CA1292962C

CA1292962C - Optimized nutrient conditions for producing microorganism secondary metabolites

Info

Publication number: CA1292962C
Application number: CA000553215A
Authority: CA
Inventors: J. Wayne Marshall
Original assignee: Individual
Current assignee: Individual
Priority date: 1986-12-03
Filing date: 1987-12-01
Publication date: 1991-12-10
Anticipated expiration: 2008-12-10

Abstract

ABSTRACT OF THE DISCLOSURE

A process for increasing the production of secondary metabolites during culture of a selected fungi comprises a pulse feeding technique to enhance continued production of the desired secondary metabolites. The selected fungi is cultured in an initiating media containing assimilable sources of carbon, nitrogen and phosphates. The fungi is cultured in the media through its growth phases to produce the secondary metabolites. The culture is pulse fed during secondary metabolite production by periodically supplementing the media with an additional source of carbon. Such pulse feeding has resulted in a significant production of secondary metabolites, whereas in normal culture feeding programs the production of secondary metabolites tends to fall off after a certain length of time.

Description

2~
`' OPTIMIZED NUTRIENT CONDITIONS FOR PRODUCING
MICROORGANISM; SECONDARY METABOLITES
_ . .
FIELD OF THE INVENTION
This invention relates to the culture of selected fungi to enhance the production of secondary metabolites.
BACKGROUND OF THE INVENTION
In the field of biotechnology, a variety of fungi are cultured in numerous types of media to produce secondary metabolites having beneficial properties particularly in the field of health care, for example, antibiotics.
Secondary metabolites are diverse natural products which appear to be unnecessary for the growth of the organism producing them. Metabolites have a restricted taxonomic distribution with respect to microorganisms. They are most prevalent among bacteria such as actinomycetes and also among filamentous fungi.
A secondary metabolite, which has become very important in the field of organ transplants, is cyclosporin. This medicine is a secondary metabolite derived from the aerobic cultures of the fungus Tolypocladium inflatum Gams. Since the discovery of the i~muno suppressi~e properties of this metabolite, cyclosporin has become a very valuable medicine where its use lies in the area of transplants, auto-immune disorders and parasitology. The discovery of cyclosporin type A
antiparasitic activity has taken on worldwide significance in view of the discovery of its effectiveness against schistosomiasis and maleria which is thought to affect over one billion people worldwide. A variety of types of cyclosporins are produced by the culture of T. inflatum under proper conditions. The cyclosporins are typified by the initials CsA, CsB, CsC, CsD, CsE, CsF, CsG and CsH. A
summary of these cyclosporins are found in Kobel and Traber (1982) Directed BiosY ~ losporins, Eur.
J. Appl. Microbiol. Biotechnol. 14, 237.
A number of publications disclose media for use in culturing T. inflatum to optimize the production of the cyclosporin metabolites. The disclosed media contain sources of carbon, nitrogen, phosphate and trace elements 2 129Z9~2 to enhance culture. A typical media contains glucose, sodium caseinate, ammonium phosphate, magnesium sulphate, hydrate, potassium dihydrogenphosphate, sodium nitrate, potassium chloride, iron sulphate hydrate in distilled water. It has been discovered that to direct the biosynthesis of various cyclosporins, for example A, B, C, D and G, the media may be supplemented with the amino acid which distinguishes cyclosporins B, C, D, and G from cyclosporin A. It is generally understood that acceptable production rates of cyclosporins by culturing T. inflatum results in a volumetric production (maximum~ in the range of 1.63 mg/L/h. The fungus is cultured in the media normally for a period of approximately 12 to 14 days, beyond which time production of the secondary metabolites falls off considerably.
SUMMARY OF THE INVENTION
According to an aspect of the invention, a process for increasing the production of secondary metabolites during culture of a selected fungi comprises initiating culture of the selected fungi in a media containing assimilable sources of carbon, nitrogen and phospha~e.
The selected fungi is cultured in the media through its growth phases to produce the secondary metabolites. The culture is pulse fed during secondary metabolite production from the selected fungi by periodically supplementing the media with additional source of carbon to enhance thereby continued production of the secondary metabolites.
BRIEF DESCRIPTION OF THE DRAWINGS
.
Preferred embodiments of the invention are shown in the drawings, wherein:
Figure 1 represents kinetic data from a typical fermentation of T. inflatum;
Figure 2 represents production of CsA by T. inflatum (wild type) cultured in sorbose medium supplemented with maltose or citric acid;
Figure 3 represents kinetic data in production of CsA
and CsC in a maltose medium with supplementation of the medium with additional maltose;

