EP1885843A2

EP1885843A2 - Peptide composition for growing and/or culturing micro-organisms and/or cells

Info

Publication number: EP1885843A2
Application number: EP06747548A
Authority: EP
Inventors: Dirk Penninga; Harry Laan
Original assignee: Stichting tot Innovatie van Koolzaadprodukten
Current assignee: Stichting tot Innovatie van Koolzaadprodukten
Priority date: 2005-05-17
Filing date: 2006-05-17
Publication date: 2008-02-13
Also published as: WO2006123926A2; NL1029059C2; WO2006123926A3

Abstract

The present invention relates to a peptide composition for culturing and/or growing micro-organisms and/or cells on the basis of at least one vegetable protein source, wherein at least 20% of the peptides present in the peptide composition have a length of 1 to 20 amino acids, to a method for the preparation thereof and to the use of the peptide composition for diverse applications.

Description

PEPTIDE COMPOSITION FOR GROWING AND/OR CULTURING MICROORGANISMS AND/OR CELLS

The present invention relates to a peptide composition for growing and/or culturing micro-organisms and/or cells, a method for the preparation thereof, and to the use of the peptide composition for several applications .

For optimal growth micro-organisms and cells depend on several nutrients in the growth medium. The known nitrogen (N) -containing nutrients such as peptides and amino acids, originating for instance from yeast extracts or proteolytic digests of casein (and other proteins) , and vitamins are however relatively expensive and moreover do not always have the correct composition for achieving optimal growth.

Yeast extract is used as a peptide source in many industrial fermentation processes. However, in these industrial fermentations this yeast extract is the most expensive factor of the total cost price. The introduction of cheaper peptide sources, such as fish meal, potato water or cornsteep liqour for industrial fermentations has heretofore resulted in processes with a lower performance, efficiency and quality. In the use of peptide compositions of animal origin there is the further risk of them being contaminated with pathogens, such as for instance viruses, or other disease-related substances. This can eventually cause problems when such raw materials are used as nutrients for fermentation processes for the preparation of food products for humans, animal feed or pharmaceuticals.

There is therefore a continuing need for alternative peptide compositions which are inexpensive and safe, and which furthermore have an optimal composition for the growth of diverse micro-organisms and/or cells.

The object of the present invention is to provide a peptide composition which is very suitable for use as peptide source for the growth of micro-organisms and/or cells, in particular for the growth of micro-organisms in fermentation processes.

This object is achieved by the invention by providing a peptide composition for culturing and/or growing cells on the basis of at least one vegetable protein source, wherein at least 20%, preferably at least 30%, more preferably at least 40%, most preferably at least 50% of the peptides present in the peptide composition have a length of 1 to 20 amino acids.

In a preferred embodiment of the invention at least respectively 20%, 30%, 40% or 50% of the peptides have a length of 1 to 15 amino acids, preferably of 1 to 13 amino acids .

In the research which has resulted in the present invention it has been found, surprisingly, that when the peptide composition according to the invention is used as a peptide source in the growth of diverse microorganisms and/or cells, comparable and/or better results were obtained compared to the known peptide compositions, such as for instance yeast extract. This is for instance shown in the V_max and lag-phase (see also Tables 2 and 5) .

In a particular embodiment of the invention the protein source comprises rapeseed, more particularly a rapeseed protein hydrolysate. Through preparation of a rapeseed protein hydrolysate the composition comprises sufficient peptides in a suitable form, such that the relevant micro-organisms and/or cells exhibit an optimal growth and/or activity. In a further embodiment the protein source comprises wheat, more particularly a wheat protein hydrolysate. use is preferably made of the water-soluble non-gluten protein fraction. A hydrolysate of these soluble proteins is used as peptide source.

In a further suitable and inexpensive embodiment the peptide composition comprises caraway seed as protein source, in particular a caraway seed protein hydrolysate .

In a particularly suitable embodiment the peptide composition according to the invention comprises a combination of one or more of the above stated protein sources, in particular a combination of rapeseed and wheat, more in particular a combination of rapeseed protein hydrolysate and wheat protein hydrolysate. Tests have shown that, to obtain growth with a V_mar comparable to 1% (w/v) yeast extract as peptide composition, both the yeast extract and rapeseed protein hydrolysate can be replaced by up to 90% with wheat protein hydrolysate. A synergistic effect moreover occurs here: for instance for Lactobacillus spp . , a combination of rapeseed protein hydrolysate and wheat-protein hydrolysate in a ratio of 1:1 achieves a roughly 10% higher growth speed than rapeseed protein hydrolysate alone, and with the use of this combination an increase was also observed in the formed biomass (yield) of about 15% relative to the biomass formed after use of rapeseed protein hydrolysate alone .

