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Title:
PROTEIN FRACTION OF BARLEY SPENT GRAINS AS A MEDIUM FOR GROWTH, SPORULATION AND SELECTION OF ACTINOMYCETES AND RELATED BACTERIA
Document Type and Number:
WIPO Patent Application WO/2004/053108
Kind Code:
A1
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.

Inventors:
Gamian, Andrzej (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Pawlik, Krzysztof (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Szponar, Bogumila (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Szwajcer-dey, Estera (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Application Number:
PCT/PL2003/000142
Publication Date:
June 24, 2004
Filing Date:
December 11, 2003
Export Citation:
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Assignee:
Gamian, Andrzej (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Pawlik, Krzysztof (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Szponar, Bogumila (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
Szwajcer-dey, Estera (ul. Rudolfa Weigla 12, Wroclaw, PL-53-114, PL)
International Classes:
C12N1/20; (IPC1-7): C12N1/00
Foreign References:
EP0588145A21994-03-23
CA1317413C1993-05-04
EP0443813A11991-08-28
Other References:
ISHIWAKI, N. ET AL.: "Development of High Value Uses of Spent Grain by Fractionation Technology" MASTER BREWERS ASSOCIATION OF THE AMERICAS TQ, vol. 37, no. 2, 2000, pages 261-265, XP009028336 cited in the application
Attorney, Agent or Firm:
Witek, Rafal (Witek, Twardowska Sniezko, Rzecznicy Patentowi-sp.p, ul. Rudolfa Weigla 12 Wroclaw, PL-53-114, PL)
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Claims:
CLAIMS
1. Application of the protein fraction of brewer's spent grain for the production of microbiological culture medium.
2. The application according to provision 1, characterized in that the microbiological medium produced is used in the culturing of Grampositive soil bacteria of the Actinomycetes series.
3. The application according to provision 2, characterized in that the microbiological medium produced is used to culture bacteria of the genus Streptomyces.
4. The application according to provision 1, characterized in that the medium produced contains a 2% solution of protein fraction extract (PFE) from spent grain.
5. The application according to provision 1, characterized in that the medium produced is used as a culture medium for industrial cultivation, advantageously the industrial cultivation of bacterial strains of the genus Streptomyces, in particular under controlled pH conditions.
6. The application according to provision 1, characterized in that the medium produced is used as a medium for the isolation of Actinomycetes strains from the environment, advantageously of new strains of Actinomycetes.
7. The application according to provision 1, characterized in that the medium produced is used as a medium for sporulation and/or the production of secondary metabolites.
8. The application according to provision 1, characterized in that the medium produced is used in the search for new, biologically active compounds and/or in monitoring environmental changes.
9. The microbiological medium for the culture of Grampositive soil bacteria of the Actinomycetes series, characterized in that it contains the protein fraction of spent grain.
10. The medium according to provision 9, characterized in that it contains a 2% solution of protein fraction extract (PFE).
11. The medium according to provision 9, characterized in that it has a pH value of from 6 and 8, advantageously of approximately 7, in particular of 7.2.
12. The medium according to provision 9, characterized in that as a solid medium it contains an addition of agar, advantageously in a quantity of approximately 2% w/v.
13. The medium according to provision 9, characterized in that as a liquid medium it contains 20 mM Tris/HCl, pH 7.2.
Description:
Protein fraction of barley spent grains as a medium for growth, sporulation and selection of actinomycetes and related bacteria

The invention involves an application of the protein fraction of spent grain as the main component of a new microbiological culture medium. The protein fraction described is a byproduct of the brewing process. The culture medium of this invention represents an excellent means of cultivation of Gram-positive soil bacteria of the genus Actinomycetes, particularly the species of Streptomyces. Use of the protein fraction extract (PFE) in solution is highly economical and allows one to obtain bacteria cultures which generate secondary metabolites.

In microbiological practice, many culture media based on ingredients originating from waste materials of the food industry are employed in addition to the well-defined culture media. One of the byproducts of the beer brewing process is fermented barley. This product presents a great problem in utilization.

In the brewing process (Fig. 1), dried malted barley is mashed to make non-hopped brewer's wort. Water is added to the dried malt and the temperature is slowly raised, and enzymatic hydrolysis of the carbohydrates and proteins takes place. After hydrolysis is complete, the dregs are filtered out of the wort and the filtered wort is then used in the subsequent processes to obtain beer. The dregs are called spent grain (SG), and represent waste material. Grinding and fractioning the spent grain results in three fractions: the coarse, fiber fraction (FF), with high fiber content; the medium fraction; and the protein fraction (PF), a fine fraction containing much protein (Ishiwaki et al. , 2000). Spent grain consists mainly of the seed vessels and hulls of barley, and it contains 28% protein, 17% lipids and 55% fiber (Hasson, 1993). It is widely used as an additive to the feed of ruminants and in the baking industry. The protein fraction is also used as a means to enhance the properties of creams and sauces.

