DD298658A5 - PROCESS FOR PRAEPARATION OF PENICILLINNAEHOLE SOLUTIONS - Google Patents

Abstract

The invention relates to a method for the preparation of particle-containing, polymeric nitrogen substrates, which are obtained as Sekundaerrohstoffe in the food industry for microbiological high-performance processes in order to achieve rapid, technically relevant, primaere growth phases with independent of the degree of milling of the shot used, standardized starting conditions. According to the invention, the object of the invention is achieved by performing a proteolytic maceration of meal mashes within or outside of a high performance microbial process by adding crude enzyme mixtures, acidic, neutral, alkaline or / and thermally stable proteases to the mashed macaques corresponding to the required degree of digestion of the high polymer and particulate nitrogen substrates and thus, upon completion of the substrate proteolysis, there is a medium permitting a consistently high performance, robust, and control-sensitized microbial growth or product formation process. {Preparations Method; Penicillinnaehrloesungen; Nitrogen substrates, polymers; Maceration, proteolytic; Schrotkorngroeszenverteilung; High performance process, microbial; Rohenzymgemische}

Description

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Field of application of the invention

The invention is applicable to the standardization of start conditions of high performance, fermontative processes in biotechnology. In particular, it finds application in the fermentative production of benzylpenicillin. Since the avoidance of unproductive loss times due to delayed primary growth phases is generally desirable, the field of application of the invention can also be extended to large-scale fermentative processes with other product-forming microorganisms.

Characteristic of the known state of the art

For economic reasons, complex organic nitrogen sources such as soybean and peanut extraction batches, cottonseed meal, corn steep liquor, yeast extract, fish meal and other secondary raw materials are used for industrial fermentation media. For special high-performance processes with genotypically changed microbial producers, subsequently refined, organic nitrogen substrates such. Pharmamedia (Buckeye Cellulose Corporation, Memphis, Tennessee 38108, USA) was used to ensure the maximum process limits of microbial fermentation in the long term. The complex nitrogen substrates mentioned contain not only the proteins but also mineral components, of which the polymer-bound phosphorus is of particular importance (PF Stanbury, A. Whitaker, Principles of Fermentation Technology, Pergamon Press, 1986, Chapter 4: "Media for Industrial Fermentations"). The use of post-processed secondary raw materials from the food industry with guaranteed quality criteria

like ζ. As amino acid, mineral and vitamin content (Pharmamedia) burden of course, the cost of large-scale fermentation in addition. However, the availability and cost of these nutrient substrates are limiting factors that still require the use of milled extraction slugs in large-scale, high-performance fermentation. The economic gain on this basis, however, has its price due to the lack of standardization due to widely varying particle size distributions and non-constant fractionation levels of the soluble proteins of such extraction soybean and peanut meal. However, the particle size distribution (Figure 1), the fractionation level of the proteins, and the sterilization conditions significantly determine the medium configuration, in their entirety for the constant rate of primary growth with the expression of product formation and the speed of expected immediate responses due to measurement signals during the potential production phase.

As is known, complex nitrogen substrates can be digested by acid hydrolysis down to the amino acids. However, acid hydrolysis has the disadvantages that it requires a particularly acid-fast container and, with high ion concentrations, it stresses the fermentation, the chemical work-up and the wastewater.

Object of the invention

The invention aims to improve the particle size distribution of pellets and the degree of fractionation of the soluble proteins in such a way that powerful, constant and externally signal sensitized microbial product formation processes are possible even using secondary raw materials from the food industry. The aim of the development is equally to use every possible productive hour of the subsequent highly potent product formation phase with maximum specific product formation rates in the technical fermentation process through rapid primary growth processes to predefined substrate limitation.

The conclusion of the essence of the invention

The object of the invention is to improve and standardize heterogeneous, batch-dependent extraction pellets by targeted proteolytic maceration in such a way that a constantly high-performance, robust but sensitive to microbiological product formation process is possible. According to the invention, based on a histogram of the particle size distribution and / or the fractionation degree of the nitrogen fractions of shot, which are obtained as a secondary raw material in the food industry, the particle size distribution and the degree of fractionation of the proteolytic activity of a microbiological growth and product formation process; ngepaßt. The particle size distribution of extraction slices is eliminated. This will allow standardization of the selected nitrogen substrates for a high performance microbial process.

The polymeric protein fraction of a selected nitrogen substrate is removed in favor of the oligomeric protein fraction or monomeric amino acids according to the process requirement by using crude enzyme mixtures, alkaline, acidic, neutral or thermostable proteases at the beginning of the medium batch within or outside a microbiological growth and / or product formation process.

