FOOD AND INDUSTRIAL MICROBIOLOGY

Module 9. Microbial process strategies

Lesson 30
BACTERIOCINS AND ANTIBIOTICS

30.1 Penicillin Fermentation

Organisms

In the early days of penicillin production, when the surface culture method was used, a variant of the original culture of Penicillium notatum discovered by Sir Alexander Fleming was employed. When however the production shifted to submerged cultivation, a strain of Penicillium chrysogenum designated NRRL 1951 (after Northern Regional Research Laboratory of the United States Department of Agriculture) discovered in 1943, was introduced. A ‘super strain’ was produced from a variant of NRRL 1951 and designated X 1612. By ultraviolet irradiation of X-1612, a strain resulted and was named WISQ 176 after the University of Wisconsin where much of the stain development work was done. On further ultra violet irradiation of WISQ 176, BL3-D10 was produced. Present-day penicillin producing P. chrysogenum strains are far more highly productive than their parents. They were produced through natural selection, and mutation using ultra violet irradiation, x-irradiation or nitrogen mustard treatment. It was soon recognized that there were several naturally occurring penicillins, viz., Penicillins G, X, F, and K. Penicillin G (benzyl penicillin) was selected because it was markedly more effective against pyogenic cocci. Furthermore, higher yields were achieved by supplementing the medium with phenylacetic acid, analogues (phenylalanine and phenethylaninie) of which are present in corn steep liquor used to grow penicillin in the United States.

Penicillin has since been shown to be produced by a wide range of organisms including the fungi Aspergillus, Malbranchea, Cephalosporium, Emericellopsis, Paecilomyces, Trichophyton, Anixiopsis, Epidermophyton, Scopulariopsis, Spiroidium and the actionomycete, Streptomyces.

Fermentation for penicillin production

The inoculum is usually built up from lyophilized spores or a frozen culture and developed through vessels of increasing size to a final 5-10% of the fermentation tank. As the antibiotic concentration in the fermentation beer is usually dilute the tanks are generally large for penicillin and most other antibiotic production. The fermentors vary from 38,000 to 380,000 liters in capacity and in modern establishments are worked by computerized automation, which monitor various parameters including oxygen content, Beta-lactam content, pH, etc.

The medium for penicillin production now usually has as carbohydrate source glucose, beet molasses or lactose. The nitrogen is supplied by corn steep liquor. Cotton nitrogen source is sometimes exhausted towards the end of the fermentation and it must then therefore be replenished. Calcium carbonate or phosphates may be added as a buffer. Sulfur compounds are sometimes added for additional yields since penicillin contains sulfur. The practice nowadays is to add the carbohydrate source intermittently, i.e. using fed-batch fermentation. Lactose is more slowly utilized and need not be added intermittently. Glucose suppresses secondary metabolism and excess of it therefore limits penicillin production. The pH is maintained at between 6.8 and 7.4 by the automatic addition of H₂SO₄ or NaOH as necessary. Precursors of the appropriate side-chain are added to the fermentation. Thus if benzyl penicillin is desired, phenylacetic acid is added. High yielding strains of P. chrysogenum resistant to the precursors have therefore been developed. 30-32°C was found suitable for the trophophase and 24°C for the idiophase. Aeration and agitation are vigorous in order to keep the components of the medium in suspension and to maintain yield in the highly aerobic fungus.

Penicillin fermentation can be divided into three phases

The first phase (trophophase) during which rapid growth occurs, lasts for about 30 hours during which mycelia are produced.

The second phase (idiophase) lasts for five to seven days; growth is reduced and penicillin is produced.

In the third phase, carbon and nitrogen sources are depleted, antibiotic production ceases, the mycelia lyse releasing ammonia and the pH rises.

Extraction of penicillin after fermentation

At the end of the fermentation the broth is transferred to a settling tank. Penicillin is highly reactive and is easily destroyed by alkali conditions (pH 7.5-8.0) or by enzymes. It is therefore cooled rapidly to 5-10°C. A reduction of the pH to 6 with mineral acids sometimes accompanied by cooling helps also to preserve the antibiotic. The fermentation broth contains a large number of other materials and the method used for the separation of penicillin from them is based on the solubility, adsorption and ionic properties of penicillin. Since penicillins are monobasic carboxylic acids they are easily separated by solvent extraction as described below.

The fermentation beer or broth is filtered with a rotary vacuum filter to remove mycelia and other solids and the resulting broth is adjusted to about pH 2 using a mineral acid. It is then extracted with a smaller volume of an organic solvent such as amyl acetate or butyl acetate, keeping it at this very low pH for as short a time as possible. The aqueous phase is separated from the organic solvent usually by centrifugation. The organic solvent containing the penicillin is then typically passed through charcoal to remove impurities, after which it is back extracted with a 2% phosphate buffer at pH 7.5. The buffer solution containing the penicillin is then acidified once again with mineral acid (phosphoric acid) and the penicillin is again extracted into an organic solvent (e.g. amyl acetate). The product is transferred into smaller and smaller volumes of the organic solvent with each successive extraction process and in this way, the penicillin becomes concentrated several times over, up to 80-100 times.