Industrially useful microorganisms are typically searched for during the process of testing the characteristics of soil samples derived from various ecological niches. Bacteria of the genus Streptomyces are unusually rich sources of biologically active compounds.

Only a few media are suggested in the literature for the culture of Streptomyces (Kieser et al. , 2000). Such substances as L-arginina (El-Nkeeb, Lechevalier, 1963), humic acid (Hayakawa, Nonomura, 1987), and trimethoprim with nalidixic acid (Hayakawa et al., 1996) have been proposed for improving the selective characteristics of cultures of the Actinomycetes species.

A goal of the invention is to propose a means of utilizing brewery waste material, in particular spent grain. A further goal of the invention is to propose a new culture medium for microorganisms, in particular a selective medium for the culturing of bacteria of the genus Actinomycetes and related ones, which would be easily accessible and would assure a high production level of secondary metabolites.

The above-described problems were unexpectedly solved thanks to this invention.

An object of the invention is the application of the protein fraction from spent grain to produce a microbiological culture medium. It its advantageous realization of the invention, the microbiological culture medium produced is used for the culture of Actinomycetes Gram-positive soil bacteria. The microbiological culture medium may be used in accordance with the invention for the culture of bacteria of the genus Streptomyces.

Advantageous is that the medium produced contain a 2% solution of protein fraction extract (PFE) of spent grain. The culture medium produced may be employed as an industrial medium, advantageously in the industrial culture of bacterial strains of the genus Streptomyces, in particular under conditions of controlled pH and/or as a culture medium for the isolation of Actinomycetes strains from the environment, advantageously of new strains of Actinomycetes and/or as a medium for the sporulation and/or production of secondary metabolites and/or as a culture medium for searching for new biologically active compounds and/or monitoring environmental changes.

An object of the invention is also a microbiological culture medium for the culture of Gram-positive soil bacteria of the genus Actinomycetes, characterized in that it contains the protein fraction from spent grain. Advantageous is that the medium according to the

invention contain a 2% solution of the protein fraction extract (PFE) and/or has a pH value of from 6 to 8, advantageously of approximately 7, in particular of 7.2. In the advantageous realization, the medium according to the invention is a solid medium which contains an addition of agar, advantageously in a proportion of 2% w/v. In another advantageous realization, the medium according to the invention is a fluid medium containing 20 mM Tris/HCl, pH 7.2.

Employing the spent grain (SG) fraction as the main component of the new, economical microbiological medium has been shown to have application in the isolation, culture and sporulation of bacteria of the genus Streptomyces and related ones. The following tests were conducted to fully characterize the medium described: the growth of various species of bacteria, the generation of spores (sporulation), the production of secondary metabolites, and studies of the growth rates of liquid cultures.

The PFE microbiological culture medium prepared from waste materials of the beer brewing process possesses the essential attributes for biotechnical applications. It may be used as an inexpensive, economical medium for industrial culture, as a medium for the isolation of Actinomycetes from the environment, and as a medium for sporulation and the production of secondary metabolites. An important attribute of the PFE medium is the selective growth stimulation of bacteria of the genus Actinomycetes, which allows the identification of unknown strains isolated from the environment. The PFE medium is an inexpensively and easily prepared product won from waste materials, so it may be used on a broad scale, by controlled pH conditions, in the industrial culture of bacterial strains of the genus Streptomyces. It is possible to apply this medium in the search for new, biologically active compounds and in monitoring environmental changes.

The presented figures represent a complementary part of this description.

Fig. 1 presents a diagram of the brewing process and the production of waste materials.

Fig. 2 presents spore formation of the strain Streptomyces coelicolor on PFE solid medium observed after a) 43 hours and b) 72 hours.

Fig. 3 presents undecylprodigiosin production by St7^eptomyces coelicolor on solid PFE medium observed after a) 43 hours and b) 72 hours.

Fig. 4 presents actinorhodin production by Streptomyces coelicolor observed on solid PFE medium observed after a) 43 hours and b) 72 hours.

Fig. 5 presents the growth curve of Streptomyces coelicolor and the number of metabolite pigments produced in liquid medium, with culture on a) SMM and b) PFE medium.

In this description, the following abbreviations are used: BSG-brewer's spent grain (fermented barley), FF-fiber fraction, PF-protein fraction, PFE-protein fraction extract, and SMM-supplemented minimal medium.

Example 1. The preparation of PFE culture medium The culture medium was prepared as follows: 2 g of dried protein fraction (PF) of brewer's spent grain was added to 100 ml of distilled water and autoclaved for 30 min at 121°C, cooled, and filtered through Watman #1 filter paper. After removal of solid particles, the solution was than brought to a pH of 7.2 using 1 M of NaOH, and autoclaved for a further 25 min. For a solid medium, 2% w/v agar was added. The liquid medium contained 20nM Tris/HCl, pH 7.2.

