Evaluation of peptones from chicken waste as a nitrogen source for micro-organisms

In this study, chicken peptone was produced by hydrolysing inedible parts derived from chickens using endo-protease and exo-protease. The usefulness of chicken peptone as a nutrient source for bacteria was evaluated in comparison with other commercially produced peptones (animal, soy and casein-derived peptone). Escherichia coli and Bacillus subtilis were used as test strains to determine the effect of peptones from different sources on their growth ability. Both bacteria were successfully cultured in chicken peptone solution, which is similar to peptone solution containing commercial peptones apart from animal peptone. In chemical analysis, chicken peptone contained 12·0% nitrogen; this was similar to the nitrogen content from other commercial peptone sources, except for the 9·0% nitrogen found in soy peptones. The molecular weight of the peptone was determined by gel filtration chromatography, and those of all peptone, except animal-derived peptone, were found to be <5000 Da. In addition, when B. subtilis was cultured in a medium containing chicken peptone, it was shown that the protease activity was highest as compared with other commercial peptones. From these results, it is suggested that chicken peptone can be utilized for microbial culture, and this is an effective method to reuse chicken waste.     

Peptones from diverse sources: pivotal determinants of bacterial growth dynamics

Aims: In view of the major problems encountered by microbiologists in obtaining reproducible data on growth dynamics in complex media, we studied the effects of different peptones made from different biological sources and produced by numerous manufacturers. Methods and Results: Peptones (including casein, gelatin, meat, soy and yeast) were assessed as a constituent of the pre‐enrichment broth buffered peptone water (BPW). Generation times (g) and yields of Salmonella serovar Typhimurium were significantly affected by the type of peptone employed with yeast peptones generating yields of 7·04 × 10⁹ CFU ml^?1 and gelatin peptones producing 0·81 × 10⁹ CFU ml^?1. Medium sterilization was also found to have significant effects (P = 0·000) upon subsequent bacterial growth. Filter sterilization of BPW media produced lower generation times compared with those obtained after sterilization by autoclaving. Finally, it was observed that some peptones which produced good growth when inoculated with healthy organisms, showed relatively poor growth when inocula were sublethally injured by heating. Conclusions: Variation in peptone as a constituent of BPW has a significant effect on growth and enumeration of bacteria. Significance and Impact of the Study: Increased consideration with respect to culture media may significantly improve bacterial growth and experimental reproducibility.     

Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970

Several varieties of peptone supported growth of A. culbertsoni to different extents reaching a maximum cell density of 1-2 X 10(6)/ml. Proteose peptone and tryptone also yielded good growth when combined with thiamine and vitamin B12. A combination of proteose peptone with glucose, yeast extract and salts promoted excellent growth of A. culbertsoni with cell density reaching 1-2 X 10(7) cells/ml; tryptone and one of the indigenous peptones also yielded comparable growth when substituted for proteose peptone in this medium. Casamino acids also supported good growth of amoebae and requirement of yeast extract could be met by a combination of thiamine, vitamin B12 and biotin. Bacto peptone did not support good growth of this amoeba but supplementation of peptone with casamino acids or amino acid mixture improved the growth supporting capacity of the medium. Development of several media with or without glucose will aid in cultivation of A. culbertsoni, studies on its metabolism as well as screening of potential drugs.     

Hydrophobic peptide auxotrophy in Salmonella typhimurium.

The growth of a pleiotropic membrane mutant of Salmonella typhimurium with modified lipopolysaccharide composition was found to be strictly dependent on the peptone component of complex media. Nutritional Shiftdown into minimal media allowed growth for three to four generations. Of 20 commercial peptones, only enzymatic digests supported growth to varying degrees. Neither trace cations, amino acids, vitamins, carbohydrates, lipids, glutathione, polyamines, carbodimides, nor synthetic peptides stimulated growth; however, cells still metabolized carbohydrates, and amino acid transport systems were shown to be functional. A tryptic digest of casein was fractionated into four electrophoretically different peptide fractions of 1,000 to 1,200 molecular weight which supported growth to varying degrees. The best of these was further fractionated to two highly hydrophopic peptides. N-terminal modifications eliminated biological activity. Fluorescein-conjugated goat antibody to rabbit immunoglobulin G was used as a probe to detect antipeptide antibody-peptide complexes on membrane preparations. Cells grown on peptone distributed the peptide into both inner and outer membranes. The peptide could be removed with chaotropic agents, and cells had to be pregrown in peptone-containing media to bind the hydrophobic peptide. The gene (hyp) responsible for peptide auxotrophy was mapped at 44 to 45 units by conjugation.     