The histogram of the grain size distribution (Fig. 1) of a relatively well milled soybean meal shows that 46.5% of the grinding has a grain size = 0.60 mm, 17.2% has a grain size of 0.60 to 1.20 mm and 36.2% has a grain size of 1.20mm. During a decontamination by the action of temperature of t = 12O ⁰ CfUr 1 hour, the fractionation and the particle size distribution is not significantly changed. Only by the addition of corn steep liquor or other substrates with a higher proportion of protein-soluble fractions can the fractionation of the nitrogen substrates in the medium be influenced in a targeted manner. However, this addition is limited by the high Phosphatphosphorgehalt (30 to 45 mg 'g dry matter) of the corn steep liquor.

It has surprisingly been found that after the proteolytic maceration of soybean meal or peanut meal, the ratio of phosphate phosphorus to polymer-fixed phosphorus, which is preferably 0.12 to 0.29, remains unchanged in said nitrogen substrate. Of particular importance is the ratio of soluble phosphorus to bound phosphorus. Both have a negative effect on the biosynthesis of secondary metabolites in many microorganisms (for review: A.Torriani-Gorini, FGRothman, S.Silver, A.Wright, E. Yagil, Phosphate Metabolism and Cellular Regulation in Microorganism, American Society for Microbiology , Publication Sales, 1913,!. Street, NW, Washington, DC 20006, 1987).

The free and readily available soluble phosphorus is metabolized during the primary growth phase in the medium, the consumption of the polymer-fixed phosphorus content by the product-forming microorganism can be controlled by a previously proposed method. The particle size distribution of the available vegetable meal and flours, the degree of fractionation of the soluble proteins, oligopeptides and amino acid, the NH ₄ -N and sugar concentration and the physiological condition of the used precultures determine the rate of the primary growth phase and a technologically manageable, on a Substratlimitationsgrenze accruing concentration of biomass. Utilization of maximum biosynthetic potency expressed up to the time of sugar or / and phosphorus limitation is determined during a benzylpenicillin fermentation essentially by a sugar dosage rate adapted to the physiological status, the retarded amino acid flux and the ratio between amino acid and NH 2 -N uptake. Since the maximum specific product formation rate is a time function (optimum between the 20th and 100th hour), it is important to allow the primary growth phase and the switch to secondary metabolite formation during the sugar limitation (specific growth rate) to proceed quickly and safely. The increase in the rate of primary growth and switching process and the standardization of start conditions were maximized by proteolytic maceration of soybean meal with the well-known grain size distribution histogram (Figure 1). With this proteolytic maceration of available shot, the addition of

Substrates with low molecular weight nitrogen fractions for the purpose of correcting and adjusting the total Stickstofffraktionierungsgrades the medium to the process requirements are drastically reduced. This also reduces the additional entry of soluble phosphate phosphorus from the low molecular nitrogen substrates (corn steep liquor) into the medium. The soybean meal or peanut meal is digested in a 3 m ³ fermentor separately prior to completion of the medium or in a stirred tank outside the microwave process with a crude enzyme mixture or with an acidic, neutral, alkaline and temperature-stable protease. In principle, each Enzymrohgemisch (preferred concentration 0.3 to 2 g per liter of mash impaction tent 3 to 15 hours) and each protease is suitable for the preparative standardization of soybean or peanut meal. The selection is determined once by the availability, on the other hand by the intended result of the degree of digestion. The conditions during the enzymatic maceration are dictated by the conditions of maximum enzyme activity (temperature, chT, concentration). The enzymatic maceration and proteolysis of Extraktlonsschroten be preferably between 2 and 15 hours at einerTemperaturvon30 ° Cbi8 80 ° C and an enzyme activity in the mash of 2 10 ~ ² pmol / mg · min to 1 (^ mol / mg · min. For the Benzylpenicillin fermentation, the proteolytic maceration was performed so far that the particle size distribution either eliminated or the 0.60-mm particle fraction is almost 100% At the same time the soluble protein content and the oligomeric peptide fraction increases in the medium 80% ethanol solution (1: 6 fraction) based on soluble proteins and amino acids, crude enzyme mixtures such as brewing enzyme containing beta-glucanase, neutral protease and alpha-amylase are particularly suitable because they are available, inexpensive and in addition to proteolytic maceration digest the residual starch contained in the pellets into C substrate monomers t. Before enzyme treatment the soy or Erdnußschrotmaische was 1 hour at 100 ⁰ C precooked after setting a ⁺ cH of pH = 8.0, in order to increase by structural relaxation of the particles, the enzyme action and thus to save Enzymsubetanz. Chemically-analytically, the precooking process has no consequence (Example 2). After proteolytic maceration, the digestion medium is completed with the other substrates and sterilized for 1 hour at 12O ⁰ C with direct and jacket steam. At the same time the complete medium is deactivated. The procedure will be explained in more detail with examples.