Example 2. Employing PFE media for the culture of various microorganisms To determine the characteristics of PFE culture medium, its usefulness in culturing various microorganisms, in particular the Actinomycetes, was investigated.

Materials: The protein fraction (PF) and fiber fraction (FF) of spent grain originated from Dr.

Bo Löfquist, BoMill AB, Sweden. Sixteen Actinomycetes strains and six other strains were cultured on PFE medium. The strain Streptomyces coelicolor A3 (2) M145 (SCP1-, SCP2-) (Kieser et al. , 2000), was obtained from H. Kieser (Norwich, UK). The following strains, obtained from the Polish Collection of Microorganisms (PCM), were used in the experiments: Nocardia asteroides PCM 2254T, Nocardia otitidiscaviarum PCM 2143, Rhodococcus equi PCM 559T, Rhodococcus erythropolis PCM 2150, Rhodococcus rhodochrous PCM 909, Gordonia bronchialis PCM 2167 T, Dietzia 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 freundii PCM 2346, Salmonella enterica subsp. salamae ser. Erlangen PCM 2533, Shigella flexneri PCM 1793, Pseudomonas aeruginosa PCM 2058, and Staphylococcus aureus PCM 2101.

Methods: Determining growth curves for Streptomyces coelicolor S. coelicolor growth was determined on two media: supplemented minimal media (SMM) (Kieser et al. , 2000, with modifications by Dr. E Takano, personal correspondence) and PFE medium. In both cases, culturing was carried out in 250 ml beakers with springs.

The beakers and springs were silicon coated. 50 ml of medium was inoculated with 5X106 spores and cultivated for 48 hours at 28°C on a shaker set at 220 rmp.

The concentrations of the antibiotics were determined as follows: The concentration of actinorhodin was determined in the culture medium. 0.75 ml of medium was added to 0.75 ml 2M KOH, stirred, and centrifuged after 30 minutes for 10 min at 20'000 x g. Then absorbance at 633 nm was measured and the actinorhodin concentration was calculated using the molar coefficient of g=25'320. To determine the undecyloprodigiosin concentration, 10 ml of culture was centrifuged for 10 min at 3000 x g, then the cells underwent extraction in 1 ml methanol for 24 hours under gentle stirring. The extract was acidified with 1 ml 0. 5M HCI, centrifuged, and the absorbance of the supernatant was measured at 530 nm. Undecylprodigiosin concentration was calculated using the molar coefficient of £=100'500.

Test of secondary metabolite production by S. coelicolor Two sets of plates were prepared: 2% PFE medium and standard BHI medium (Difco). Spores of S. coelicolor were suspended on the plates in quantities of 1x101 to lux108. The production of secondary metabolite pigments was observed over the following seven days. The cultivation was conducted at 28°C. Metabolite production was based on observation and evaluated on a scale of 0-3.

Preparation of soil samples Soil samples were collected in the town of Lund and environs. One gram of each soil sample was warm-air dried at 40°C for 24 hours. Then the sample was transferred to a

sterile test tube and mixed intensively for 75 min with 3 ml sterile distilled water. Fifty il of each solution was disseminated on solid PFE medium and incubated at 37°C for 7 days.

Bacterial growth was studied by microscope, the unaided eye, the production of pigments, and spore production.

Tests of collection strains The growth of the strains from the collection was carried out on BHI and PFE media at 37°C for 48 hours. Gram-negative bacteria were also cultured on solid MacConkey medium at 37°C for 24 hours.

Results: The media tested prepared based on: spent grain (SG), the fiber fraction (FF) of spent grain, and the protein fraction (PF) (Table 1).

Table 1. The growth of microorganisms from three different soil samples on media based ondifferent fractions of spent grain Category of microorganisms (%) Medium contents Soil sample spore-forming non-spore-forming strains strains SG 2%-6% A 30 70 0 K 30 70 0 M 20 40 40 FF2%-6% A 10 30 60 K 25 40 35 M 25 15 60 PF 2%-4% A 80 20 0 K 90 10 0 M 90 5 5 Samples taken from three different places are designated as A, K and M. SG-brewer's spent grains, PF-protein fraction, FF-fiber fraction. The number of particular colonies found on the plates are expressed in percents.