The peptide composition according to the invention can further comprise vitamins, such as vitamin Bl, B2, biotin, choline, and minerals. For good growth a suitable carbon source must further be added if essential for the micro-organisms in question.

The composition according to the invention is particularly suitable for use in culturing and/or growing various prokaryotes, in particular various micro-organisms such as bacteria, yeasts and fungi. The composition according to the invention can however also be used in the culture medium of eukaryotic cells, for instance in the culture of diverse cell lines, including hybridomas .

The present invention further relates to a method for preparing the peptide composition, comprising of

(a) providing a suspension with the protein source;

(b) centrifuging the suspension;

(c) incubating the supernatant with a protease for at least 30 minutes at a temperature between 50° C and 100⁰C, preferably between 55 and 65^° C, more preferably about 60 ^°C.

In a preferred embodiment of the invention the protease is a protease which splices a protein at random and preferably acts at a higher temperature (60-100 "C). Examples of such proteases are Neutrase and Alcalase (Novozymes, Denmark), and/or proteases from the group of TLPste (Thermolysin-Like Protease from Bacillus stearothermophilus) , including for instance Boilysin.

The invention further relates to the use of the above described peptide composition as peptide source for one or more micro-organisms in a fermentation medium.

The micro-organism is preferably selected from the group consisting of bacteria, yeasts and fungi.

According to the invention the peptide composition can be used for diverse bacteria. The composition according to the invention is particularly suitable for use in growing anaerobic bacteria.

The peptide composition is preferably applied for lactic acid bacteria such as inter alia Lactococcus lactis , Lactobacillus plantarum, Lactobacillus casei Shirota, Lactobacillus, Lactobacillus sakei, Lactobacillus bulqaricus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. bulqaricus , Bifidobacterium lonqum. Streptococcus thermophilus, and/or Leuconostoc mesenteroides . The composition according to the invention can however also be used in suitable manner for diverse other bacteria, such as among others Escherichia coli, Pseudomonas spp . , Clostridium spp . , Bacillus spp. Gluconobacter spp. and/or Corynebacterium spp.

The peptide composition can also be used in suitable manner for growing diverse yeasts such as for instance, among others, Hansenula polvmorpha and/or Saccharomvces cerevisiae, and fungi such as for instance, among others, Penicillium spp.

In a further embodiment of the invention the peptide composition is used as peptide source in a culture medium, for instance for culturing and/or growing eukaryotic cells, such as diverse cell lines in tissue culture.

The invention is further illustrated by the following Figure and Examples, wherein

Fig. 1 shows the relation between the peptide source concentration (in %% of the total volume) and the maximum growth speed (V_max) . THE: wheat protein hydrolysate; YE: yeast extract; KZKH: rapeseed protein hydrolysate.

EXAMPLES

EXAMPLE 1 - Preparation of the peptide composition according to the invention Preparation of rapeseed protein hydrolysate:

Rapeseed is pressed in order to remove the oil therefrom. The remaining rapeseed cake is then ground by means of rapidly rotating blades to a fineness with particle size < 0.2 mm. A 20-30% suspension (200 g per L) in demineralized water or buffer (pH +/- 6) of 30- 60^° C is then made. The suspension is stirred for (at least) 1 hour by means of an overhead stirring means with an impeller (diameter 10 cm) at 400-700 rpm. The temperature herein decreases from 45 to 35 "C.

The dissolved material (supernatant) is then separated from non-dissolved material by means of for instance filtration or centrifugation in portions at 6000 rpm (GSA rotor) for 15 minutes at 25^° C, and the decanting of supernatant over a coarse filter (cheesecloth) .

The supernatant is then incubated with 1/1000 volume Alcalase AF 2.4 L (Novozymes) at about 60 ^'C for about 60 minutes, after which it is cooled on ice and stored at -20^"C.