embodiments

example 1

76kg of soy flour according to the histogram of particle size distribution are precooked in one cubic meter of tap water (= 40g soy flour / l culture solution) in a 3-m ³ fermentor for one hour at 100 ° C after setting a cH ⁺ of pH = 8.0. For this change in pH from the starting pH = 6.5, 400 to 500 ml of technical sodium hydroxide (30%) are required. After cooling (jacket cooling) to 45 ⁰ C, this approach, which now has a CH ⁺ of pH = 7.5, 1 kg ßraueei eienzym (1 g / l mash containing 60 to 80g of soybean flour) in 51 tap water was dissolved at a temperature of 20 to 3O ⁰ C was added. The enzyme treatment takes place at 45 ° C to 5O ⁰ C and a Ch ⁺ before "oH = 7.5, with stirring (50 min" ¹⁾ without aeration for 4 hours, maximum of 5 hours. Before any samples for analytical examination or control is 5 min stirred at 100 min " ¹ , then pulled the sample and again set the Ausgangsrührbedingung. After the enzyme treatment, the completion of the medium is carried out according to the recipe and the pH correction. The sterilization of the entire medium batch is carried out with direct steam for 60 min at 12ü ° C. The analysis of the first batch free of particles after four hours of proteolytic maceration is shown in the table below (Table 1). The values for organic nitrogen are good for amino acid analysis.

1: 6 fraction: all proteins and amino acids soluble in 80% ethanol solution. Before starting the analysis, all samples were treated with an Ultraturrax for three times a minute.

Table 1

Primary intake: 76kg soy flour / 1m ³ tap water, 1kg brewing enzyme TGL 29166/01 Analysis: mg / ml

Original 1: 6 GN NH ₄ -N Norg. PO ₄ -P 1. Soybean meal suspension (SM) 76g / l, pH = 8.0 without enzyme Original 1: 6 6,09 0,18 0.035 0.0 6.05 0.13 0.085 2. ¹ h 100 ⁰ C SO ⁰ CO. h enzyme addition (ezf.) pH = 7.5 Original 1: 8 5.38 1.34 0.065 0.015 5.32 1.32 0,090 3. 2 hours after Ezf. 5O ⁰ CpH = 7.5 Original 1: 6 5.38 1.53 0.065 0.020 5.32 1.50 0,090 4. 4 hours after Ezf. 5O ⁰ CpH = 7.5 Original 1: 6 5.38 1.56 0.065 0.020 5.32 1.54 0,090 5. 5 hours after Ezf. 5O ⁰ CpH = 7.5 5.98 1.56 0.060 0.015 5.82 1.54 0,090

Result: After two hours of proteolytic maceration, 24.8% of the organic nitrogen of the already particle-free preparation (sieving £ 0.6 mm) is present in the 80% ethanol fraction. Further treatment for up to 5 hours only increases this value by 0.65%. The PO «P concentration is constant throughout the treatment until the end.

Belsplnl 2

This primary treatment was macerated in a 80 l gross volume sterilizer with 2.8 kg soy flour / 491 tap water (57 g SM / I mash). The preparation was again with a precooking process for 60min at 100 ⁰ C and a Ch ⁺ begun by pH = 8.0, after the starting pH value of 6.2 with 150 ml of 3 N NaOH was adjusted to pH = 8.0. The proteolytic maceration was carried out with 0.5 g of brewery enzyme / I mash. The conditions correspond to those in Example 1. The analysis for this preparation is again shown in the following tables (Tables 2 and 3).

Result: After the 4-hour enzyme treatment 32.1% of the organic nitrogen or 34.5% of the amino acid nitrogen (AS-N) of the present at that time particle-free preparation in the 80% ethanol fraction are present. The PO ₄ -P concentration is consistent until the end of the proteolytic maceration, and so is the relation of polymer-fixed phosphorus

remained constant to phosphate phosphorous.

