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Scientific
Publications - Work Done by Microbiology Reader
Becton, Dickinson and Company, 2004, 63 pp
BD Biopharmaceutical Production. Peptone
technical manual
The BD Difco™ and BBL™ Peptone Technical Manual has been provided to assist you in your selection of BD products for use in cell culture and microbial fermentation production, from Research and Development to the final finished product. In this age of globalization, a peptone supplier needs the ability and resources to serve customers all over the world. It is our commitment to innovation and product consistency as well as our broad access to the raw material market that makes BD (Becton, Dickinson and Company) such a strong global supplier and partner. In over 142 countries worldwide, BD offers a full line of peptones and media, for the biotechnology, pharmaceutical, animal and human vaccine, and bioremediation markets. BD offers products for both cell culture and microbial fermentation production, as well as applications for industrial research, QA/QC and environmental monitoring. When developing a new medium formulation, care should be taken in choosing the peptones for the new formulation. Individual experimentation with a variety of peptones is suggested to select the optimum peptone or combination of peptones. Figures 1 and 2 demonstrate such a preliminary screen for multiple peptones and two different organisms. The peptones are each 1% solutions with 0.4% glucose and buffering salts. Growth testing was performed using the Labsystems Bioscreen C Kinetic Optical Density Reader. Compare the growth support curves of Proteose Peptone No. 3: in Figure 1, the proteose curve demonstrates the least growth support; in Figure 2, proteose gave the second best growth support. Based on these results, one would likely create a medium formulation consisting of yeast and proteose to support the growth of Enterococcus faecalis and not use proteose in a Saccharomyces cerevisiae formulation.
INTRODUCTION The BD Difco™ and BBL™ Peptone Technical Manual has been provided to assist you in your selection of BD products for use in cell culture and microbial fermentation production, from Research and Development to the final finished product.
In this age of globalization, a peptone supplier needs the ability and resources to serve customers all over the world. It is our commitment to innovation and product consistency as well as our broad access to the raw material market that makes BD (Becton, Dickinson and Company) such a strong global supplier and partner. In over 142 countries worldwide, BD offers a full line of peptones and media, for the biotechnology, pharmaceutical, animal and human vaccine, and bioremediation markets. BD offers products for both cell culture and microbial fermentation production, as well as applications for industrial research, QA/QC and environmental monitoring. ' Capability Our commitment to the media market is exemplified in our wide range of capabilities: • Largest media manufacturing plant in the world—In 1999, as part of the Difco Laboratories acquisition, BD opened a manufacturing plant in Sparks, Maryland, which is our Center of Manufacturing Excellence. With over 101,000 sq. ft. of production capacity, the center is a state-of-the-art modern facility that manufactures dehydrated culture media, prepared culture media, fermentation peptone ingredients, stains, and other products for microbiological and cell culture use worldwide. • Full line of meat peptones—Building on the reputation of the Difco meat peptones, BD continues to operate the previous Difco hydrolysation facility in Detroit as a source for the high quality Difco™ and Bacto™ brand products. This commitment to tradition carries on the quality and performance established under the Difco name, long recognized throughout the industry for superior quality. BD continues investing in Research and Development for peptone products, which continually expands our understanding of their application in cell culture and microbial fermentation. • Expanding line of non-animal origin products—As early as 1998, BD started offering non-animal products to the fermentation industry, introducing its Select APS™ (Alternative Protein Source) Super Broth, Select APS™ LB Broth, and Select Soytone. BD continues to leverage its expertise in creating high performing non-animal origin products to meet evolving customer needs in the cell culture and fermentation industry. • Custom Media Program—BD has available a custom media program to meet individual customer requirements. The program offers three levels of customization, from special packaging and QC testing requirements to full formula optimization services. It is our goal to service our customers with the highest level of technical support and manufacturing flexibility. Phytone™ Peptone Phytone™ Peptone UF Select Soytone Bacto™ TC Yeastolate TC Yeastolate UF Bacto™ Yeast Extract Yeast Extract Yeast Extract UF Yeast Extract LD Select APS™ LB Broth Select APS™ Super Broth Bacto™ Malt Extract Today, BD Offers 12 Non-Animal Products • Media Optimization Program—At the highest level of technical interaction, the Custom Media Program allows customers to outsource media optimization to BD, thus allowing them to focus resources in other critical areas. By accessing BD expertise in media development, customers can take advantage of our experience in yield enhancement and media formulation scale-up. • Dedicated non-animal equipment—With the rising concerns over bovine spongiform encephalopathies and transmissible spongiform encephalopathies (BSE/TSE), BD has met the challenge by initiating new strategies to dedicate process equipment to the production of non-animal derived products. Specific orders can be produced in this non-animal derived, product controlled environment. • Heightened Regulatory Compliance—BD plants are ISO 9001 and 13485 Certified, and regularly inspected by the FDA and USDA to conform with our cGMP manufacturing practices. We also offer the biotherapeutic industry comprehensive programs in documenting raw material origin, manufacturing change control and Drug Master Files (DMF) for key products.
History How did BD build this unmatched foundation to support the Biopharmaceutical industry? In 1955, BD acquired the Baltimore Biological Laboratory (BBL) of Baltimore, Maryland, and used its expertise to continually advance the clinical market with prepared media and diagnostic tools. In 1997, BD acquired Difco Laboratories of Detroit, Michigan. Since that acquisition, BD has merged Difco Laboratories and BBL Microbiology Systems into one division that provides customers with microbiology media, peptones, hydrolysates and extracts. Today, as a result of these acquisitions, BD is the largest microbiology company in the world, offering the widest range of microbiology and cell culture products worldwide.
Difco Contributions to the BD Product Line Beginning in 1895, Difco Laboratories produced high quality enzymes, dehydrated tissues and glandular products to aid in the digestion process. The knowledge gained from processing animal tissues, purifying enzymes and performing dehydration procedures allowed a smooth transition to the preparation of dehydrated culture media, in addition to its peptones. Meat and other protein digests were developed to stimulate growth of bacteria and fungi. Extensive research led to the development of Difco™ Bacto™ Peptone, which was introduced in 1914. Since then, Bacto™ Peptone continues to be recognized as the ‘premium quality’ standard for all other peptones. Building on this knowledge base, Difco continued to develop more peptones to add to the Bacto line of products. Bacto Proteose Peptone, Bacto Proteose Peptone No. 2, and Bacto Proteose Peptone No. 3 were created from the accumulated information that no single peptone was the most suitable nitrogen source for growing fastidious bacteria and supplementing cell culture. Today, many cell culture procedures, in addition to microbial cultures, call for the addition of a peptone to enhance the yield of the fermentation.
Combined Strengths Build the Largest Breadth of Line Today, having consolidated products from the BBL™ and Difco™ lines, BD not only offers peptones/hydrolysates manufactured from meat, animal tissue, collagen, gelatin and casein, but also products from non-animal origin materials. BD Difco™ brand yeast extracts are produced from primary grown (baker’s) yeast and provide lot-to-lot consistency that out-performs brewer’s yeast, as well as competitive products. BD is presently expanding its peptone line of products by adding additional non-animal and plant origin peptones. Along with combining the two premier manufacturers in the industry, BD has implemented a new branding strategy for peptones. While the Difco brand is the leading BD brand for production peptones, the BBL brand will remain on certain key BBL formulations. The BBL brand has become the leading BD brand for all prepared liquid media products. In addition to the main Difco™ and BBL™ brands, familiar brands such as Bacto™, BiTek™, and Select will also continue. The Difco™ Bacto™ brand has been retained for the traditional Bacto labeled products, while maintaining the integrity of the original premium Bacto formulas. The Difco™ BiTek™ brand has been continued for the production grade products where a premium product is not required. The Select brand remains for certain original non-animal BBL™ formulas. The consolidated BD BBL and BD Difco product lines provide quality products with lot-to-lot consistency, backed up by BD service, support, and custom programs to address individual requirements. All this, combined with our proactive responses to BSE/TSE concerns makes BD stand out as the best choice for fermentation and cell culture ingredients.
Product Quality In our effort to reduce BSE/TSE issues, BD sources raw materials for all products from known BSE-free countries—U.S.A., Canada, New Zealand, and Australia. All raw materials are tested upon receipt to assure that they meet BD incoming specifications. Then final products are tested prior to release to assure quality and consistency. After final release, the products are packaged and retention samples are held for stability studies and any additional testing required at a later date. Certificates of Analysis and Certificates of Origin for each product contain all the information required for complete traceability of all raw materials included in each product. For your convenience, these certificates are available from the BD web site 24 hours a day, 7 days a week, at www.bdregdocs.com.