Aqueous two-phase systems: a novel approach for the separation of proteose peptones

Poly(ethylene glycol) and dextran aqueous two-phase systems (ATPS) were developed to facilitate the separation of components of the proteose peptone fraction of bovine milk, which are mostly large casein derived peptides or glycoproteins. These have proved difficult to purify using conventional chromatographic procedures. ATPS exploit differences in hydrophobicity, size and ionic properties of the proteose peptones with a view to developing methods for future large scale preparations of the individual components of this whey protein fraction.     

Ram horn peptone as a source of citric acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis>, with a process

The present study deals with the production of citric acid from a ram horn peptone (RHP) by Aspergillus niger NRRL 330. A medium from RHP and a control medium (CM) were compared for citric acid production using A. niger in a batch culture. For this purpose, first, RHP was produced. Ram horns were hydrolyzed by treatment with acids (6 N H₂SO₄, 6 N HCl) and neutralizing solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined. RHP was compared with peptones with a bacto-tryptone from casein and other peptones. The results from RHP were similar to those of standard peptones. The optimal concentration of RHP for the production of citric acid was found to be 4% (w/w). A medium prepared from 4% RHP was termed ram horn peptone medium (RHPM). In comparison with CM, the content of citric acid in RHPM broth (84 g/l) over 6 days was 35% higher than that in CM broth (62 g/l). These results show that citric acid can be produced efficiently by A. niger from ram horn.     

Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-γ-producing CHO cell line

A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-gamma (IFN-gamma), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-gamma secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-gamma production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety.     

Microbial growth on peptones from fish industrial wastes

Industrial fish peptone was an excellent substrate for biomass production in solid and submerged fermentations. The maximal growth rates of several microorganisms were two to three times higher than those grown on beef (bacto)peptones and the final biomass concentrations were almost twice as great as those grown on beef peptones. Fish peptones did not increase the production of secondary metabolites relative to those produced on beef peptones in non-optimized media. Fish peptone has promising potential as a substrate for biomass production.     

Growth of Lactobacillus plantarum in media containing hydrolysates of fish viscera

AIMS: To compare growth of Lactobacillus plantarum on media containing hydrolysates (peptones) from cod viscera with growth on commercial media. METHODS AND RESULTS: Growth of Lact. plantarum on various fish peptones and commercial peptones/extracts was evaluated using both a Bioscreen apparatus (microtiter plates, no pH control) and fermentors (with pH control). Generally, the performance of the fish peptones was good and only beaten by the performance of yeast extract. Replacement of the 22 g l(-1) complex nitrogen source in standard MRS medium with only 5 g l(-1) fish peptone reduced the biomass yield with only 10%, whereas replacement with a mixture of 2.5 g l(-1) fish peptone and 2.5 g l(-1) yeast extract increased the biomass yield by 10%. CONCLUSIONS: Peptones derived from cod viscera support excellent growth of Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: We show that peptones derived from cod viscera are promising constituents of growth media for fastidious food bacteria such as lactobacilli. Media containing these peptones show excellent performance while problems associated with the use of meat-derived peptones (BSE, kosher status) or plant-derived peptones (genetically modified organisms) are avoided.     

Extracellular acid proteases produced by Saccharomycopsis lipolytica.

Saccharomycopsis lipolytica CX161-1B produced at least three extracellular acid proteases during exponential growth in medium containing glycerol, Difco Proteose Peptone, and mineral salts at pH 3.4 (Difco Laboratories, Detroit, Mich.). Little extracellular acid protease activity was produced with glutamic acid as the sole nitrogen source, somewhat higher levels were obtained with peptone, and much higher levels were obtained with Difco Proteose Peptone. The relative amounts of the three proteases varied during growth on Difco Proteose Peptone, which suggested that the proteases were not coordinately regulated. The proteases were purified to near homogeneity (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) by use of ultrafiltration, gel filtration, and DEAE-Sephacel and hydroxylapatite chromatography. Protease I had a molecular weight near 28,000, an isoelectric point of pH 4.9, and a pH optimum of 3.5. Protease II had a molecular weight near 32,000 and a pH optimum of 4.2. Protease III had a molecular weight near 36,000, an isoelectric point of 3.8, and a pH optimum of 3.1. All three proteases were glycoproteins; proteases I, II, and III contained 25, 12, and 1.2% carbohydrate, respectively. The proteases were inhibited by pepstatin and 1,2-epoxy-3-(4-nitrophenoxy) propane and were largely insensitive to diazoacetyl-DL-norleucine methylester and to compounds which inhibit the serine, sulfhydryl, or metallo-proteases.     