lZ9~Z

Figure 4 represents kinetic data in the production of CsA and CsC in an initial maltose medium with supplementation with sorbose;
Figure 5 represents kinetic data for the production of CsA and CsC with an initial sorbose medium supplemented with sorbose; and Figure 6 represents kinetic data for the production `
of CsA and CsC with an initial sorbose medium supplemented with maltose.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Figure 1 is typical of ~he kinetic data derived from the fermentation of T. inflatum (wild typel available from the American Type Culture Collection under deposition number 34921. The medium employed is that identified in the above publication and reproduced in following Table l except the suggested glucose was replaced by sorbose at 4%
weight per volume as the carbon source.

~
Amount Medium Component (g/L dist. water) .
Standard Glucose (C source) 50.0 3acto-peptone 10.0 KH2P4 5 . O
KCl 2.5 Cyclosporin A was produced by the third day of incubation and was essentially completed by the tenth day of incubation when 85~ of the initial carbon source sorbose had been used. Beyond ten days incubation, there was a considerable decrease in mycelial biomaqs and an apparent ; increase in pH.
An initial rapid growth phase associated with rapid uptake of carbon source was followed by a ~lower growth phase during which time most CsA production took place.

,.

- 4 lZ9Z962 The yields and production rates for this batch fermentation are summarized in following Tables 2 and 3.

5 Yieldsa Based on Batch Fermentation of T. Inflatum Utilizing SS Medium Containing 4% (w/v) Sorbose as Carbon Source Yield - Units 10 Yx/s 0.39 g biomass/g sorbose Yx/c 0.96 g bionass/g C
Yp/x 3.83 mg CsA/g biomass Yp/s 1.48 mg CsA/g sorbose Yp/c 3.70 mg CsA/g C
. _ a Yields are based on 10 day fermentations, as this is the typical incubation period. Yields based on carbon neglect the contribution of Bacto-peptone.

Volumetric Production and Utilization Rates Based on Batch aFermentation of T. Inflatum Utilizing SS
Medium Containing 4~ 7~/v) Sorbose as the Carbon Source _ _ _ Maximum-Rate Overall Rateb Units 0.26 0.19 mg CsA/L/h 30 0.12 0.05 g biomass/L/h 0.37 0.13 g sorbose/L/h 0.91 0.33 g C/L/h : . . _ _ . . . _ a Standard medium with 4~ (w/v) Sorbose as C Source.
b Overall rates are based on 10 day fermentations, as this the typical incubation period. Utilization rate of carbon, neglects the contribution of Bacto-peptone.
Although the results of this culture of T. inflatum provides production rates of CsA considerably lower than 1~9296Z

that in the published data, which is in the range of 1.63 mg CsA per liter/hour compared to the approxima~e 0.26 mg CsA per liter/hour of the example of Figure 1, the medium reported in published material may be different from media used to obtain the reported higher production rates in the range of 1.63 mg CsA per liter/hour. However, Figure 1 is representative of CsA production whereby the tenth day of culture and thereafter there is a decrease in biomass due to fragmentation and lysis of the cells, secondary metabolite production and carbon source utilization.
Hence, with the existing technology for culturing the T.
nflatum shown in Figure 1, the cyclosporin production is at the optimum by the tenth day of incubation. Continued incubation thereafter does not yield increased quantities of cyclosporin and in fact, cyclosporin concentration may fall off due to degradation of the metabolites.
It has been discovered that the approach to culturing T. inflatum may be modified to realize significant increase production of the cyclosporin secondary metabolite well beyond the normal ten day cut-off period and before harvesting of the secondary metabolite. As noted with respect to Figure 1, the production of cyclosporin CsA begins to level off after approximately the eighth day. At this point approximately 80% of the initial carbon source has been used. It has been discovered that supplying additional carbon source just as production of the metabolite commences to fall off sustains the maximum production rate of the metabolite well beyond the eighth day of fermentation. The amount of additional carbon source added may be less than the initial amount provided in the fermentation media. This results in a more cost effective process. Tests using excessive supplementations of the carbon source have demonstrated that production of cyclosporin does not exceed that experienced by using a lesser amount in the supplementation phase. The specific examples discussed with respect to Figures 2 through 6 demonstrate supplementation on the eighth day of culture. However, depending upon the characteristics of the selected fungus 6 1 Z 9 Z 9 ~ Z