Table 2

Primary intake: 2.8 kg / 49 l tap water, 0.5 g brewery enzyme / I Mash analysis: mg / ml

sample Original 1: 6 GN NH ₄ -N Norg. AS-N Total phosphorus PCV-P (SUClj) Glucose m. Hydroysis Glucose mil In vert βθ Glucose without hydrolysis Fructose (resorcinol) 1. Start before the pre-cooking process 3.89 0.13 0.060 0.028 3.83 0.097 3.10 0.058 12:40 0.026 4.20 1.90 0.10 4.41

1h100'C pH = 8.0 after precooking

Original 4.03 0.060 3.97 3.27 0.38 1: 6 0.106 0.021 0.085 0.040

0.024

1.90

0.11

4.69

4 h enzyme treatment pH = 7.5 t = 50-65 ⁰ C

Original 3.93 0.120 3.81 3.22 0.40 1: 6 1.25 0.021 1.23 1.07

0.028

1.90

0.18

4.88

1: 6 fraction: all 80% ethanol-soluble proteins and amino acids (AS)

Table 3

Primary intake: 2.8kg soya flour / 49I tap water, 0.5g brewer's enzyme / I mash

Analysis: μς / ηηΙ Amino Acid Chromatogram (Amino Acid Analyzer T339M, Microtechna Praha)

sample AS-N Amino acids (AS) pg / ml His bad Asp Thr Ser Glu Per G / Y A / a Val mead Lieu Leu Tyr Phe mg / ml Lys 592 2090 2590 702 1040 4120 1080 950 945 1110 255 1 100 1 880 770 1190 1. before Sterili 3.10 1520 sation original 13 33 59 16 17 116 A 20 25 18 A 14 25 28 34 1: 6, m.h. 0.06 21 X χ 3 χ χ 7 χ A 2 3 χ χ χ χ χ 1: 6, o.H. A X 661 1900 2760 887 1210 5020 1280 994 978 1170 244 1110 2020 798 1200 2. 1h100 ^c C 3.27 1530 pH = 8.0 original 10 23 42 9 10 88 A 12 17 8th A 6 10 16 28 1: 6, m.h. 0.04 15 X χ 3 χ χ 7 χ A 2 3 χ χ χ χ χ 1: 6, o. H. A X 3. 4 h of enzyme 663 2100 2650 634 1080 4410 1 160 982 991 1140 244 1 150 2 000 809 1250 plot 3.22 1580 original 183 458 1010 395 456 1470 477 350 420 448 43 424 767 373 522 1.6, m.h. 1.07 392 XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX 1: 6, o. H. A XX

A: Hints rn. H .: with hydrolysis o. H .: without hydrolysis1: 6-fraction: all proteins soluble in 80% ethanol solution and AS χ all AS-peaks are peptide-overlayed, therefore it is hardly possible to analyze XY very many peptides, hardly free AS.

Example 3

The conditions of this preparation, the complex nitrogen partikelhaitigen substrate (Fig. 1) enforechen those in Example 2. Instead of the Rohenzymgemisches was an alkaline protease from Bacillus licheniformis as a granulate used (activity: 254.5 TU ₀ ,, · s ~ 'at pH - 10 , 0 and t = 25 ° C. Origin: Research Center for Biotechnology, Berlin).

Table 4

Primary intake: 2.8 kg soy flour / 491 tap water, 0.1 g alkaline protease / I Mash Analysis: mg / ml

sample Original 1: 6 GN NH ₄ -N Norg. PO ₄ -P After ¹ h at 100 ⁰ C boil pH = 8.0 without enzyme Original 1: 6 4.35 0.16 0.027 0.003 4.32 0.16 0.024 0.003 1. 1 h after enzyme addition t = 50 ° C pH = 9.0 Original 1: 6 4.57 1.18 0.025 0.005 4.54 1.18 0.026 0.005 2. 2 h 50 ° C, pH = 9.0 Original 1: 6 4,79 107 0.043 0.004 4.75 1.07 0.026 0.004 3. 3 h 50 ⁰ C pH = 9.0 Original 1: 6 4.57 1.25 0.025 0.006 4.54 1.24 0.026 0.006 4. 4 h 50 ^° C pH = 9.0 4.64 1.25 0.033 0.003 4.61 1.25 0.026 0.003

Result: After a three-hour enzymatic maceration with 0.1 g of alkaline protease (56 g soybean meal / l), 28.7% of the organic nitrogens of the peptides and proteins are dissolved in the 80% ethanol fraction (1: 6). At this time, the batch is particle-free.

Example 4

The conditions of this preparation of the complex particle-containing nitrogen substrate correspond to those in Example 3 except for the following modifications. Instead of soybean meal, peanut meal was used and the thermal poultry socking by precooking was omitted. 0.1 and 0.08 g of alkaline protease according to Example 3 were used. The results are shown in Tables 5 and 6.

