are also compounds with antibiotic activity, was investigated. As shown in Figure 3a actinorodin, a dark blue dye appeared around the colonies after 72 hours of growth on plates where spores were plated at levels ranging from 10 ¹ to 10 ⁴ . After 5 days, all S. coelicolor cultures on PFE medium were dark blue stained. Actinorhodine production was not observed in the BHI control group.

The red dye undecylprodigiosin is in fact a mixture of undecylprodigiosin, butylcycloheptylprodigiosin and meta-cycloprodigiosin (Tsao et al. 1985). The production of undecylprodigiosin was observed only in the BHI control group. The cells produced the dye after 43 hours of culturing (Figures 4a and 4b). The lack of undecylprodigiosin production by cells on PFE medium may be due to the rapid rate of colony growth or the presence or absence of specific medium components. The influence of various factors present in the medium on the synthesis of secondary metabolites is known (Kang et al. 1998).

PL 199 644 B1

The suitability of PFE medium for use in industrial liquid culture was tested by comparing the growth curves and production of colored metabolites by the S. coelicolor strain, growth on PFE medium and standardized minimal medium (SMM) were compared (Figures 5a and 5b).

During the growth of S. coelicolor on the PFE liquid medium, a significant increase in acidification of the medium was observed, which could be the reason for the inhibition of the growth and decrease of the cell mass (Fig. 5b). The increase in acidity may be due to the secretion of specific serine proteases into the medium, which hydrolyze prolamines. The secretion of serine proteases by the species Streptomyces griseus has been reported. The described proteases have found application in protein sequencing and the expression of fusion proteins (Svendsen et al. 1991). It can be assumed that the described medium will be useful in the search for microorganisms secreting similar proteases.

Streptomyces bacteria show a remarkable ability to spontaneously duplicate certain DNA fragments. This property is used for molecular monitoring of soil contamination. The level of expression and the presence of a marker gene located on the chromosome of Streptomyces violaceoruber and introduced into the test environment is a determinant of contamination. (Wellington et al. 1990, Cresswell et al. 1991). The requirements for the substrates in these tests are perfectly met by the PFE medium, which allows for the selective growth of Streptomyces bacteria and rapid DNA isolation.

The work was financed under the State Committee for Scientific Research grant number 4PO5A 073 19, and by the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences as part of its statutory activity.

Literature

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El-Nakeeb, M.A., Lechevalier, H.A., 1963. Selective isolation of aerobic Actinomycetes. Appl. Microbiol. 11, 75-77.

Hassona H.Z., 1993. High fiber bread containing brewer's spent grains and its effect on lipid metabolism in rats. Nahrung, 37, 576-582.

Hayakawa, M. & Nonomura, H., 1987. Efficacy of artificial humic acid as a selective nutrient in HV agar used for the isolation of soil actinomycetes. J. Ferment. Technol. 65, 609-616.

Hayakawa M .; Takeuchi T., Yamazaki T., 1996. Combined use of trimethoprim with nalidixic acid for the selective isolation and enumeration of actinomycetes from soil. Actinomycetologica, 10, 80-90.

Kang S.G., Jin W., Bibb M., Lee KJ. (1998) Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3 (2) grown in continuous culture. FEMS Microbiol Lett, 168, 221-226.

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Wellington E.M.H, Cresswell N. and Saunders V.A. (1990) Growth and survival of streptomycete inoculants and the extent of plasmid transfer in sterile and non-sterile soil. Appl. Environ. Microbiol. 56, 1413-1419.

Claims (9)

Patent claims 1. Use of a protein fraction from spent grains for the production of a microbiological medium for the cultivation of gram-positive soil bacteria from the order Actinomycetes, the produced medium containing a 2% solution of the extract protein fraction (PFE) from spent grains. 2. The use according to claim 1 The process of claim 1, characterized in that the produced medium is used as a medium for industrial cultivation, preferably industrial cultivation of strains of bacteria of the genus Streptomyces, especially under controlled pH conditions. 3. Use according to claim 1 2. A method according to claim 1, characterized in that the produced medium is used as a medium for the isolation of Actinomycetes strains from the environment, preferably new Actinomycetes strains. 4. The use according to claim 1 The method of claim 1, characterized in that the produced medium is used as a medium for sporulation and / or production of secondary metabolites. 5. Use according to claim 1 The method of claim 1, characterized in that the produced substrate is used as a substrate for searching for new biologically active compounds and / or monitoring environmental changes. 6. A microbiological medium for the cultivation of Actinomycetes gram-positive soil bacteria, characterized in that it contains a 2% solution of the protein fraction extract from spent grains. 7. The substrate according to claim 1 6. The process according to claim 6, characterized in that it has a pH of 6 to 8, preferably about 7, especially 7.2. 8. The substrate according to claim 1 The solid support according to claim 6, characterized in that the solid support is added agar, preferably in an amount of about 2% w / v. 9. The substrate according to claim 1 The process of claim 6, wherein the liquid medium is 20 mM Tris-HCl pH 7.2.