Service BD maintains inventory in our BD Distribution Centers in Sparks, Maryland, and Temse, Belgium. With multiple manufacturing locations, BD is prepared to provide products and support to handle any need or situation. For global organizations, BD offers a formal global interacting capability to manage a single contract for multiple locations on a global basis. Please contact your local BD representative for details of the Global Key Account Program, or our web site at www.bd.com/industrial. At BD, we are continually adding new products. Please contact us if you have a need for a product that you do not find in this Peptone Technical Manual. Our Technical Services Group and Research and Development team are available to work with you and support your media requirements.
Using This Manual As presented in this manual, BD offers a wide range of peptones in the following categories: • Meat Peptones and Media • Casein Peptones • Non-Animal Peptones, Yeast Extracts and Media The manual provides insight into both cell culture and fermentation applications. Each product description contains data on physical, chemical analysis and amino acid distribution. A complete listing of regulatory services is provided and an alphabetical listing of products appears in the back of the manual. Thank you for your past and continued business.
HYDROLYSIS TO HYDROLYSATE Proteins, molecules essential to the structure and function of all living organisms, are made up of chains of any number of amino acids linked by peptide bonds and folded in a variety of complex structures. Proteins may be broken down into amino acids and peptides by hydrolysis using strong acids or proteolytic enzymes such as pepsin, papain, or pancreatin, which contains trypsin and chymotrypsin. These protein hydrolysates are called peptones. The starting materials for peptones vary from animal to vegetable. Protein sources include meat, casein (milk protein), gelatin, soybean, yeast and grains.1 Enzyme sources include animal organs (pancreatin and pepsin), papaya (papain), fig (ficin), pineapple (bromelain) and microbes.2 Acid hydrolysis is a harsh process, usually carried out at high temperature, which attacks all peptide bonds in the protein substrate, destroying some of the individual amino acids liberated. Tryptophan is usually totally lost in an acid hydrolysis. Cystine, serine and threonine are partially broken down and asparagine and glutamine are converted to their acidic forms. Vitamins are mostly destroyed by acid hydrolysis. Proteolytic enzymes hydrolyze proteins more gently than acids, do not require the high temperature used for acid hydrolysis, and usually are specific to the peptide bonds they will break. The resulting material from a proteolytic digestion is a mixture of amino acids and polypeptides of varying lengths. The enzyme pepsin will cut an amino acid chain where there is a phenylalanine or leucine bond.3 Papain will cut the chain adjacent to arginine, lysine and phenylalanine,4 and pancreatin shows activity at arginine, lysine, tyrosine, tryptophan, phenylalanine and leucine bonds.5 Microbial proteases, proteolytic enzymes secreted by microorganisms, are becoming more widely used in peptone production. Proteases from bacterial, algal, fungal and yeast sources cover a wide variety of enzyme activities, can be produced in large scale, and usually require only simple purification.6 The raw materials and manufacturing conditions for protein hydrolysis are controlled to produce consistent peptone products. Ingredients used
Meat Peptones and Media: Beef Extract, Powder Bacto™ Proteose Peptone Bacto™ Beef Extract, Desiccated BiTek™ Proteose Peptone Bacto™ Brain Heart Infusion Bacto™ Proteose Peptone No.2 Bacto™ Brain Heart Infusion, Porcine Bacto™ Proteose Peptone No.3 Gelysate™ Peptone BiTek™ Proteose Peptone No.3 Bacto™ Neopeptone Peptone Bacto™ Proteose Peptone No.4 Bacto™ Peptone Thiotone™ E Peptone BiTek™ Peptone Bacto™ Tryptose Peptone Polypeptone™ Peptone
Casein Peptones: Acidicase™ Peptone Trypticase™ Peptone Bacto™ Casamino Acids Bacto™ Tryptone Peptone BiTek™ Casamino Acids BiTek™ Tryptone Peptone Bacto™ Casitone Peptone Biosate™ Peptone
Soy Peptones: Phytone™ Peptone Select Soytone Phytone™ Peptone UF (Ultra Filtered) Bacto™ Soytone
Yeast Extracts: Bacto™ TC Yeastolate Yeast Extract TC Yeastolate UF Yeast Extract UF Bacto™ Yeast Extract Yeast Extract LD (Low Dusting)
Alternative Protein Source (APS)/Non-Animal Origin Products: Phytone™ Peptone Yeast Extract Phytone™ Peptone UF Yeast Extract UF Select Soytone Yeast Extract LD Bacto™ TC Yeastolate Select APS™ LB Broth TC Yeastolate UF Select APS™ Super Broth Bacto™ Yeast Extract Bacto™ Malt Extract Peptones by Category for peptone manufacture, including the protein, agent of hydrolysis, and any buffering agents used, are selected based on specific purity and quality standards. The conditions of the hydrolysis, such as the amount of enzyme used, the time for digestion, and the pH and temperature at which hydrolysis is conducted, determine the degree of hydrolysis and the quality of the hydrolysate. Therefore, these conditions must be carefully controlled thoughout the manufacturing process. Purification, concentration and drying steps are carefully regulated due to their bearing on the characteristics of a peptone. Finally, each batch of protein hydrolysate is tested for an array of physical, chemical, analytical and growth support tests to ensure product quality and lot-to-lot consistency.
References 1. Bridson and Brecker. 1970. Design and formulation of microbial culture media. In Norris and Ribbons (ed.), Methods in microbiology, vol. 3A, p. 230. Academic Press, New York. 2. Bridson and Brecker. 1970. Design and formulation of microbial culture media. In Norris and Ribbons (ed.), Methods in microbiology, vol. 3A, 236-7. Academic Press, New York. 3. Dixon and Webb. 1979. Enzymes, 3rd ed. p. 892. Longman Group Limited, London. 4. Dixon and Webb. 1979. Enzymes, 3rd ed., p. 890. Longman Group Limited, London. 5. Dixon and Webb. 1979. Enzymes, 3rd ed., p. 884-886. Longman Group Limited, London. 6. Cowan. 1991. Industrial enzymes. In Moses and Cape (ed.), Biotechnology, the science and the business, p. 314. Harwood Academic Publishers GmbH, Chur, Switzerland.
GELYSATE™ PEPTONE
Product Description Bacto™ Neopeptone Peptone is an enzymatic digest of protein. Neopeptone contains a wide variety of peptide sizes in combination with vitamins, nucleotides and minerals.
Applications Neopeptone is recommended for use in media for detection of fungi.1 Apodaca and McKerrow2 used Neopeptone for the cultivation of Trichophyton rubrum for study of its proteolytic activity. Neopeptone has been cited as a component of culture media used for cultivation of human pathogens, notably, Bordetella pertussis and group A streptococci. Neopeptone has also been reported to provide nutrients for support of spirochetes and protozoa. Wyss et al.3 used Neopeptone as a component of a medium for cultivation of Treponema maltophilum sp. nov., a fastidious oral anaerobe. Ifediba and Vanderberg4 reported that Neopeptone, in addition to calf serum, was used as an inexpensive replacement for human serum in cultivation of Plasmodium falciparum, the causative agent of human malaria. Cushion and Ebbets5 utilized Neopeptone in their investigations of various media for cultivating Pneumocystis carinii without feeder cells; optimal replication of P. carinii separated from host fungi cells was observed in media with Neopeptone and N-acetylglucosamine at low pH.
Physical Characteristics Bacto™ Neopeptone is a tan, free-flowing, homogeneous powder.
Availability Bacto™ Neopeptone 211681, 500 g Bacto™ Neopeptone 211680, 10 kg
References 1. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and wastewater, 20th ed., 9-131-137. American Public Health Association, Washington, DC. 2. Apodaca and McKerrow. 1990. Expression of proteolytic activity by cultures of Trichophyton rubrum. J. Med. Vet. Mycol. 28:159-171. 3. Wyss, Choi, Schupbach, Guggenheim and Gobel. 1996. Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions. Int. J. Syst. Bacteriol. 46:745-752. 4. Ifediba and Vanderberg. 1980. Peptones and calf serum as a replacement for human serum in the cultivation of Plasmodium falciparum. J. Parasitol. 66:236-239. 5. Cushion and Ebbets. 1990. Growth and metabolism of Pneumocystis carinii in axenic culture. J. Clin. Microbiol. 28:1385-1394.
BACTO™ NEOPEPTONE PEPTONE
Product Description Bacto™ Peptone is an enzymatic digest of animal protein. Bacto™ Peptone was first introduced in 1914 and became the standard Peptone for the preparation of bacteriological culture media. The nutritive value of Bacto™ Peptone is largely dependent on the amino acid content that supplies essential nitrogen. Bacto™ Peptone contains only a negligible quantity of proteoses and more complex constituents. BiTek™ Peptone is an enzymatic digest of animal protein. BiTek™ Peptone has growth properties similar to Bacto™ Peptone, but was developed to offer a cost-effective ingredient for scale-up to production processes.