Preparation of wheat protein hydrolysate:

Wheat is ground into flour and a coarse ground fraction. The flour is then separated into starch and gluten, the valuable wheat protein, via a so-called gluten-starch separation. The starch is hydrolyzed with amylase into glucose and a non-gluten protein. This non- gluten protein is converted into water-soluble peptides by treatment with neutral proteases (such as for instance Neutrase 0.5 L; 7 μl/10 g) , prior to the starch deliquescence, for 1 hour at pH 6.7, 45 "C.

The samples are then pasteurized (30 min 80° C) and centrifuged at 6500 g (15 min) for the removal of undissolved material. The fraction with undissolved material is applied as peptide source for fermentation. Preparation of caraway protein hydrolysate:

Caraway oil is recovered via steam distillation. The processed seed is dried and sold as cattle feed. This by-product contains 20-25% protein. Before drying and after cooling this proteinaceous by-product is treated with protease, such as for instance neutral proteases (for instance Neutrase 0.5 L; 7 μl/10 g) , in order to manufacture a protein hydrolysate. The water- soluble fraction of this hydrolysate is separated from the insoluble debris. The debris can be used for cattle feed, the water-soluble fraction is used as peptide source for fermentation. The water-soluble fraction can be further concentrated or dried. It is then pasteurized (for instance 30 min 80 'C) .

Composition of the peptide preparation according to the invention :

For analysis of the peptide composition according to the invention a number of experiments/analyses were carried out :

1. freeze-drying (production freeze-dried samples and determining of dry weight)

2. heating and autoclaving (determining effect of heat on hydrolysate)

3. protein determination of samples

4. polyacrylamide gel-electrophoresis (determining size of protein fragments)

5. additional growth experiments with the heated hydrolysate

6. sugar/protein degradation for the purpose of HPLC analysis

7. HPLC (High-Performance Liquid Chromatography) for analysis of sugar content and by-products 8. LCMS (High-Performance Liquid Chromatography-Mass Spectrometry) for analysis of the formed peptides.

1. Freeze-drying

Three samples (together 740 ml) were freeze-dried for the production of a number of dried samples and in order to determine the dry weight. The dry weight was determined at 8.3 % (+/- 0.2 %) .

2. Heating and autoclaving

In order to determine the effect of heat on the hydrolysate, samples were incubated for 15 minutes at 70, 80, 90 and 121'C. In the case of samples of 80°C and higher an increasing quantity of precipitation occurred as temperature increased. The samples were then applied in growth experiments with L. lactis and analysed (protein determination and polyacrylamide gel- electrophoresis) .

3. Protein determination

The protein content (Bradford) of the following samples was determined (see table 1) :

Table 1. Protein determination of rapeseed protein hydrolysate samples

For determination purposes the samples were centrifuged (1 min. 12000 g) . The determination showed that the measured protein content of the samples after suspension and centrifugation before and after hydrolysis was about 20 mg/ml (2.0%) . The hydrolysis step did not result in any loss of protein in the Bradford determination. Nor did the heating at 70 ^'C result in any protein loss. With heating at 80 ^'C and higher however, dissolved protein was increasingly lost (precipitation) , up to more than 75% after autoclaving at 121 "C. The peptide content of the samples remained about the same before and after heating.

4. Polyacrylamide gel-electrophoresis

For visualization and determination of the size of the protein fragments a polyacrylamide gel- electrophoresis (12.5%) was carried out (results not shown) . The results show that the hydrolysis of the rapeseed protein results in a shift in the protein from large to small. The hydrolysate contained mainly protein of a maximum of about 30 kDa . The effect of the heat incubation was shown mainly with the sample which was

121 ^"C and contained considerably less protein.

5. Additional growth experiments heated hydrolysate

In order to monitor the quality of the heated samples the growth experiments were carried out with a reference organism (Lactococcus lactis) (Table 2) . Table 2. Growth experiments with diverse hydrolysates and L. lactis.

Table 2 shows that the growth parameters in the different hydrolysates do not differ much. This is remarkable since it has been shown that the hydrolysates do display great differences in protein concentration. The cells do not however absorb proteins but the degradation products thereof, the peptides. The samples apparently do not vary significantly in the quantity of peptides .