and the medium used, pulse feeding by way of supplementation of the carbon source may be adjusted and may be done on a sequential basis, such as on the eighth day, fourteenth day, twentieth day, etc.
It has been discovered that the preferred manner of pulse feeding is to supplement the media with the same carbon source as used in initiating the culture. However, where improved biomass production is desired in the first phase before supplementation, a carbon source can be selected which optimizes biomass production. Thereafter, a different carbon source, which favors production of the metabolite, can be used in the supplementation phase.
The methodology used in culturing the selected fungus is principally the same for the prior art cultures and those according to this invention, except for changes in the sources of carbon and the pulse feeding in Figures 2 through 5. Each fermentation is conducted by the submerged cultivation technique. The innoculum for the media consists of mycelial suspensions of T. nflatum obtained by addin~ sterile distilled water to cultures (less than 4 months old) on sl~nts of malt-yeast-agar medium in screw top tubes. The agar surfaces of the slants are scraped with sterile spatulas to remove the mycelial. The mycelial are suspended and introduced to the flask containing malt yeast medium. Cultures are incubated in these flasks for approximately 72 hours at 27C with rotational speeds of 200 rpm. From the prepared innocula, 10 mls are introduced into larger flask containing 100 mls of media. Incubations are maintained at 27C and at 200 rpm for the shake flask system for 10 days or whatever other period indicated in the following Examples.
With respect to the data reported in these examples, the following determinations were made:
Biomass determination was made from the dry cell weight of filtered cultures. Sartorius cellulose nitrate filter papers, available from Sartorius GmbH, Goettingen, Federal Republic of Germany. The filter papers are dried for 4 hours at 105C and weighed for use in filtration.

7 12~29~2 After filtration, the residual fungal material was dried at 105C to a constant weight which usually took about 16 hours.
Carbohydrate determinations were made to establish consumption of the carbon source. The determinations were by a color metric anthrone method adapted from the procedures of Roe, J.H. (1955) The Determination of Suqar in Blood and Spinal Fluid with Anthrone Reagent J. Biol.
Chem. 212, 355 and Weiner, J. (1978) Determination of Total Carbohydrates in Beer J. Inst. Brew. 84, 222. The amount of hexose units in a sample was determined by the conversion of hexoses to furfural derivatives in strong acid. These derivatives react with anthrone to form a blue green colored compound which absor~s 625 nm light.
The reagent for use in this test was prepared by adding 850 mls of concentrated sulfuric acid to 220 mls of distilled water. 1.00 g of anthrone was added to the solution. To improve the stability of the reagent 10 g of thiourea was added. The reagent was cooled and stored at approximately 4C in accordance with known methodology.
Samples containing carbohydrates were treated with the reagent and the absorbancy of each sample was determined in a spectrometer at a wavelength of 625 nm. Curves for standard concentrations of carbohydrates were prepared and used as references for carbohydrate determination in the culture sample.
Cyclosporin determination was accomplished by a high performance liquid chromatographic system, such as that available from Waters Scientific Ltd., Mississauga, Ontario, Canada. Samples of the fermentation media were extracted from time to time to determine the concentration of cyclosporin produced. The isolated cultures were macerated in accordance with standard techniques to release the cyclosporin for analysis by HPLC technique.
Useful sources of carbon to initiate and supplement the fermentation of the T. inflatum may be selected from the group consisting of: sugar alcohols, ketoses, aldoses, organic acids, disaccharides, polysaccharides and mixtures thereof. The aldoses may include galactose, 8 1 ~ 9 Z ~ 6 Z