Applications Bacto™ Peptone is used as an organic nitrogen source in microbiological culture media for cultivation of a variety of bacteria and fungi. For example, Iwanaga et al.1 utilized Bacto™ Peptone for production of cholera toxin by Vibrio cholerae O1 El Tor. Benkerroum et al.2 reported using Bacto™ Peptone in a selective medium developed for isolating Leuconostoc spp. from food samples. Bacto™ Peptone was used in a culture medium for two anaerobic, extremely thermophilic Archaea, Thermococcus celer and Pyrococcus woesei, by Blamey et al.3 Bacto™ Peptone has also been utilized as a nitrogen source in cell culture media formulations. Taylor et al.4 used Bacto™ Peptone to supplement serum-free medium for several mammalian cell lines and reported the solubility of Bacto™ Peptone as very good at 10 g/100 mL water. Sakoda and Fukusho5 also utilized Bacto™ Peptone in serum-free culture for maintaining porcine kidney epithelial cells. Bacto™ Peptone is also useful as a supplement in cell culture with serum. Researchers uncovered estrogenic activity associated with Bacto™ Peptone when including the peptone in medium for culture of yeast; the estrone contained in Bacto™ Peptone was converted to estrodiol by Saccharomyces cerevisiae. These findings suggest that adding estrogens to a medium containing Bacto™ Peptone for studies of estrodiol production by yeast may confound results.6,7 BiTek™ Peptone is used as a nitrogen source in microbiological culture media. BiTek™ Peptone was developed to serve the biotechnology/pharmaceutical industry in providing a lower-cost alternative to Bacto™ Peptone, offering the same lot-to-lot consistency and similar growth characteristics to Bacto™ Peptone.
Physical Characteristics Bacto™ Peptone appears as tan, free-flowing, granules. BiTek™ Peptone is a tan, free-flowing, homogeneous powder.
Availability Bacto™ Peptone 211840, 100 g Bacto™ Peptone 211677, 500 g Bacto™ Peptone 211820, 2 kg Bacto™ Peptone 211830, 10 kg BiTek™ Peptone 254820, 10 kg
BACTO™ PEPTONE BITEK™ PEPTONE
References 1. Iwanaga, Yamamoto, Higa, Ichinose, Nakasone and Tanabe. 1986. Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol. Immunol. 30:1075-1083. 2. Benkerroum, Misbah, Sandine and Elaraki. 1993. Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products. Appl. Environ. Microbiol. 59:607-609. 3. Blamey, Chiong, Lopez and Smith. 1999. Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei. J. Microbiol. Methods 38:169-175. 4. Taylor, Dworkin, Pumper and Evans. 1972. Biological efficacy of several commercially available peptones for mammalian cells in culture. Exp. Cell Res. 74:275-279. 5. Sakoda and Fukusho. 1998. Establishment and characterization of a porcine kidney cell line, FS-L3, which forms unique multicellular domes in serum-free culture. In Vitro Cell. Dev. Biol. Anim. 34:53-57. 6. Feldman and Krishnan. 1995. Estrogens in unexpected places: possible implications for researchers and consumers. Environ. Health Perspect. 103 Suppl 7:129-133. 7. Miller, Bottema, Stathis, Tokes and Feldman. 1986. Unexpected presence of estrogens in culture medium supplements: subsequent metabolism by the yeast Saccharomyces cerevisiae. Endocrinology 119:1362-1369.
Product Description Polypeptone™ Peptone is a mixture of peptones made up of equal parts of pancreatic digest of casein and peptic digest of animal tissue. Polypeptone™ Peptone includes the high content of amino acids and small polypeptides characteristic of pancreatic digest of casein and the larger polypeptides characteristic of peptic digest of animal tissue.
Applications Researchers have found Polypeptone™ Peptone to meet nutritional requirements of various bacteria, fungi and mammalian cells, where a single source of casein or meat peptones has been unsatisfactory. Polypeptone™ Peptone has been utilized in culture media for the production of trypsin inhibitor by Cephalosporium sp.;1 the production of bacterial cellulose by Acetobacter sp. A9;2 production of succinic acid from whey by Anaerobiospirillum succiniciproducens;3 mass production of luciferase-bacterial magnetic particles by recombinant Magnetospirillum magneticum AMB-1;4 and the production of a novel tumor-killing factor by human macrophage-monocyte hybridomas.5
Physical Characteristics Polypeptone™ Peptone is a tan, free-flowing, homogeneous powder.
Availability Polypeptone™ Peptone 211910, 454 g Polypeptone™ Peptone 297108, 25 lb
References 1. Tsuchiya and Kimura. 1978. Production of trypsin inhibitor by a Cephalosporium sp. Appl. Environ. Microbiol. 35:631-635. 2. Son, Heo, Kim and Lee. 2001. Optimization of fermentation conditions for the production of bacterial cellulose by a newly isolated Acetobacter sp. A9 in shaking cultures. Biotechnol. Appl. Biochem. 33(Pt 1):1-5. 3. Lee, Lee, Kwon, Lee and Chang. 2000. Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey. Appl. Microbiol. Biotechnol. 54:23-27. 4. Yang, Takeyama, Tanaka and Matsunaga. 2001. Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1. Enzyme Microbiol. Technol. 29:13-19. 5. Taniyama, Yoshida and Furuta. 1988. Demonstration of a novel tumor-killing factor secreted from human macrophage-monocyte hybridomas. J. Immunol. 141:4061-4066.
POLYPEPTONE™ PEPTONE
Product Description The Bacto™ Proteose Peptones are enzymatic digests of protein. Studies of peptic digests of animal tissue prepared under varying digestion parameters led to the development of Proteose Peptone, Proteose Peptone No. 2 and Proteose Peptone No. 3. Data accumulated during these studies demonstrated that no one peptone is the most suitable nitrogen source for every microbiological application. Bacto™ Proteose Peptone No. 4 is a spray-dried version of Bacto™ Proteose Peptone. BiTek™ Proteose Peptone and BiTek™ Proteose Peptone No. 3 are enzymatic digests of protein, developed to offer cost-effective alternatives to the Bacto™ Proteose Peptones for scale-up to production applications.
Applications Bacto™ Proteose Peptone is used in preparing microbiological culture media and in producing bacterial toxins. Bacto™ Proteose Peptone was originally developed to produce a diphtheria toxin of high and uniform potency from cultures of Corynebacterium diphtheriae. Studies support the use of Proteose Peptone for production of diphtheria toxin, toxin-antitoxin mixtures and toxoid.1,2 Proteose Peptone is also valuable in the production of other bacterial toxins: Clostridium botulinum toxin;3 toxin from Clostridium perfringens;4 toxin of hemolytic streptococci;5 pneumococcus toxin;6 and toxin from Salmonella pullorum.7 Many factors account for the suitability of Proteose Peptone for the culture of fastidious pathogens, including the nitrogen components, buffering range and the high content of proteoses. These elements create an environment beneficial to the maintenance of virulence and the elaboration of bacterial by-products, thus stock cultures are well preserved on media containing Bacto™ Proteose Peptone.
BACTO™ PROTEOSE PEPTONE BITEK™ PROTEOSE PEPTONE BACTO™ PROTEOSE PEPTONE NO. 2 BACTO™ PROTEOSE PEPTONE NO. 3 BITEK™ PROTEOSE PEPTONE NO. 3 BACTO™ PROTEOSE PEPTONE NO. 4
Bacto™ Proteose Peptone may be used in culture medium for a variety of applications, including production of substances from the culture of bacteria, fungi and mammalian cells. Proteose Peptone has been utilized in a medium for producing glycosidases from Bacteroides fragilis,8 and to stimulate amyloglucosidase production by Aspergillus sp.9 It has been used to cultivate halophilic bacteria isolated from soil in Egypt for production of polymers.10 Jan et al.11 reported that Proteose Peptone as supplementation to defined medium resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Proteose Peptone has also been used to provide nutrients for axenic culture of amoeba.12 BiTek™ Proteose Peptone was developed to provide a product priced for the biotechnology/pharmaceutical market with growth characteristics to match Bacto™ Proteose Peptone. Bacto™ Proteose Peptone No. 2 is used in preparing microbiological culture media. It was originally developed for use in media for the production of diphtheria toxin. Bunney and Thomas13 reported good yield of diphtheria toxin with Proteose Peptone No. 2 in a simple peptone-sugar-sodium acetate medium.