6. Sugar/protein degradation

In order to determine the concentration of starch in the samples and the HPLC analysis a sample was incubated with Thermamyl (a thermostable alpha-amylase; Novo) at 100 and 90 'C (2 hours) and with San Super (an amyloglucosidase, Novo) at 55^°C (2 hours). This method is highly suitable for hydrolysing to glucose molecules the starch polymers and sugar oligomers that are present. Because rapeseed cake contains 8% starch, a maximum concentration in the hydrolysate of 1.6% can be expected at a 20% suspension in water. See table 3.

7. HPLC analysis

In order to determine the sugar content and byproducts an HPLC analysis was carried out on a number of samples, as shown in Table 3. The samples were analysed using a Polyspher OA HY column and an RI detector. The column was eluated with 0.01 N H₂SO₄ at 0.6 ml.min-1. This method is very suitable for analysis of small organic acids and sugars.

Table 3. Overview of the HPLC analysis of a number of hydrolysate samples.

T = thermamyl; SS = San Super

Table 3 shows only substances which were detected in the samples. No lactose, acetic acid, ethanol, butyric acid or lactic acid were detected in the samples. These sugars and organic acids are often found in this type of sample and were therefore included as a reference. Sucrose and maltose were however detected and an unknown substance with a retention time of 15.3 min. was detected. The concentrations of these substances were however relatively low (2-10 inM) .

After incubation with Thermamyl and San Super the concentration of glucose increased by 10 mM. This increase seems to be the result of starch degradation. The concentration of starch can hereby be calculated at a maximum of 0.2 % .

In addition to proteins, peptides and amino acids, the samples comprise mainly glucose (50 mM = 0.9%) and galactose (60 mM = 1%) .

8. LCMS

The composition of the peptide preparation according to the invention was analysed on the basis of LCMS. The compositions of rapeseed protein hydrolysate and wheat protein hydrolysate were compared to yeast extract. The results are shown in Table 4 below.

Table 4. Mass distribution of the peptides from a hydrolysate of respectively rapeseed, wheat and yeast extract, analysed by means of LCMS.

The mass distribution on the basis of LCMS is converted to masses of peptides with an average amino acid length on the basis of the average amino acid mass. It is surprising that the hydrolysates on the basis of rapeseed protein contain a relatively large amount of peptides (50%) with a length of on average a maximum of 13 amino acids. Conversely, yeast extract and wheat hydrolysate contain far fewer peptides in this range (20%) . Because the rapeseed protein hydrolysate contains a relatively larger amount of low-molecular peptides, it is more favourable as a nutrient for fermentation. It is for instance known of Lactococcus lactis that peptides having a size of a maximum of (and preferably fewer than) 10 amino acids can be absorbed efficiently by the cell. On the basis of these data the application of rapeseed protein hydrolysate seems to be a considerable improvement relative to wheat hydrolysate alone and yeast extract in an application as readily absorbable nitrogen source for micro-organisms.

Example 2 - Growth experiments with rapeseed hydrolysate

Growth conditions and media:

In order to carry out the growth experiments pre- culture took place in a medium with 1% glucose with 0.3% yeast extract and 1% β-glycerophosphate (pH 6.2) . For growth experiments on micro-scale 96 well microtiter plates with an effective volume of 200 μl per well were applied. The speed of the change in the optical density at a wavelength of 600 nm was taken as a measure for the growth .

Different media were used for the growth experiments. The hydrolysate concentration was varied between 1/20 to 1/3 part. This corresponds with 0.1 to 0.6% (ds/vol) (dry substance/vol) of protein. The yeast extract concentration was varied in the same way (between 0.2 and 0.6% (ds/vol) of protein). Also added to the media was 3% β-glycerophosphate (pH 6.5) for the purpose of buffering acidification in the microtiter plate- spectrophotometer . The media were sterilized or pasteurized (hydrolysate) before use. The results are summarized in the table below (Table 5) :

Table 5. Overview of growth experiments with industrial micro-organisms .

nd = not determined

Table 5 shows two growth parameters, V_max and the Lag phase, of 11 different micro-organisms grown on rapeseed hydrolysate with yeast extract as supplement. For both supplements a quantity was added such that saturation occurred. The maximum V_max and the minimum lag phase were taken and put in Table 5.

The highest possible number is important for V_ma:: since it does after all say something about the growth speed of the organism, and therefore the increase in biomass per unit of time. It can be seen in the table that, for 10 organisms, rapeseed protein hydrolysate produced a higher V_max (and therefore growth speed) than yeast extract. No growth on yeast extract was observed in these experiments for Lactobacillus spp. For two organisms hdwever, it is. precisely the reverse: i.e. for Lactobacillus casei Shirota and Escherichia coli. The elements magnesium and manganese are possibly necessary: this can influence the growth parameters.