The non-nutritional performance characteristics of peptones made from rendered protein

Economic considerations require the use of inexpensive feedstocks for the fermentative production of moderate-value products. Our previous work has shown that peptones capable of supporting the growth of various microorganisms can be produced from inexpensive animal proteins, including meat and bone meal, feather meal, and blood meal, through alkaline or enzymatic hydrolysis. In this work, we explore how these experimental peptones compare to commercial peptones in terms of performance characteristics other than chemical make-up; these characteristics can impact fermentation operating cost. It is shown that experimental peptone powders produced through enzymatic hydrolysis are highly hygroscopic and that their physical form is not stable to humid storage conditions; those produced through alkaline hydrolysis and commercial peptones are less hygroscopic. When used in growth medium, all peptones contribute haze to the solution; experiments show that the source of haze is different when using enzyme- versus alkali-hydrolyzed peptones. Alkali-hydrolyzed peptones and all peptones made from blood meal are stronger promoters of media foaming than the commercial peptones; some enzyme-hydrolyzed peptones support very little foam formation and are superior to the commercial peptones in this sense. Alkali-hydrolyzed peptones are roughly equivalent to commercial peptones in the coloration they contribute to media, while enzyme-hydrolyzed peptones contribute intense coloration to media. No peptone caused a significant change in the viscosity of media. The experimental peptones studied here may be acceptable low-cost substitutes for commercial peptones, but none is equivalent to the commercial products in all respects.     

Effect of medium on the bacteriocin production byKlebsiella pneumoniae

The influence of growth media and media constituents on bacteriocin production byKlebsiella pneumoniae was studied. Among the standard laboratory media used trypticase soy broth (TSB) showed the maximum production and poor yields resulted from growth in peptone water and nutrient broth. A number of peptones differed in their bacteriocin production. Best yields were observed in tryptone and proteose peptone water. Addition of 1 % yeast extract to TSB further stimulated bacteriocin production. However, activity was low when glucose, glycine, sodium mercaptoacetate or bile salt mixture were added to the medium. Supression of synthesis by certain agents as well as inhibition of formed bacteriocin by the others appears to affect the bacteriocin yield. No proteinase activity was detected during the entire incubation period.     

P-Factor,a New Peptidic Growth Factor for Pediococcus soyae

The P-factor a new growth factor for Ped. soyae, is found in partial acid and enzymic hydrolysates of Hammersten’s milk casein, and is therefore, supposed to be a kind of peptide. The well-known peptidic growth promoting substance for Lact. casei, strepogenin and its relating substances, are quite ineffective to the author’s organism. P-factor exists along with the S-factor which is another growth promoting-factor for this lactic acid bacterium, in soy mash juice and in the water extract of Aspergillus sojae mycelium. The former substance is also found in various kinds of commercial peptones. The factor was not substitutable with all of the 19 synthetic peptides tested. S-factor is effective, only in the presence of the P-factor.     

Optimization of Cultivation Medium for the Production of Bacillus intermedius 3-19 Glutamyl Endopeptidase

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase.     

Effects of medium composition on the recovery of bacteria from sea water

Water samples were collected from offshore and inshore localities at various depths off the Connecticut coast over a two-year period. Spread plates for bacterial counts at 20 °C were made on a variety of complex solid media. Counts on Difco-Marine Agar were controls in all cases with counts on test media related to these in ratio form. Initially, nine media were used and represented some from the literature as well as personal formulations. Differences between inshore and offshore samples were greatest with media containing the highest peptone concentrations. Two media containing the peptones Gelysate and Trypticase showed the highest overall counts. A second phase concerned a comparative study of these peptones varying in concentration from 0.1 to 10.0 g/l in a constant basal medium. None of the media invariably gave counts greater than the control, but peptone concentrations of 10.0 and 5.0 g/l resulted in the lowest comparative counts. Considering all samples, peptone levels of 0.1 and 1.0 g/l showed the highest counts. Counts for both inshore and offshore water samples decreased as peptone concentration increased. Qualitatively, high peptone media showed large, mucoid, confluent colonies which made the counting of smaller ones difficult. Pigmented colonies were more frequent on low peptone media. Bacteria were isolated from all media and from all stations; the percentage of various groups varied with peptone concentration and source of sample.Media containing three fish peptones in varying concentrations have also been investigated. None produced overall counts greater than Difco-Marine Agar and counts decreased with increasing peptone levels: there was a trend towards higher counts in offshore waters with fish extracts. Quantitative and qualitative aspects of the work are discussed.     