alpha-d-glucose, arabinose, xylose, beta-d-glucose, manose and ribose. Ketoses may include fructose, sorbose;
disaccharides may include cellobiose, sucrose, maltose and lactose. Typical polysaccharides includes dextrin. Sugar alcohols include myo-inositol, sorbitol, manitol and glycerol. Other acceptable carbohydrates include citric acid, pyruvic acid, rhamnose and sodium acetate.
Examples of suitable sources of nitrogen for the media include Bacto-peptone, Bacto-soytone and corn steep liquor. The sources of these constituents for the nitrogen component may be purchased from Bifco Labs., Detroit, Michigan. Bacto-peptone is the preferred source of nitrogen.
A suitable source of phosphates for the medium is potassium dihydrogenphosphate.
The preferred aspects of the process, when applied to T. inflatum, demonstrates the preferred embodiments of the invention. The fungus used is that of the wild type available from American Type Culture Collection deposition no. 34921. A mutant thereo which i~ particularly useful in the production o cyclosporins i8 deposited at American Type Culture Collection deposition no. 20798. The improved aspects of this mutant are disclosed in assignee's copending application S.N 540,705 filed June 26, 1987.
In accordance with the prescribed methodology, T.
inflatum (wild type~ is cultured in the medium of Table 1 containing sorbose 5% weight per volume as the initiating source of carbon. After 8 days of fermentation, the media is supplemented with additional carbon source in the form of maltose at 2% wlv or citric acid at 2% w/v. The addition of the supplemental source of the carbon component, noting that neither7additional sources of trace elements nitrogen or phosphates are added, as shown in Figure 2, results in an increased production of cyclosporin beyond that achieved under normal culture conditions with the same fungus. As shown in Figure 1, the maximum production of cyclosporin is approximately 50 mg per liter. Whereas Figure 2 shows that by the feed "

9 1;~9Z9~iZ
pulsing of the carbon source, the concentration of cyclosporin at the end o~ the sixteen day fermentation period is increased to approximately 95 mg/l. There is a lag period after the addition of the supplemental carbon source which may range from 2 to 4 days. The lag period results from the supplemental carbon source being different from the initiating carbon source. As a result, it is necessary for the T. inflatum to adjust its metabolism to the new carbon source before resuming increased production of the secondary metabolites.
Further investigations were conducted on the mutant of T. inflatum ATCC 20798 to optimize the conditions for pulse fermentation involving the use of various carbon sources. Several twenty-one day fermentations were conducted to determine the effect of carbon source on-the production of CsA and CsC. The medium used is the standard SS medium of Table 1 with variations on the initiating carbon source and the carbon source supplemented on the eighth day of fermentation. A summary of the yields based on twenty-one day fermentations of the mutant ATCC 20798 are summarized in the following Table 5.

Yields Based on 21 Day ~ermentations of M6 Mutant Incubation Conditions Yx/c ypa/x ypc/c Ypa/c YpC/c M 0~9313.706.9012.68 6.20 MM 0.6339.1918.0024.60 11.30 MS 0.069.454.27 5.78 2.55 S 1.237.581.69 9.23 2.06 SS 0.826.253.42 5.16 2.82 SM 0.6315.096.759,45 4,23 . _ Key:
M-Standard SS Medium (Table 1) with 3% (w/v) Maltose*as C
Source.
MM-Same as M but supplemented on day 8 of incubation with an additional 2% (w/v) Maltose.
MS-Same as M but supplemented on day 8 of incubation with 2% ~w/v) Sorbose.*
~, .
~! *trade marks 1292~2 S-Standard SS Medium (Table 1) with 3~ (w/v) Sorbose as C
Source.
SS-Same as S but supplemented on day 8 of incubation with an additional 2~ (w/v) Sorbose.
SM-Same as S but supplemented on day 8 OL incubation with 2% (w/v) Maltose.
Yx/c - g biomass/g C
Yp/x - mg cyclosporin/g biomass Yp/c - mg cyclosporin/g C
a - CsA
c -- CsC
The volumetric production rates based on the 21 day fermentation of the mutant is summarized in the following Table 6.

Volumetric Production Rates* Based on 21 Day Fermentation of M6 Mutant Incubation Cyclosporin A Cyclosporin C
Conditions Overa~rl MaximumOveraL1 ~-~h~
-- ---M 0.27 0.63 0.14 0.83 MM 0.92 5.10 0.45 2.76 MS 0,21 0.63 0.09 0.83 2S -S 0.19 0.39 0,08 0.24 SS 0.19 0.39 0.11 0.24 SM 0.34 1.06 0.15 0.50 Key - See Key, Table 5 *mg cyclosporin/L/h Figures 3, 4, 5 and 6 show in graph form the effect of the supplementation with various carbon sources. In addition to CsA and CsC production, pH, biomass and carbon utilization are also recorded. Only four of the selected carbon sources are shown in the attached drawings. Figure 3 represents incubation conditions MM. Figure 4 represents incubation conditions MS. Figure 5 represents incubation conditions SS and Figure 6 represents 11 ~292~62 .