The lag phase is the time required for the organism to start growing (before exponential growth is observed) . This time (measured in hours) is preferably as short as possible. In practically all cases the lag phase is shorter with rapeseed protein hydrolysate than with yeast extract .

In addition to growth speed and lag phase, the finally formed biomass is also important (yield) . This is shown in table 6.

Table 6. Maximum biomass (measured as OD₆₀₀) formed in growth experiments with industrial micro-organisms.

The highest biomass values were found in the application of rapeseed hydrolysate as peptide composition, except for Lb. casei Shirota, wherein no significant difference was observed.

Figure 1 shows the relation between the maximum achieved growth speed (V_maκ) of L. lactis subsp. lactis on medium with rapeseed protein hydrolysate, wheat protein hydrolysate and yeast extract. This shows that, with an increasing concentration of hydrolysate, the V_ma;, increases to a maximum and then decreases again at an even higher concentration. This maximum varies for the different hydrolysates; 4.8 for yeast extract; 8.3 for wheat; and 8.7 for rapeseed. From this example it can be concluded that the maximum growth speed of L. lactis on a hydrolysate of rapeseed protein is a factor of 1.8 higher than on yeast extract as peptide source. This difference is also approximately observed when a hydrolysate of wheat protein is used as peptide source. It was further notable that, during the stationary phase, no decrease in biomass, or hardly any, due to lysis was detected when rapeseed protein hydrolysate was used. This was however the case with yeast extract, sometimes up to a 20% decrease in 10 hours after reaching the stationary phase.

Claims

1. Peptide composition for culturing and/or growing micro-organisms and/or cells on the basis of at least one vegetable protein source, wherein at least 20% of the peptides present in the peptide composition have a length of 1 to 20 amino acids.

2. Peptide composition as claimed in claim 1, wherein at least 30% of the peptides present in the peptide composition have a length of 1 to 20 amino acids.

3. Peptide composition as claimed in claim 2, wherein at least 40% of the peptides present in the peptide composition have a length of 1 to 20 amino acids.

4. Peptide composition as claimed in claim 3, wherein at least 50% of the peptides present in the peptide composition have a length of 1 to 20 amino acids.

5. Peptide composition as claimed in any of the claims 1-4, wherein the peptides have a length of 1 to 15 amino acids.

6. Peptide composition as claimed in claim 5, wherein the peptides have a length of 1 to 13 amino acids .

7. Composition as claimed in any of the claims 1-6, wherein the protein source comprises rapeseed.

8. Composition as claimed in claim 1, wherein the protein source comprises a rapeseed protein hydrolysate.

9. Composition as claimed in any of the claims 1-6, wherein the protein source comprises wheat.

10. Composition as claimed in claim 9, wherein the protein source comprises a wheat protein hydrolysate.

11. Composition as claimed in any of the claims 1-6, wherein the protein source comprises caraway seed.

12. Composition as claimed in claim 11, wherein the protein source comprises a caraway seed protein hydrolysate.

13. Composition as claimed in any of the foregoing claims 1-12, wherein the protein source comprises a combination of rapeseed protein hydrolysate and wheat protein hydrolysate.

14. Composition as claimed in any of the claims 1- 13, wherein the cells are eukaryotic cells.

15. Method for preparing a protein composition as claimed in any of the claims 1-13, comprising of:

(a) providing a suspension with the protein source;

(b) centrifuging the suspension;

(c) incubating the supernatant with a protease for at least 30 minutes at a temperature between 50° C and 100 "C, preferably between 55 and 65 "C, more preferably about 60 ^°C.

16. Use of the peptide composition as claimed in any of the claims 1-14 as peptide source for one or more micro-organisms in a fermentation medium.

17. Use as claimed in claim 16, wherein the microorganism is selected from the group consisting of bacteria, yeasts and fungi.

18. Use as claimed in claim 17, wherein the bacteria are anaerobic bacteria.

19. Use as claimed in claim 16 or 17, wherein the bacteria are lactic acid bacteria.

20. Use of a peptide composition as claimed in any of the claims 1-14 as peptide source in a culture medium for culturing and/or growing eukaryotic cells.