The medium prepared from spent grain promoted bacterial growth, that from the fiber fraction favored fungal growth. The fiber fraction contains mainly cellulose, and fungi are know producers of cellulase. The medium, prepared from the protein fraction proved to be selective with regard to Streptomyces and other bacteria of the Actinomycetales series. Except for two Nocardiopsis strains, i. e. N. dassonvillei and N.

alborubida, which are strains similar to Nocardia belonging to the Actinomycetes series, all the strains studied grew on solid PFE medium. Streptomyces strains growing on PFE medium produce spores after the same time period as when growing on the organically rich BHI medium. Weak growth on PFE medium was observed for the selected strains of the other Gram-positive and Gram-negative bacteria (Table 2).

Table 2. Cultures of strains growing on 2% (w/v) PFE medium The actinomycetes and related strains Other strains Nocardia asteroides PCM 2254T Gram-negative: Nocardia brasiliensis Citrobacter freundii PCM2346 Nocardia otitidiscaviarum PCM2143 Salrnonella enteriea subsp. salamae ser. Erlangen PCM 2533 Rhodococcus equi PCM 559T Shigellaflexneri PCM 1793 Rhodococcus erythropolis PCM 2150 Pseudomonas aeruginosa PCM 2058 Rhodococcus rhodochrous PCM 909 Gram-positive: Gordonia bronchialis PCM 2167T Staphylococcus aureus PCM 2101 Dietzia maris PCM 2292 Streptomyces griseus PCM 2448 Streptomyces albus PCM 2319 Streptomycesflavoscleroticus PCM 2303 Streptomyces exfoliates PCM 2367 coryneform clinical isolate CI

The growth of organisms not belonging to the genus Actinomycetes was weaker on solid PFE medium than on solid BHI medium. Because the soil and similar ecological niches are dominated by Streptomyces strains and related organisms, PFE medium was shown to be a good for the selection of strains producing new, hitherto unknown, biologically active substances. Research on further growth properties of PFE medium were carried out using the stain Streptomyces coelicolor A3 (2) as a model for the Streptomyces genus. S. coelicolor has been the subject of much research and is a well characterized Streptomyces strain (Kieser et al. , 2000). For test comparisons, organically rich BHI medium and liquid SMM were used.

One of the main problems of cultivating Streptomyces strains under industrial conditions is obtaining reproducible growth conditions which lead to sporulation. In the course of growth and sporulation, the components of the medium play a key role. We inoculated PFE and BHI media with S. coelicolor A3 (2) spores at various concentrations.

On PFE medium, the colonies produced at first white, later gray, polyketide pigmented spores (Fig. 2a and 2b). On BHI medium, Streptomyces did not produce spores.

Two polyketide pigment compounds, actinorhodin and undecyloprodigiosin (Ishizuka et al. , 1992) were investigated, these also being compounds of highly antibiotic activity. As presented in Fig. 3a, actinorhodin, a dark blue pigment, appeared around the colony after 72 hours of growth on plates which had been seeded with quantities of 101 to 104 spores. After 5 days all S coelicolor cultures on PFE medium were colored gray. In the control group on BHI medium, actinorhodin production was not observed.

The red pigmented undecyloprodigiosin is in reality a mixture of undecyloprodigiosin, butylocycloheptyloprodigiosin, and meta-cycloprodigiosin (Tsao et al. , 1985). Production of undecyloprodigiosin was observed only in the control group on BHI medium. The cells already produced pigment after 43 hours of culture (Fig. 4a and 4b). The lack of undecyloprodigiosin production by cells cultured on PFE medium may have resulted from the enormous rate of colony growth or the presence or lack of specific ingredients of the medium. The influence of various medium factors on the synthesis of secondary metabolites is known (Kang et al. , 1998).

The usefulness of PFE medium in industrial liquid cultures was investigated by comparing growth curves and metabolite pigment production by S. coelicolor strains on PFE medium and SMM (Fig. 5a and Sb).

During S. coelicolor growth on PFE medium, a significant increase in the acidity of the medium was observed, which may be the reason for the growth retardation and decrease in the cell mass (Fig. 5b). The increase in acidity may be caused by the secretion of specific serine proteases, which hydrolyze prolamins. Serine proteases secreted by the species Streptomyces griseus has been reported. The proteases described find application in protein sequencing and the expression of fusion proteins (Svendsen et al. , 1991). One may assume that the described medium will be useful in the search for microorganisms which secrete similar proteases.

The Streptomyces bacteria display an unusual capability of spontaneously duplicating certain fragments of DNA. This property is exploited in the molecular monitoring of soil impurities. The expression level and the appearance of a marker gene in

the chromosome of Streptomyces violaceoruber and its introduction to the environment under study is an indication of impurity (Wellington et al. , 1990, Cresswell et al. , 1991).

The requirements set for the medium in these investigations are excellently fulfilled by PFE medium, which allows one to select Streptomyces growth and fast DNA isolation.

This work was financed within grant No. 4POSA 073 18 of the Polish Committee for Scientific Research (KBN), and by the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences (PAN) within the framework of its statutory activity.

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