Optimization of microbial transglutaminase production using experimental designs

In prokaryotes, transglutaminase (TGase) has been found only in actinomycetes from the genus Streptoverticillium. The role of this TGase, as well as the mechanism regulating the enzyme expression, are still unknown. In order to improve TGase production by Streptoverticillium cinnamoneum CBS 683.68 and simultaneously elucidate the relationship between growth and TGase activity, we decided to study these two responses using different designs of statistical analysis. Among the five factors tested, casein, glycerol, peptones, yeast extract and oligoelements, only oligoelements were found to have no effect either on growth or on TGase production in a complete factorial design. The two factors casein and glycerol were found to have a highly significant effect on both dry weights and TGase activity in a Box-Behnken design used to improve the model. Finally, the TGase activity was increased three times to reach 0.331±0.038 U/ml with optimum concentrations of casein (38.4 g/l) and glycerol (31.2 g/l) calculated with the help of a composite design. In the course of these experiments, the two responses varied in the same way, demonstrating that growth and TGase production were tightly correlated under the conditions described. However, TGase was produced during the stationary phase of growth in optimized medium, indicating that the enzyme production could be induced. Received: 23 July 1997 / Accepted: 25 August 1997     

Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones

Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations<18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.     

Crude peptones from cowtail ray (Trygon sephen) viscera as microbial growth media

In the processing of cowtail ray (Trygon sephen) in Indonesia, viscera (up to 20% body weight) is wasted together with the head, frame and skin. A series of studies have been carried out to investigate the utilization of the viscera, and the present paper reports the conversion of the viscera into microbiological peptones. Ensilation using 3% (v/w) mixture of propionic and formic acids (1:1, v/v), followed by vacuum evaporation, has been used to prepare crude liquid peptones from cowtail ray viscera. These peptones were compared to three commercial peptones in supporting the growth of microorganisms. Mixed populations of microorganisms from foods (beef, egg and milk) and selected pure microorganisms (Aspergillus flavus, Bacillus subtilis, Saccharomyces cerevisiae, Escherichia coli and Staphylococcus aureus) were grown on liquid media containing 0.5 g test peptones/100 ml and the optical densities were monitored. The biomass produced was measured at the end of incubation period. The results show that crude peptones from cowtail ray viscera performed similar to or even better than commercial peptones as nitrogen sources for microorganisms growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Peptones and mycological reproducibility.

The pH reactions, ultraviolet spectra and phosphorus content of solutions of a variety of commercially available peptones all indicated, predictably, considerable differences in the chemical composition of the peptones. The effects of these differences on the outcome of experiments with Candida albicans grown in different peptone media was investigated. The fungus produced germ tubes equally effectively on all such media provided that the inoculum was kept to 10(6) blastospores/ml or less. However, expression of inducible enzyme activities in C. albicans varied extensively from peptone to peptone; there was, for example, an inverse relationship between the inorganic phosphorus content of peptones and the amount of acid phosphomonoesterase detectable in intact blastospores. The results indicated that use of different peptones in "Sabouraud's" media by different laboratories may account for some, but not all, published instances of irreproducibility of experiments with C. albicans.     

Assimilation of peptides and amino acids and dissimilation of lactate during submerged pure cultures of Penicillium camembertii and Geotrichum candidum

The behavior of Penicillium camembertii and Geotrichum candidum growing in submerged pure cultures on simple (glutamate) or complex (peptones) substrates as nitrogen and carbon sources and an lactate as a second carbon source was examined. Similar to the behavior previously recorded on a simple substrate (glutamate), a clear differentiation between the carbon source and the energy source was also shown on peptones and lactate during P. camembertii growth, since throughout growth, lactate was only dissimilated, viz., used for energy supply by oxidation into CO2, whereas peptides and amino acids from peptones were used for carbon (and nitrogen) assimilation. Because of its deaminating activity, G. candidum preferred peptides and amino acids to lactate as energy sources, in addition to being assimilated as carbon and nitrogen sources. From this, on peptones and lactate, G. candidum grew faster than P. camembertii (0.19 and 0.08 g/l/h, respectively) by assimilating the most readily utilizable peptides and amino acids; however, owing to its lower proteolytic activity, the maximum biomass was lower than that of P. camembertii (3.7 and 5.5 g/l, respectively), for which continuous proteolysis and assimilation of peptides were shown.