Bacto™ Proteose Peptone No. 3 is used in preparing microbiological culture media. It is a modification of Proteose Peptone adapted for use in the preparation of chocolate agar for propagation of Neisseria species and chocolate tellurite agar for Corynebacterium diphtheriae. While investigating the nutritional values of the Proteose Peptones, Difco Laboratories found that Proteose Peptone No. 3 provides superior nutrition for fastidious microorganisms. It supports growth of streptococci, staphylococci, pneumococci, gonococci and other organisms that require a highly nutritious substrate. For example, Ifediba and Vanderberg14 report that Proteose Peptone No. 3 in addition to calf serum was used as an inexpensive replacement for human serum in cultivation of Plasmodium falciparum, the causative agent of human malaria. Recently, because of the benefit of lower endotoxin levels, cell culture manufacturers have found significant yield improvements in using Proteose Peptone No. 3. BiTek™ Proteose Peptone No. 3 was developed to provide a product priced for the biotechnology/pharmaceutical market with growth characteristics to match Bacto™ Proteose Peptone No. 3. Bacto™ Proteose Peptone No. 4 is a spray-dried version of Bacto™ Proteose Peptone. It offers the same beneficial nutrients as Proteose Peptone for growth promotion and toxin production with a wide range of fastidious microorganisms.
Physical Characteristics Bacto™ Proteose Peptone appears as tan, free-flowing granules. BiTek™ Proteose Peptone is a tan, free-flowing, homogeneous powder. Bacto™ Proteose Peptone No. 2 appears as tan, free-flowing granules. Bacto™ Proteose Peptone No. 3 appears as tan, free-flowing granules. BiTek™ Proteose Peptone No. 3 is a tan, free-flowing, homogeneous powder. Bacto™ Proteose Peptone No. 4 is a tan, free-flowing, homogeneous powder.
Availability Bacto™ Proteose Peptone 211684, 500 g Bacto™ Proteose Peptone 212010, 10 kg Bacto™ Proteose Peptone 212030, 50 kg BiTek™ Proteose Peptone 253310, 10 kg Bacto™ Proteose Peptone No. 2 212120, 500 g Bacto™ Proteose Peptone No. 2 212110, 10 kg Bacto™ Proteose Peptone No. 3 211693, 500 g Bacto™ Proteose Peptone No. 3 212220, 2 kg Bacto™ Proteose Peptone No. 3 212230, 10 kg Bacto™ Proteose Peptone No. 3 211692, 50 kg BiTek™ Proteose Peptone No. 3 253720, 25 kg Bacto™ Proteose Peptone No. 4 211715, 10 kg 24 B D B I O P H A R M A C E U T I C A L P R O D U C T I O N
References 1. Kirkbride, Berthelsen and Clark. 1931. Comparative studies of infusion and infusion-free diphtheria toxin in antitoxin production and in standardization by the flocculation, subcutaneous, and intracutaneous tests. J. Immunol. 21:1-20. 2. Hazen and Heller. 1931. Further studies upon the effect of various carbohydrates on production of diphtheria toxin with special reference to its flocculating titer and final pH. J. Bacteriol. 23:195-209. 3. Nelson. 1927. The relationship between the intracellular globulin and the toxin of C. botulinum. J. Infect. Dis. 41:9-12. 4. Mollby and Holme. 1976. Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D. J. Gen. Microbiol. 96:137-144. 5. Kirkbride and Wheeler. 1926. Studies of the toxins of the hemolytic streptococci associated with scarlet fever. J. Immunol. 11:477-497. 6. Kneeland and Dawes. 1932. Studies on the common cold: V. The relationship of pathogenic bacteria to upper respiratory diseases in infants. J. Exp. Med. 55:735-744. 7. Hanks and Rettger. 1931. Bacterial endotoxin; search for a specific intracellular toxin in S. pullorum. J. Immunol. 22:283-314. 8. Berg, Nord and Wadstrom. 1978. Formation of glycosidases in batch and continuous culture of Bacteroides fragilis. Appl. Environ. Microbiol. 35:269-273. 9. Mamo and Gessesse. 1999. Production of raw-starch digesting amyloglucosidase by Aspergillus sp. GP-21 in solid state fermentation. J. Ind. Microbiol. Biotechnol. 22:622-626. 10. Hezayen, Rehm, Eberhardt and Steinbuchel. 2000. Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor. Appl. Microbiol. Biotechnol. 54:319-325. 11. Jan, Jones, Emery and Al-Rubeai. 1994. Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture. Cytotechnol. 16:17-26. 12. Shukla, Kaul and Mehlotra. 1989. Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970. Indian J. Exp. Biol. 27:785-791. 13. Bunney and Thomas. 1936. Diphtheria toxin-production on broths made from dried complete media. J. Immunol. 31:95-102. 14. Ifediba and Vanderberg. 1980. Peptones and calf serum as a replacement for human serum in the cultivation of Plasmodium falciparum. J. Parasitol. 66:236-239.
Product Description Thiotone™ E Peptone is an enzymatic digest of animal tissue. Thiotone™ E Peptone contains a wide range of peptide sizes, including the large molecular weight peptides which support fastidious organisms.
Applications Thiotone™ E Peptone is a nitrogen source in microbiological culture media. It has been recommended for use in blood agar formulae for hemolysis studies with pneumococci and streptococci. Thiotone™ E Peptone is high in sulfur amino acids and can be used in media to detect hydrogen sulfide production. Tortora1 utilized Thiotone™ E Peptone as the nitrogen source in a medium promoting sporulation of Clostridium perfringens strains. Thiotone™ E Peptone is recommended for use in media for testing water samples for coliforms.2 Kwinn3 utilized Thiotone™ E Peptone as a supplement to her medium for Corynebacterium glutamicum to make the cells electrocompetent for transformations. Thiotone™ E Peptone has also been cited as an ingredient in media for non-bacterial organisms; Thiotone™ E Peptone is used in Modified HL5 Medium, one of the main media used for culturing the cellular slime mold Dictyostelium discoideum.
Physical Characteristics Thiotone™ E Peptone is a tan, free-flowing, homogeneous powder.
Availability Thiotone™ E Peptone 212302, 500 g
References 1. Tortora. 1984. Alternative medium for Clostridium perfringens sporulation. Appl. Environ. Microbiol. 47:1172-1174. 2. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, DC. 3. Kwinn. 2001. Regulation of acetyl-CoA carboxylase in Corynebacterium glutamicum: isolation and cloning of the upstream region of the accBC gene. Bug Journal, Biology Department, Massachusetts Institute of Technology 4:193-200.
THIOTONE™ E PEPTONE
Product Description Bacto™ Tryptose Peptone is a mixed enzymatic hydrolysate with distinctive nutritional properties. The digestive process of Tryptose results in assorted peptides of higher molecular weight suitable for long-chain amino acid requirements.
Applications Bacto™ Tryptose Peptone was originally developed as a peptone particularly adapted to the growth requirements of Brucella. Tryptose is very useful for cultivation of streptococci, pneumococci, meningococci and other fastidious organisms, and was found to be superior to meat infusion peptone media previously used for these organisms.1,2 Mobley et al.3 reported that Tryptose Broth was the preferred medium for strains of Bordetella bronchiseptica in studies of phosphatase activity. Tryptose has been reported as beneficial for cell culture applications. Litwin4 found Tryptose suited to supplementing a serum-free medium for growing human diploid fibroblasts. Vaughn and Fan5 established that Tryptose provided free amino acids necessary for growth of Spodoptera frugiperda and Lymantria dispar insect cell lines. Tryptose Peptone is often used as a biomass enhancer for recombinent E. coli production. Tryptose is the major ingredient and only peptone in the formulation, Tryptose Phosphate Broth, an often-used medium for various culture applications. Hata and Kojima6 have shown Tryptose Phosphate Broth (TPB) to be a useful supplement in culturing the nematode, Angiostrongylus cantonensis, in vitro. TPB was also reported as a supplement to a medium for cultivating a protozoan parasite, which parasitizes vectors of Chagas’ disease, on its insect cell host.7 Spodoptera frugiperda, a cotton pest in Argentina8 and several tick cell lines have also been grown using a TPB supplemented medium.9 Tryptose Phosphate Broth has been reported as a suitable supplement for growth of baby hamster kidney cells10 and porcine kidney cells.11
Physical Characteristics Bacto™ Tryptose Peptone appears as tan, free-flowing granules.