incubation conditions SM. From the results shown in Figure 3, it is apparent that the incubation conditions which most suit the mutant are an initiating source of carbon at 3% w/v of maltose with a supplementation on day 8 of fermentation with an additional 2% w/v maltose. The overall rates of production of mg of cyclosporin/L/h reaches a maximum of 5.10 for the MM media as shown in Table 6 which well exceeds the maximum reported production rates of (1.63 mg/CsA per L/h) CsA from T. inflatum wild type. As shown in the Tables and Figures, there is, however, a marked improvement in the production of CsA by the use of other types of carbon sources such as sorbose, as the initiating and supplementary source of carbon, or, the combined use of maltose with sorbose as the initiating source of carbon. In the medium used in these conditions, the source of phosphates is potassium dihydrogen orthophosphate phosphate. The medium contains approximately 0.5~ by weight of the source of phosphorous.
The source of nitrogen is Bacto-peptone where the medium contain~ approximately 1% by weight per volume thereof.
With respect to Figures 2 through 6, it is noted that the carbon source supplement is introduced to the media on the eighth day which also corresponds to an update of approximately 80% of the initiating source of carbon. In using the process of this invention, with other types of fungus, similar guidelines based on uptake of carbon can be used to determine the times when supplementation of carbon source is desirable. In accordance with this method, only the carbon source is supplemented without any requirement to add any other nutrients of nitrogen, phosphorous or trace elements.
Accordingly, this invention requires the use of low concentration additions of carbon source to the fungus over an extended fermentation period. Such "pulse"
fermentation circumvents the possible carbon catabolite repressive effects of high carbon concentration. The overall efficiency of carbon to cyclosporin conversion is greatly increased and higher yields of cyclosporin are attained by adopting this feeding strategy with a simple 12 1~92~3~2 batch fermentation. Cyclosporin yields achieved by pulse fermentation are more than ten times the levels officially produced by T. inflatum (wild type) cultivation. The present production level of in excess of 500 ml CsA/L is significant commercially since the CsA titre isolated from the culture media are relatively pure which results in reduced associated recovery and purification of the CsA.
The highest volumetric production of cyclosporin produced by T. inflatum (wild type) at a level of lOS ml/L
of broth was obtained on a semi-synthetic medium containing:
1) 3% w/v sorbose:
2) 1~ w/v Bacto-peptone;
3) 0.5% w/v potassium dihydrogen ortho phosphate;
and 4) 0.25% w/v potassium chloride The media was adjusted to a pH of 5.3. Cultures were incubated for 10 days at 27C with agitation.
By using a media containing an initial concentration %o of maltose equal to 3% w/v supplemented on the eighth day o incubation with an additionàl 2% w/v maltose and using the T. inflatum mutant ATCC 20798, the highest production of CsA and C5C was obtained at 537 mg CsA per liter of broth and 250 mg of CsC per liter of broth respectively.
By using the pulse feeding mode, a more efficient approach to process development than simple batch fermentation is realized. The overall efficiency of carbon to cyclosporin conversion is increased by more than twofold for pulse fermentation compared to simple batch fermentation.
With reference to Figure 3, supplementing the fermentation with additional maltose at 2~ w/v on the eighth day of fermentation stimulated cyclosporin productivity to a maximum of 5.10 mg CsA per L/h and 2.76 mg CsC per L/h. The maximum titre of Cs~ and CsC occurred on the sixteenth day of fermentation at 537.5 mg per liter and 250 mg per liter respectively. A slight decrease in cyclosporin titre occurred after the sixteenth day of fermentation. There was no lag in cyclosporin production when the maltose medium was supplemented with additional 13 12929~2 maltose. However, when the medium was supplemented with sorbose, there was a four day lag in production as shown in Figure 4. This lag is probably due to the fungus not having the metabolite machinery in place to utilize sorbose when initiated in the presence of maltose.
However, the fungus recovers and the overall yields are far beyond those obtained under standard batch fermentation conditions.
The highest yield of biomass at 1.23 g biomass per gram of carbon was attained in cultures grown in 3% w/v sorbose medium as shown in Table 5. The maximum titre of cyclosporin A produced in this medium was only 93 mg per liter of broth after 21 days incubation. This indicates that the T. _nflatum mutant does not utilize sorbose as effectively as the wild type, because 10 day fermentations of the wild type on the same medium produced greater than 100 mg CsA per liter. Therefore, it appears that the mutant utilizes sorbose for growth and thus biomass formation, whereas the ~ild type produces considerably less biomass but increased output of cyclosporin. This is conirmed as shown in Figure 5 where supplementing the sorbose medium with the addition of 2% w/v sorbose was directed towards the produc*ion of more biomass instead of increased production of cyclosporin.
As shown in Table 6 and Figure 6, the addition of maltose to the sorbose medium on the eighth day of fermentation resulted in biomass productivity approaching zero and after a short lag, cyclosporin productivity increased to a maximum of 1.06 mg CsA per liter/h.
Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made ~- ~ - thereto without departing from the spirit of the invention or the scope of the appended claims.