Table 5

Primary Rate: 2.8kg peanut flour / 49I (57g / l), 0.1g alk. Protease / I Mash Analysis: mg / ml

sample Original 1: 6 GN NH ₄ -N Norg. PO ₄ -P 1. Before adding enzyme pH = 9.0 Original 1: 6 4.50 0.34 0.023 0.0 4.48 0.34 0,018 2. 1 h after addition of enzyme pH = 9.0 5O ⁰ C Original 1: 6 4,65 0,99 0.032 0.005 4,63 0,98 0.016 3. 2h pH = 9.0 50 ° C Original 1: 6 4.50 0.99 0.026 0.003 4.47 0.99 0,021 4. 3hpH = 9.0 5O ⁰ C Original 1: 6 4.94 0.96 0.023 0.0 5.13 0.96 0.019 5. 4hpH = 9.0 50 ° C Original 1: 6 4.94 0.82 0.026 0.006 4.91 0.82 0,021 6. 4 dog heat up to 12O ⁰ CfUr 15min. 4.79 0.87 0.022 0.017 4.77 0.85

Result: After three hours of enzymatic maceration with 0.1 g of alkaline protease (57 g peanut flour / I), 21.4% of the organic nitrogen is present in the 80% ethanol fraction. The approach is particle-free at this time.

Table β

Primary intake: 2.4kg peanut flour / 481 (50g / l), 0.08g alkaline protease / I mash Analysis: mg / ml

sample Original 1: 6 GN NH ₄ -N Norg. PO ₄ -P 1. Before enzyme treatment pH = 9.0 Original 1: 6 4.48 0.13 0.037 0.002 4.44 0.13 0,022 2. 1 h after enzyme treatment pH = 9.0, EO ⁰ C Original 1: 6 4.40 0.84 0.031 0.0 4.37 0.84 0,025 3. 2hpH = 9.0 5O ⁰ C Original 1: 6 3.06 0.83 0.017 0.0 3.94 0.83 0,022 3 h after enzyme treatment 15 min 12O ⁰ C 3.50 0.89 0.049 0.0 3.45 0.83 0.023

Result: After three hours of enzymatic maceration with 0.08 g of alkaline protease (50 g peanut flour / I), 18.7% of the organic nitrogen is present in the 80% ethanol fraction. The PG <-O concentration remains unchanged, the approach is particle-free at this time.

Claims (8)

A process for the preparation of penicillin broths, characterized by adjusting grain size distribution and fractionation level of proteolytic activity of a microbial growth and product formation process from a histogram of grain size distribution and fractionation level of the nitrogen fractions of shot, eliminating the grain size distribution of extraction slices, that the polymeric protein fraction of a selected nitrogen substrate is eliminated by using crude enzyme mixtures, alkaline, acidic, neutral and temperature stable proteases at the beginning of the medium batch within a microbiological growth and product formation process 2. The method according to claim 1, characterized in that starting from a histogram of the particle size distribution of shot the particle size distribution and the degree of fractionation of the proteolytic activity of a microbiological growth and product formation process is adjusted. 3. The method according to claim 1, characterized gekannzeichnet that, starting from a histogram of the fractionation of the nitrogen fractions of Schroten Fractionitätsgrad the proteolytic activity of a microbiological growth and product formation process is adjusted. 4. The method according to claim 1 to 3, characterized in that the polymeric protein fraction of a selected nitrogen substrate is eliminated by at the beginning of the medium batch outside a microbiological growth and product formation process crude enzyme mixtures alkaline, acidic, neutral and temperature-stable proteases are used. 5. The method according to claim 1 to 4, characterized in that an ethanolic solution is used for the control and analytical definition of the proteolytic maceration and the proteolysis of shot and polymeric nitrogen substrates. 6. The method according to claim 1 and 2, characterized in that the enzymatically ^ Mozeration and the proteolysis of extraction meal 2 to 15 hours at a temperature of 3O ⁰ C to 8O ⁰ C and an enzyme activity in the mash of 2 · 10 ^-2 Mmol / mg.min to ΙΟμιτιοΙ / mg.min (Tyr) is performed. 7. The method according to claim 1 to 6, characterized in that the crude enzyme concentration used 0.3 g to 2 g per liter of mash and the exposure time is 3 to 15 hours. 8. The method according to claim 1 to 7, characterized in that the ratio of phosphate phosphorus to the polymer-fixed phosphorus is 0.12 to 0.29 and remains unchanged in this area.