When it is sufficiently concentrated the penicillin may be converted to a stable salt form in one of several ways which employ the fact that penicillin is an acid: (a) it can be reacted with a calcium carbonate slurry to give the calcium salt which may be filtered, lyophilized or spray dried. (b) it may be reacted with sodium or potassium buffers to give the salts of these metals which can also be freeze or spray dried; (c) it may be precipitated with an organic base such as triethylamine. When benzyl penicillin is administered intramuscularly it is given either as the sodium (or potassium) salt or as procaine penicillin. The former gives high blood levels but it quickly excreted. Procaine penicillin gives lower blood levels, but it lasts longer in

the body because it is only slowly removed from the blood. It is produced by dissolving

sodium or penicillin in procaine hydrochloride.

Nisin

Biopreservation systems in foods are of increasing interest for industry and consumers. Bacteriocinogenic lactic acid bacteria and/or their isolated bacteriocins are considered safe additives (GRAS), useful to control the frequent development of pathogens and spoiling microorganisms in foods and feed. The spreading of bacterial antibiotic resistance and the demand for products with fewer chemicals create the necessity of exploring new alternatives, in order to reduce the abusive use of therapeutic antibiotics. In this context, bacteriocins are

indicated to prevent the growth of undesirable bacteria in a food-grade and more natural way, which is convenient for health and accepted by the community. According to their properties, structure, molecular weight (MW), and antimicrobial spectrum, bacteriocins are classified in three different groups: lantibiotics and nonlantibiotics of low MW, and those of higher MW. Several strategies for isolation and purification of bacteriocins from complex cultivation broths to final products were described. Biotechnological procedures including salting out, solvent extraction, ultrafiltration, adsorption-desortion, ion-exchange, and size exclusion chromatography are among the most usual methods. The best known and most characterized bacteriocin is nisin. There are 11 genes making the nisin cluster that code for nisin production, immunity, and externalization.

Nisin is a 34-amino acid antimicrobial polypeptide produced during a fermentation of Lactococcus lactis subsp. lactis. Due to its antimicrobial activity against a wide range of Gram-positive bacteria, including several major foodborne pathogens such as Clostridium and Listeria, nisin has been used extensively in the food industry as a natural food preservative. It is the only bacteriocin that is approved for food applications by FDA and produced commercially.

Nisin production is affected by several cultural factors such as producer strain, nutrient composition of media, pH, temperature, agitation and aeration, as well as the unique characteristic of nisin production, such as substrate and product inhibition, adsorption of nisin onto the producer cells, and enzymatic degradation. A dramatic decrease in nisin level after reaching the peak value was suspected to be a result of proteolytic degradation and/or adsorption of nisin by producer cells. At pH 6.80 (controlled fermentation), more than 80% of the nisin synthesized was bound to the cells, whereas at a pH below 6.0, more than 80% of the nisin was in the culture fluid.

Removal of nisin during fermentation would reduce the chance of the nisin being degraded or adsorbed and thus increase the amount of nisin that can be recovered. Although nisin production is auto-regulated with the nisin itself acting as an inducer molecule or peptide, the presence of product inhibition caused by nisin and removal of nisin from culture broth during fermentation significantly enhanced nisin production.

Purification steps of large-scale nisin production are commercially sensitive but are suspected to include foam precipitation (frothing), sodium chloride precipitation, centrifugation or ultrafiltration, and spray or drum drying. On the other hand, laboratory scale purification of nisin includes an ammonium sulphate precipication step, followed by various combinations of ion-exchange and hydrophobic interaction chromatography, with a final reverse phase-high pressure liquid chromatography purification step. immunological-based techniques have also been developed and tested like a one-step purification of nisin A using immunoaffinity purification with the specific monoclonal antibody against nisin A.

Nisin is manufactured by controlled fermentation of L. lactis in a milk-based medium at pH 2.0. Above pH 3.0 nisin adsorbs to the producer cells, but is completely desorbed at pH 3.0 or below. Consequently, at the pH of the fermentation all nisin is released into the medium, from which it can be extracted at the end of the fermentation. Solvent extraction methods have been used and a one-step immunoaffinity chromatography method is highly efficient. However, in the industrial-scale process it is concentrated and separated by a simple low-cost foaming process. This involves merely bubbling nitrogen or air through a column of the completed fermentation medium. As nisin is a surface-active agent it accumulates in the foam at the air–aqueous interface. The foam is collected, broken mechanically and the nisin recovered. It is then spray-dried before being milled into fine particles and finally standardized by the addition of sodium chloride.