BACTO™ TRYPTOSE PEPTONE
Availability Bacto™ Tryptose Peptone 211713, 500 g Bacto™ Tryptose Peptone 211709, 10 kg
References 1. Casman. 1942. A dehydrated medium to supplement meat infusion as a base for blood agar. J. Bacteriol. 43:33. 2. Casman. 1947. A noninfusion blood agar base for neisseriae, pneumococci and streptococci. Am. J. Clin. Pathol. 17:281-289. 3. Mobley, Chengappa, Kadel and Stuart. 1984. Effect of pH, temperature and media on acid and alkaline phosphatase activity in "clinical" and "nonclinical" isolates of Bordetella bronchiseptica. Can. J. Comp. Med. 48:175-178. 4. Litwin. 1985. Further studies on a tryptose based serum-free medium for human diploid fibroblasts. Dev. Biol. Stand. 60:25-33. 5. Vaughn and Fan. 1997. Differential requirements of two insect cell lines for growth in serum-free medium. In Vitro Cell. Dev. Biol. Anim. 33:479-482. 6. Hata and Kojima. 1990. Angiostrongylus cantonensis: in vitro cultivation from the first-stage to infective third-stage larvae. Exp. Parasitol. 70:467-482. 7. Reduth, Schaub and Pudney. 1989. Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae). Parasitology 98:387-393. 8. Deutschmann and Jager. 1994. Optimization of the growth conditions of Sf21 insect cells for high-density perfusion culture in stirred-tank bioreactors. Enzyme Microb. Technol. 16:506-512. 9. Munderloh and Kurtti. 1989. Formulation of medium for tick cell culture. Exp. Appl. Acarol. 7:219-229. 10. Prodafikas and Plavsic. 2000. Effects of medium supplements on BHK-21 cell growth and bluetongue virus production. Focus 22:35. 11. Sakoda and Fukusho. 1998. Establishment and characterization of a porcine kidney cell line, FS-L3, which forms unique multicellular domes in serum-free culture. In Vitro Cell Dev. Biol. Anim. 34:53-57.
CASEIN PEPTONES The casein peptones are so named because of their starting material, milk. Milk is a very complex material, consisting of water, lactose, lipids, salts and proteins. It is the protein portion of the milk that provides the starting material for the casein peptones. After the milk has had the cream fraction removed and it is acidified (there are several methods available to accomplish this from the addition of acid to enzymatic and bacterial processes), the protein composition of milk can be separated out.1,2 The soluble portion, known as whey protein, can be found in the supernatant. The insoluble portion, which precipitates out, consists of a group of heterogeneous phosphoproteins that exist together in a colloidal particle called a micelle. This group of phosphoproteins is collectively known as casein. The casein micelle can be broken down into four component classes, based in order of their decreasing mobility when separated by electrophoresis at pH 7, designated as αs1,αs2, β and κ casein. Casein, which can make up to 3% of the total components in bovine milk, is one of the most nutritive of the milk proteins, as it contains all of the common amino acids and is rich in the essential ones. The casein recovered is known as acid casein and is insoluble in water. Generally, the acid casein is dissolved in a suitable hydroxide such as NaOH, to make it soluble in water. The resulting sodium caseinate is then used as the basis for hydrolyzed caseins. Sodium caseinate typically consists of between 87% to 90% protein.3 Hydrolyzed caseins are manufactured by one of two processes, acid hydrolysis or enzymatic hydrolysis.
Acid Hydrolysis The diagram in Figure 1 shows the basic processing steps in the manufacture of a hydrolyzed casein by acid digestion. Many acids can be utilized but hydrochloric acid is most commonly used in the process. This process leads to complete hydrolysis of the casein to amino acids and other compounds of relative chemical simplicity. However, not all of the amino acids survive intact. Tryptophan is typically destroyed in the process, as is cystine. Other amino acids, such as serine and threonine are reduced. Asparagine and glutamine, two amino acids with uncharged polar R groups, undergo hydrolysis to yield aspartic acid and glutamic acid, two amino acids which are negatively charged at pH 6 to 7.
Enzymatic Hydrolysis In animals, the stomach initiates the digestion of protein. However, it is the pancreas which carries out the majority of the protein breakdown. In the enzymatic hydrolysis of casein, the enzymes from the pancreas are utilized to manufacture these peptones. While the pancreas contains a battery of enzymes from the protease, lipase and amylase groups, it is the proteases, which are used in the hydrolysis of casein. The proteases found in the pancreas consist of trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B and elastase. However, trypsin and chymotrypsin make up the greatest percentage of the total amount of proteases and carry out the bulk of the work. Both of these proteases have the ability to digest proteins into peptides, but they do not have the ability to break the protein down into its component amino acids. The carboxypeptidases do have this ability; however, they are not present in large enough amounts to accomplish this task to any great degree. Generally, in the digestion process of animals, these peptides would be broken down into amino acids through the actions of peptidases found in the epithelial cells of the small intestine.5 Thus, one of the characteristics of pancreatic digest of casein, as opposed to the acid hydrolysis of casein, is a peptone that consists of greater amounts of peptides. The basic processing steps involved in the manufacture of pancreatic digest of casein are shown in Figure 2.
References 1. Huffman and Harper, W.J. 1999. Symposium: Marketing dairy value through technology: maximizing the value of milk through separation technologies. J. Dairy Sci. 82:2238-2244. 2. Van Boekel. 1999. Heat-induced deamination, dephosphorylation and breakdown of caseinate. Int. Dairy. J 9:237-241. 3. Dziuba, Babuchowski, Smoczynski and Smietana. 1999. Fractal analysis of caseinate structure. Int. Dairy J. 9:287-292. 4. Lehninger. 1975. Proteins: covalent backbone and amino acid sequence. In Biochemistry. 2nd ed. Worth Publishers, Inc., New York. 5. Austgen, Bowen and Rouge. 2000. Hypertexts for Biomedical Science: Pathophysiology – Digestive Systems. http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/pancreas/exocrine.html Figure 2 Acid Hydrolysates of Casein Product Description Acidicase™ Peptone is a hydrochloric acid hydrolysate of casein. The manufacturing process produces a casein hydrolysate that has a high salt content of approximately 37% and nitrogen content of approximately 8%. The hydrolysis of the casein, a milk protein rich in amino acid nitrogen, is carried out until all the nitrogen is converted to amino acids or other compounds of relative simplicity. It is deficient in cystine, because casein contains little cystine, and in tryptophan, which is destroyed by the acid treatment. Bacto™ Casamino Acids are an acid hydrolysate of casein, prepared according to the method described by Mueller and Miller.1 The method described, reduces the sodium chloride and iron content of the hydrolyzed casein. This hydrolyzed casein, supplemented with inorganic salts, growth factors, cystine, maltose and an optimum amount of iron was used by Mueller and Miller to prepare diptheria toxin. Bacto™ Casamino Acids duplicate this specially treated hydrolyzed casein. BiTek™ Casamino Acids are prepared similarly to Bacto™ Casamino Acids but are less refined in the final processes.
Applications Acidicase™ Peptone is intended for use as a nutritional supplement in vitamin assay, susceptibility testing and other laboratory media and microbial fermentation where the high salt content will not interfere. Bacto™ Casamino Acids, due to the nearly complete hydrolysis of casein and the low sodium chloride and iron content, make an excellent supplement for many media formulations where nitrogen requirements are minimal. It has been recommended as a compromise for the replacement of pure amino acids in a defined medium for the growth of Lactobacillus, thus eliminating the complexity of preparation.2 Additionally, it has been successfully used, along with Tryptone Peptone in nutritional studies to determine a bacterium’s growth requirement for peptides or amino acids.3,4 It also works well as a component in laboratory media. It has been utilized in such diverse applications as TYI-S-33 media for the parasite Entamoeba histolytica and LCM medium for the growth of a nematode-bacterium complex.5 BiTek™ Casamino Acids provides the same benefits as Bacto™ Casamino Acids, in instances where a less refined hydrolysate can be utilized.
Physical Characteristics Acidicase™ Peptone is a homogeneous, free-flowing, dehydrated powder; light beige in color. Bacto™ Casamino Acids are a homogeneous, free-flowing, dehydrated powder; very light beige in color. BiTek™ Casamino Acids are a homogeneous, free-flowing, dehydrated powder; very light beige in color.
ACIDICASE™ PEPTONE BACTO™ CASAMINO ACIDS BITEK™ CASAMINO ACIDS
Availability Acidicase™ Peptone 211843, 500 g Bacto™ Casamino Acids 223050, 500 g Bacto™ Casamino Acids 223030, 10 kg BiTek™ Casamino Acids 252920, 10 kg References 1. Mueller and Miller. 1941. Production of diphtheria toxin of high potency (100 lf) on a reproducible medium. J. Immunol. 40:21-32. 2. Van Niel and Hahn- Hägerdal. 1999. Nutrient requirements of lactococci in defined growth media. Appl. Microbiol. Biotechnol. 52:617-627. 3. Takahashi, Sato and Yamada. 2000. Metabolic pathways for cytotoxic end product formation from glutamate- and aspartatecontaining peptides by Porphyromonas gingivalis. J. Bacteriol. 182:4704-4710. 4. Attwood, Klieve, Ouwerkerk and Patel. 1998. Ammonia-hyperproducing bacteria from New Zealand ruminants. Appl. Environ. Microbiol. 64:1796-1804. 5. Strauch and Ehlers. 2000. Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures. Appl. Microbiol. Biotechnol. 54:9-13.
Product Description Biosate™ Peptone is a mixed hydrolysate comprised of casein and yeast extract.