Claims

1. A process for increasing the production of secondary metabolites during culture of a selected fungi, said process comprising initiating culture of said selected fungi in medium containing assimilable sources of carbon, nitrogen and phosphate, culturing said selected fungi in said medium through its growth phases to produce said secondary metabolites, pulse feeding said culture during secondary metabolite production from said selected fungi by periodically supplementing said media with additional source of carbon to enhance thereby continued production of said secondary metabolites.

2. A process of claim 1, wherein said fungi is selected from the species Tolypocladium inflatum.

3. A process of claim 2, wherein the selected strain of said species comprises T. inflatum ATCC 34921 or ATCC
20798.

4. A process of claim 3, wherein said produced secondary metabolites are cyclosporins.

5. A process of claim 4, wherein said cyclosporins are types A and C.

6. A process of claim 1, 2 or 3, wherein said carbon source used to initiate and supplement said culture is selected from the group consisting of sugar alcohols, ketoses, aldoses, organic acids, disaccharides and polysaccharides.

7. A process of claim 5, wherein said carbon source used to initiate and supplement said culture is selected from the group consisting of sugar alcohols, ketoses, aldoses, organic acids, disaccharides and polysaccharides.

8. A process of claim 7, wherein said selected carbon source used to initiate and supplement said culture is selected from the group consisting of glucose, maltose and sorbose.

9. A process of claim 8, wherein said selected carbon source used to initiate culture is maltose and said selected carbon source used to supplement culture is maltose.

10. A process of claim 8, wherein said fungi is T.
inflatum ATCC 20798.

11. A process of claim 1, 5 or 10, wherein said additional carbon source is the same as said carbon source used to initiate said culture.

12. A process of claim 10, wherein said source of nitrogen is Bacto-peptone.

13. A process of claim 12, wherein said medium contains approximately 1% by weight/volume of said source of nitrogen.

14. A process of claim 10, wherein said source of phosphorous is potassium dihydrogen orthophosphate (KH2PO4).

15. A process of claim 14, wherein said medium contains approximately 0.5% by weight/volume of said source of phosphorus.

16. A process of claim 10, wherein said medium contains approximately 3% by weight/volume of maltose to initiate culture and an additional approximately 2% by weight/volume is added to said media to supplement culture.

17. A process of claim 1, wherein said pulse feeding comprises supplementing said culture once production of a desired secondary metabolite beings to fall off.

18. A process of claim 17, wherein said culture is supplemented with approximately one-half to two-thirds of the initiating amount of carbon source.

19. A process of claim 5, 7 or 10, wherein said pulse feeding comprises supplementing said culture once production of said cyclosporins begin to fall off in the eighth day of culture.

20. A process of claim 17, wherein said culture medium is supplemented when approximately 80% of said initiating source of carbon has been used.

21. A process of claim 16, wherein said media is supplemented on the eighth day of culture when approximately 80% of said initiating source of carbon has been used, said initiating medium also comprising:
(i) approximately 1% by weight/volume of Bacto-peptone;
(ii) approximately 0.5% by weight/volume of potassium dihydrogen phosphate;
(iii) approximately 0.25% by weight/volume of potassium chloride, culturing said medium at a temperature in the range of 27°C with agitation and pH adjusted to 5.3.