Applications Biosate™ Peptone can be used as a component in microbiological media or in fermentation applications. The synergistic effect of two or more types of hydrolysates is well documented and has been utilized for decades in culture media formulation. The combination of pancreatic digest of casein and yeast extract provides nutritional benefits that are not provided by the components alone. It has been reported that the combined use of these two peptones has shown improved toxin production in clostridia.1,2 Additionally, the combination of pancreatic digest of casein and yeast extract has been used successfully as components in media which supported the hatching and culture of Giardia spp. from cysts and the first-time culturing of a nematode without the need of its symbiotic bacteria
Physical Characteristics Biosate™ Peptone is a homogeneous, free-flowing, dehydrated powder, yellow-tan in color.
Availability Biosate™ Peptone 211862, 454 g Biosate™ Peptone 294312, 25 lb
References 1. Artemenko, Ivanova, Nenashev, Kuznetsova and Ochkina. 1985. Use of experimental analytical method for equilibrating nutrient broths for Clostridium perfringens type A growth and toxin production. Zhurnal Mikrobiologii, Epidemiologii, i Immunobiologii. 11:37-41. 2. Siegel and Metzger. 1980 Effect of fermentation conditions on toxin production by Clostridium botulinum type B. Appl. Environ. Microbiol. 40:1023-1026. 3. Ponce, Martínez and Alvarez. 1989. Excystation and culture of Giardia spp. from human source. Archivos de Investigacion Medica 20:123-127. 4. Dorsman and Bijl. 1985. Cultivation of free-living stages of Trichostrongylus colubriformis in media without bacteria, animal tissue extract, or serum. J. Parasitol. 71:200-203.
BIOSATE™ PEPTONE
Enzymatic Digests of Casein Product Description Bacto™ Casitone Peptone is a pancreatic digest of casein. The manufacturing process for an enzymatic digest of casein is not as destructive as an acid hydrolysis. Thus, the casein is not broken down as completely into its constituent components. In many cases this makes for a more nutritious hydrolysate, especially for those organisms that prefer peptides to amino acids. Trypticase™ Peptone is a pancreatic digest of casein and is the primary nitrogen source in Trypticase™ Soy Broth and Agar. Bacto™ Tryptone Peptone is a pancreatic digest of casein. It was developed by Difco Laboratories while investigating a peptone particularly suitable for the elaboration of indole by bacteria. It is also notable for the absence of detectable levels of carbohydrates. BiTek™ Tryptone Peptone is prepared similarly to Bacto™ Tryptone Peptone but the final product goes through fewer refinement steps during processing.
Applications Bacto™ Casitone Peptone can be used as a component in microbiological media or in fermentation applications. A recent publication has also reported that the stability of lyophilized influenza virus vaccine was augmented by the addition of 2% Casitone.1 Trypticase™ Peptone is recommended for use in media formulations, where good growth of fungi and bacteria is required. It is referenced in Official Methods of Analysis of AOAC International and meets the USP specifications for pancreatic digest of casein. Bacto™ Tryptone Peptone has been used in conjunction with Casamino Acids in nutritional studies to determine amino acids vs. peptide utilization.4,5 It is included in standard methods manuals applications and is listed in the “Reagent” section of The United States Pharmacopeia, as meeting the specifications for pancreatic digest of
BACTO™ CASITONE PEPTONE TRYPTICASE™ PEPTONE BACTO™ TRYPTONE PEPTONE BITEK™ TRYPTONE PEPTONE casein, a component in many of the media listed.2,3,6-9 The European Pharmacopoeia also lists pancreatic digest of casein as a component in many of the recommended media.10 Bacto™ Tryptone Peptone also works well in fermentation applications. It has been used successfully with commonly used organisms such as Escherichia coli,11 as well as uncommon organisms such as the diatom Nitzschia laevis.12 BiTek™ Tryptone Peptone provides some of the same benefits as Bacto™ Tryptone in instances where a less refined hydrolysate can be utilized.
Physical Characteristics Bacto™ Casitone Peptone is a homogeneous, free-flowing, dehydrated powder, tan in color. Trypticase™ Peptone is a homogeneous, free-flowing, dehydrated powder; very light beige in color. Bacto™ Tryptone Peptone is a homogeneous, free-flowing, dehydrated powder; light beige in color BiTek™ Tryptone Peptone is a homogeneous, free-flowing, dehydrated powder; beige in color. Availability Bacto™ Casitone Peptone 225930, 500 g Bacto™ Casitone Peptone 225910, 10 kg Trypticase™ Peptone 211921, 454 g Trypticase™ Peptone 211923, 25 lb Bacto™ Tryptone Peptone 211705, 500 g Bacto™ Tryptone Peptone 211701, 10 kg BiTek™ Tryptone Peptone 251420, 10 kg
References 1. Yannarell, Goldberg and Hjorth. 2001 Stabilizing cold-adapted influenza virus vaccine under various storage conditions. J. Virol. Methods (in press). 2. Horowitz. (ed.). 2000. Official methods of analysis of AOAC international. 17th ed. AOAC International, Gaithersburg, Md. 3. United States Pharmacopeial Convention, Inc. 2001. The United States Pharmacopeia 25/The national formulary 20—2002. United States Pharmacopeial Convention, Inc., Rockville, Md. 4. Takahashi and Yamada. 2000. Metabolic pathways for cytotoxic end product formation from glutamate- and aspartate-containing peptides by Porphyromonas gingivalis J. Bacteriol. 182:4704-4710. 5. Nagel, Oostra, Tramper and Rinzema. 1999. Improved model system for solid-substrate fermentation: effects of pH, nutrients and buffer on fungal growth rate. Process Biochem. 35:69-75. 6. Downes and Ito. (ed.).2001. Compendium of methods for the microbiological examination of foods. 4th ed. American Public Health Association. Washington D.C. 7. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC International, Gaithersburg, Md. 8. Clesceri, Greenberg and Eaton. (ed.). 1998. Standard methods for the examination of water and wastewater. 20th ed. American Public Health Association, Washington D.C. 9. Marshall (ed.). 1993. Standard methods for the examination of dairy products. 16th ed. American Public Health Association. Washington D.C. 10. European Directorate for the Quality of Medicines within the Council of Europe, Strasbourg. 2001 Supplement European Pharmacopoeia 3rd ed. Council Of Europe, Strasbourg. 11. Sivakesavs, Chen, Hackett, Huang, Lam, Lam, Siu, Wong and Wong. 1999. Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system. Process Biochem. 34:893-900. 12. Wen and Chen. 2001. Optimization of nitrogen sources for heterotrophi production of eicosapentaenoic acid by the diatom Nitzschia laevis. Enzyme Microbia Technol. 29:341-347.
NON-ANIMAL PEPTONES, YEAST EXTRACTS AND MEDIA The need for non-animal derived products has increased due to the concerns about BSE/TSE and their link to animal derived products. In response to this need, BD has developed a broad offering of soy peptones, yeast extracts and animal-free media. Along with the leading non-animal supplement categories of soy and yeast, many new plant-based products are being tested on the market. Wheat, pea and potato have become interesting experimental candidates. These plant-based materials offer a new supplementation source and performance characteristics. In an effort to aggressively expand our non-animal product offering, BD has created a Developmental Peptone Sample Program. The program identifies new plant-based peptone candidates and creates a process for you to sample them, in exchange for your feedback on their performance in your system. Soy Peptones, the first successful plant-based peptone to be optimized into cell culture, are processed in several different ways to provide various nutrient mixes. Yeast Extracts and Tissue Culture (TC) Yeastolates offer a different range of nutritional choices to enhance production in both bacterial fermentation and as a supplement in cell culture.
Soy Peptones Soy Peptones are manufactured in the more traditional peptone manner as described in the “Hydrolysis to Hydrolysate” section and the “Meat Peptone” section. The BD Difco™ Soy Peptones are all enzymatic digests of soy flour. Since soy contains a tryptic inhibitor, before digestion begins the soy flour is thoroughly washed to eliminate the inhibitor. Soy flour, the principle substrate in Soy Peptone, is rich in high-quality protein, carbohydrates, calcium and B vitamins.1 The enzymes used in the digestion of soy flour should be from animal-free sources or sourced from microorganisms that have been grown in animal-free media. Sometimes the organism from which the enzymes have been sourced has been stored with bovine serum albumin (BSA). All of these factors should be taken into consideration when selecting for animal-free peptones.
Individual Animal-Free Media Components Yeast Products Yeast extract is defined in the USP as “a water-soluble, peptone-like derivative of yeast cells (Saccharomyces).”2 Yeast extract is readily available in the U.S. as a spray-dried powder. In Europe, pharmaceutical companies use it as a liquid or paste, as well as in the powdered form. Yeast extract is used by the health food industry as an inexpensive source for many of their vitamins. Yeast extract has long been recognized as a major source of B-complex vitamins. Yeast extract, as a substrate in a media formulation, supplies not only vitamins, but also proteins, carbohydrates and some micronutrients. There are many kinds of yeast extract. The two principle sources of yeast extract are “brewer’s” yeast and “baker’s” yeast. Brewer’s yeast is a by-product from the brewing industry. It requires de-bittering (removal of hop resins) before it is suitable for fermentation use.3 A wide variety of strains and growth processes have been used in the manufacture of brewer’s yeast, thus precluding any inconsistency of the final product. Baker’s yeast (Saccharomyces cerevisiae) is defined as a primary yeast because the yeast is grown for the specific purpose of being used as a substrate in a bioprocess or as a food product/flavoring. Manufacture of baker’s yeast is a reproducible and controlled process. The yeast organism is grown on a molasses-based medium optimized for the specific yeast.4 Commercial yeast fermentations are always fed-batch type fermentations lasting from 12-20 hours.5 Commercial baker’s yeast manufacturers have found that the more highly aerated a culture, the higher the final product yield.5 The process of manufacturing baker’s yeast extract is unique compared to the manufacture of other peptones. Yeast extract is an autolysate; i.e. cell hydrolysis is performed by the endogenous enzymes of the Saccharomyces organism. Autolysis is usually begun by either a controlled temperature shock or, for the food industry, an osmotic shock, which causes the yeast cells to expire. The temperature shock is not high enough to inactivate the proteases of the yeast cell, which proceed to degrade the cell. Autolysis can proceed from 10 to 60 hours. After autolysis, soluble material is separated from insoluble by means of centrifugation and several filtration steps.6 The final filtration product is concentrated and then spray dried, or can be left in the concentrated paste form, which contains approximately 60-80% solids. Temperature, pH, addition of other enzymes, type of medium substrate for the growth of the Saccharomyces and duration of autolysis are all variables that create the large variety of yeast extracts available.
References 1. Human Nutrition Service, U.S. Department of Agriculture. 1986. Composition of foods: legume and legume products. Agriculture handbook, No. 8-16, revised. U.S. Department of Agriculture, Washington, D.C. 2. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/ The national formulary 20–2001. p.2337. United State Pharmacopeial Convention, Inc., Rockville, Md. 3. Bridson and Brecker. 1970. Design and formulation of microbial culture media. In Methods in microbiology, volume 3A, Academic Press, p.252-56. 4. http://www.ohly.ed/sommer.htm. 5. Reed and Nagodawithana. Yeast technology, 2nd ed., p.264-267. Van Nostrand Reinhold, New York.
Product Description Bacto™ Malt Extract is the water-soluble portion of malted barley. The extraction process breaks down the polysaccharides into simple sugars. After the malting process is complete the extract is prepared from the malted barley by cracking the grain in a mill and then extracting the grain with a warm liquor. The resulting “wort” is filtered and evaporated or dried under vacuum.1,2
Applications Bacto™ Malt Extract is used in the culture of yeasts and molds. Bacto™ Malt Extract is very high in carbohydrate content.3 This product is suitable for the culture of yeasts and molds because of the high concentration of reduced sugars, especially the maltoses. Malt extract in the agar form is recommended for the detection and isolation of yeasts and molds from dairy products and foods and as a medium for stock culture maintenance.
Physical Characteristics Bacto™ Malt Extract is a medium tan, free-flowing, homogeneous powder.
Availability Bacto™ Malt Extract 218630, 500 g Bacto™ Malt Extract 218610, 10 kg
References 1. Bridson and Brecker. 1970. In Norris and Ribbons (ed.), Methods in microbiology, vol. 3A, p. 256. Academic Press, New York. 2. http://www.briess.com/HomebrewNew/hbhow.htm 3. Cote. 1999. In Flickinger and Drew (ed.), Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation, p. 1652. John Wiley & Sons, Inc., New York.
BACTO™ MALT EXTRACT
Product Description All of the Difco™ and BBL™ brand soy peptones are enzymatic digests of soybean meal. They are recommended for use in media for the cultivation of a large variety of organisms, including fungi. The nitrogen source in the soy peptones contains naturally occurring high concentrations of vitamins and carbohydrates of soybean.
Applications Phytone™ Peptone is an animal-free soy peptone. Phytone™ Peptone retains the high vitamin and high carbohydrate content of the soy plant tissue. It is an excellent plant peptone for the cultivation of fungi and fastidious types of bacteria, such as members of the Clostridium and Neisseria genera.1 It has been used in cell culture applications due to its high carbohydrate content.
PHYTONE™ PEPTONE PHYTONE™ PEPTONE UF SELECT SOYTONE BACTO™ SOYTONE Phytone™ Peptone UF is an ultra-filtered peptone that was developed specifically for the tissue culture market. Its nitrogen content combined with the naturally occurring vitamins has demonstrated remarkable growth support with monoclonal antibodies and protein expression. It has an endotoxin level of less than or equal to 500 EU/g. Select Soytone demonstrates excellent growth support for E. coli. Select Soytone is also used in Select APS™ Super Broth. Subtle differences in the hydrolysation process give Select Soytone improved performance in cell culture (see page 8). Bacto™ Soytone was found to be effective in the recovery of stressed E. coli.2 It was found that Bacto™ Soytone with the addition of 7 vitamins replaced yeast extract as an economical alternative for the production of lactic acid by Lactobacillus rhamnosus.3 It should be noted that Bacto™ Soytone utilizes an enzyme in the digestion of the soy flour that has been sourced from animal.
Physical Characteristics Phytone™ Peptone is a tan, free-flowing, homogeneous powder. Phytone™ Peptone UF is a tan, free-flowing, homogeneous powder. Select™ Soytone is a tan, free-flowing, homogeneous powder. Bacto™ Soytone is a tan, free-flowing, homogeneous powder.
Availability Phytone™ Peptone 211906, 454 g Phytone™ Peptone 298147, 5 lb Phytone™ Peptone 292450, 10 kg Phytone™ Peptone UF 210931, 500 g Phytone™ Peptone UF 210936, 10 kg Select™ Soytone 212488, 500 g Select™ Soytone 212489, 10 kg Select™ Soytone 292725, LitrePak™ 20 x 1.0 L Bacto™ Soytone 243620, 500 g Bacto™ Soytone 243610, 10 kg
References 1. Power (ed.), 1988. Manual of BBL® products and laboratory procedures, 6th ed., p.293. Becton Dickinson Microbiology Systems, Cockeysville, Md. 2. Chou and Cheng. 2000. Recovery of low-temperature stressed E. coli O157:H7 and its susceptibility to crystal violet, bile salt, sodium chloride and ethanol. Int. J. Food Microbiol. 61:127-136. 3. Kwon, Lee, Lee, Chang, Keun and Chang. 2000. Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate. Enzyme Microb. Technol. 26:209-215.
Product Description Both TC Yeastolate products are animal-free water-soluble portions of autolyzed yeast or S. cerevisiae. Each TC Yeastolate is a mixture of peptides, amino acids, carbohydrates, simple and complex as well as vitamins. TC Yeastolate, UF has been ultra-filtered at a 10,000 MWCO. It has an endotoxin value under 500 EU/g.
Applications Both TC Yeastolate products are intended as nutritional supplements for bacterial, insect and mammalian cell culture. For years, TC Yeastolate has been used in insect cell nutrition. TC Yeastolate was found to be a very versatile supplement to enhance growth and production characteristics of Sf9 and High-Five cells.1-5
Physical Characteristics Bacto™ TC Yeastolate is a free-flowing, spray-dried powder. TC Yeastolate, UF is a free-flowing, spray-dried powder.
BACTO™ TC YEASTOLATE TC YEASTOLATE, UF
Availability Bacto™ TC Yeastolate 255772, 100 g Bacto™ TC Yeastolate 255771, 10 kg TC Yeastolate, UF 292804, 500 g TC Yeastolate, UF 292805, 10 kg
References 1. Chan, Greenfield and Reid. 1998. Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system. Biotechnol. Bioeng 59:178-188. 2. Nguyen, Jarnagin, Williams, Chan and Barnett. 1993. Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system. J Biotechnol. 31:205-217. 3. Ikonomou, Bastin, Schneider, Agathos. 2001. Design of efficient medium for insect cell growth and recombinant protein production. In Vitro Cell Dev. Biol. Anim. 37:549-559. 4. Bedard, Kamen, Tom and Maassie. 1994. Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures. Cytotechnology 15:129-138. 5. Donalson and Shuler. 1998. Low-cost serum-free medium for the BTI-TN5B1-4 insect cell line. Biotechnology Prog. 14:573-579.
Product Description All of the BD Yeast Extracts are concentrates of the water soluble portion of S. cerevisiae cells that have been autolyzed. BD Yeast Extracts are derived from primary grown baker’s yeast. Yeast extract is considered a non-animal product and is used extensively for many non-animal formulations for bacterial, fungal, mammalian and insect cell culture. Difco™ Yeast Extracts have been considered one of the most complete and versatile of the fermentation bionutrients available.
Applications BD Yeast Extracts are animal-free products to be used as multi-functional nutritional supplements in cell culture, microbial fermentation and insect cell culture applications. Bacto™ Yeast Extract will provide essential water soluble vitamins, amino acids, peptides and carbohydrates to any medium formulation.
BACTO™ YEAST EXTRACT YEAST EXTRACT YEAST EXTRACT, UF YEAST EXTRACT, LD Yeast Extract, UF is ultra-filtered and specifically designed for tissue culture applications. With its low endotoxin level and high content of naturally occurring B vitamins, it is an ideal substitute for fetal bovine serum. It has an endotoxin level of less than or equal to 500 EU/g. Yeast Extract, LD was created to eliminate the problem of dust inhalation when handling large quantities of yeast extract. Yeast Extract, Yeast Extract UF, and Yeast Extract LD are processed from the same culture of Saccharomyces. Yeast Extract was developed to provide a product priced for the biotechnology/ pharmaceutical market with growth characteristics to match Bacto™ Yeast Extract.
Physical Characteristics All of the BD Yeast Extracts are free-flowing, spray-dried powders.
Availability Bacto™ Yeast Extract 212750, 500 g Bacto™ Yeast Extract 212720, 2 kg Bacto™ Yeast Extract 212730, 10 kg Yeast Extract 211929, 454 g Yeast Extract 211930, 5 lb Yeast Extract 211931, 25 lb Yeast Extract, UF 210929, 500 g Yeast Extract, UF 210934, 10 kg Yeast Extract, LD 210933, 500 g Yeast Extract, LD 210941, 10 kg
Product Description The Select APS™ media line is manufactured from non-animal origin ingredients. These Alternative Protein Source (APS) media are nutrient-rich formulations designed to outperform classical meat-based formulations (see growth curves below).
Applications Select APS™ Super Broth is a molecular genetic medium that will grow E. coli to a high cell density. There is no glucose in the formulation thus preventing acetate build-up in the fermentation of the organism.1 Select APS™ LB Broth is an excellent all-purpose growth medium for the propagation and maintenance of E. coli in molecular biology procedures.
SELECT APS™ SUPER BROTH SELECT APS™ LB BROTH
Physical Characteristics Select APS™ Super Broth is a tan, free-flowing powder. Select APS™ LB Broth is a tan, free-flowing powder.
Availability Select APS™ Super Broth 212485, 500 g Select APS™ Super Broth 212486, 10 kg Select APS™ Super Broth 292713, LitrePak™ 20 x 1.0 L Select APS™ LB Broth 292438, 500 g Select APS™ LB Broth 212484, 10 kg Select APS™ LB Broth 292724, LitrePak™ 20 x 1.0 L
Reference: 1. Swartz. 2001. Advances in Escherichia coli production of therapeutic proteins. Curr. Opinion Biotechnology 12:195-201.
NOTES
NON-ANIMAL PEPTONE AND YEAST EXTRACTS TYPICAL ANALYSES Analytical tests used in data gathering for this manual are described below. The AN/TN ratio gives an estimate of the degree of protein hydrolysis. Ash values were measured after a minimum of 4 hours of heating at 600°C. Ash values refer to the non-combustible portion of the sample and roughly correspond to the mineral content of the sample. Carbohydrate percentage was calculated by the AOAC method.1 Chloride, Sulfate and Phosphate percentages were determined by ion chromatography. Elemental analysis was determined by ICP (Inductively Coupled Plasma) using a Thermo Jarrell Ash instrument. Endotoxin values were determined by a quantitative kinetic chromogenic method. Free Amino Acids are defined as amino acids that are not part of a protein or peptide chain. The amino acids were measured using the Waters AccQ•TagTM Method. The AccQ•TagTM Method is based on the derivatizing reagent 6-aminoquinolyl-Nhydroxysuccinimide- activated heterocyclic carbamate. Labsystems BioScreen C is a 200-well incubating kinetic optical density reader. Media were inoculated with approximately 100 CFU per 200 µL fill in each well. OD readings were averaged from 4 duplicate wells. L.O.D. (Loss on Drying) test procedure is based on the method described in The United States Pharmacopeia.2 There are some modifications to the procedure. Molecular weight distribution was determined by size-exclusion chromatography using a silica-based column and a SDS/phosphate buffer mobile phase. pH was measured in a 2% solution after autoclaving. Sodium Chloride was determined by silver nitrate/potassium thiocyanate titration method. Total Amino Acids were measured by the same method as the Free Amino Acids after an acid hydrolysis at 110°C for 20 hours. Cystine and tryptophan are destroyed during the hydrolysis. The cystine and tryptophan values are not reported for Total Amino Acids.
References: 1. Horowitz (ed.). 2000. Official methods of analysis of AOAC International, 17th ed, vol II, section 50, pg. 18. AOAC International, Gaithersburg, Md. 2. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/The national formulary 20—2002. United States Pharmacopeial Convention, Inc., Rockville, Md.
DEFINITION OF METHODS
REGULATORY DOCUMENTATION BD prides itself on the investment it has made in regulatory compliance, based on government agency guidance and customer feedback. Our strong commitment to quality products, reliably delivered with the appropriate documentation, has resulted in the provision of the following services.
Certificates of Analysis/Certificates of Origin Certificates of Analysis (C of A) are available on all production products and include Certificates of Origin (C of O) when appropriate (see exhibit 1). As a leader in manufacturing and sourcing meat-based products, BD has invested in a very intensive documentation program. In order to streamline the communication and transmission of C of A and C of O information, BD operates a Certificates On-line System on the Internet, 24 hours a day, 7 days a week. With specific lot number information, you can access certificates on the BD web site at www.bdregdocs.com.
Drug Master Files (DMF) BD maintains Drug Master Files (DMF) on certain key proprietary products used in the manufacture of bio-therapeutics. A DMF is a submission to the Food and Drug Administration (FDA) that contains confidential information on the manufacturing, process and packaging of a raw material used in the production of a drug. The information contained in the DMF may be used to support an Investigational New Drug Application (IND). The FDA reviews DMF information upon written request by the DMF holder in support of another regulatory application. For more information on DMF availability and permission to reference, please contact your local BD representative.
Change Notification Program BD offers an Automated Change Notification Program to customers who require notification of agreed-upon manufacturing and process changes. The program provides greater assurances that these changes are occurring under our ISO-certified Quality Systems. To request a Change Notification Program packet, please e-mail ProductInfo@bd.com.
Certificates of Suitability BD participates in the European Pharmacopeia program for Certificates of Suitability, for animal derived products. Under the procedure, based on Resolution of the Public Health Committee (Partial Agreement, Resolution AP-CSP (99) 4), BD has applied for certificates concerning: evaluation of the suitability of the control of the chemical purity and microbiological quality of the substance according to the corresponding specific monograph; or the evaluation of reduction of Transmissible Spongiform Encephalopathy (TSE) risk. For a complete list of products certified or under application, please contact your local BD representative.
PRODUCT LISTING Product Name 500 g 454 g 10 kg 25 kg 25 lb Acidicase™ Peptone 211843 Beef Extract, Desiccated, Bacto™ 211520 Beef Extract, Powder 212303 Biosate™Peptone 211862 294312 Brain Heart Infusion, Bacto™ 237500 237300 Brain Heart Infusion, Porcine, Bacto™ 256120 256110 Casamino Acids, Bacto™ 223050 223030 Casamino Acids, BiTek™ 252920 Casitone, Bacto™ 225930 225910 Gelysate™ Peptone 211870 Malt Extract, Bacto™ 218630 218610 Neopeptone Peptone, Bacto™ 211681 211680 Peptone, Bacto™ 211677 211830 Peptone, BiTek™ 254820 Phytone™Peptone 211906 292450 Phytone™ Peptone, UF 210931 210936 Polypeptone™ Peptone 211910 297108 Proteose Peptone No. 2, Bacto™ 212120 212110 Proteose Peptone No. 3, Bacto™ 211693 212230 Proteose Peptone No. 3, BiTek™ 253720 Proteose Peptone No. 4, Bacto™ 211715 Proteose Peptone, Bacto™ 211684 212010 Proteose Peptone, BiTek™ 253310 Select APS™ LB Broth 292438 212484 Select APS™ Super Broth 212485 212486 Select Soytone 212488 212489 Soytone, Bacto™ 243620 243610 TC Yeastolate, Bacto™ 255771 TC Yeastolate, UF 292804 292805 Thiotone™ E Peptone 212302 Trypticase™ Peptone 211921 211923 Tryptone, Bacto™ 211705 211701 Tryptone, BiTek™ 251420 Tryptose, Bacto™ 211713 211709 Yeast Extract, Bacto™ 212750 212730 Yeast Extract 211929 211931 Yeast Extract, LD 210933 210941 Yeast Extract, UF 210929 210934
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