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Mathematic modeling of Rennin Production by Mucor miehei
Abstract
Rennin is the most popular enzyme in the food industry as a coagulator of milk to produce cheese. An efficient large-scale fermentation for rennin production by Mucor miehei is developed by the mathematic equations to combat the need for rennin not derived from infant ruminants. Scaling-up of the fermentation process, involving calculations to derive industrial parameters from the laboratory are used extensively to determine the feasibility of the developed process. Limitations and recommendations of the design are discussed to point out the complications of assumptions and to grasp engineering ingenuity by circumnavigating such problems. Ultimately we show that using aerobic liquid fermentation in a continuously fed fermenter, 1,580,000 grams of rennin per annum (an estimated 10% of market supply) can be produced with three 80,000 liter bioreactors after inoculation with 5.2% (v/v) M. miehei. After the fermentation process, important rennin separation and purification processes are discussed: filtration, ammonium sulfate precipitation, anion-exchange chromatography and crystallization.
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The aim of this work was to study the e ffect of carbon and nitrogen sources in submerged fermentation, and the casein effect on solid-state fermentation on rennin production by Mucor miehei. Biomass peaks reached 6.7; 8.1 and 8 g/L and enzymatic production peaks of 1,066; 857 and 480 Soxhlet Units (S.U.) for glucose concentration of 18; 25 and 35 g /L respectively. Flasks with b affles s howed biomass peaks of 6.7; 8.3 and 1 0 g /L and enzyme activity peaks of 648; 279 and 300 S.U. for the same glucose concentration. The values of 923 and 667 S.U. were obtained when corn steep liquor and Proflo (Cottonseed Nutrients from Traders ®) were used. In SSF system the enzymatic activities were 414, 264 and 167 S.U., when using HCl 0.2 N, 0.3 N and 0.4 N respectively. SSF experiments using 1 and 2 g of casein/gram wheat bran (10% moisture) showed an increase in the enzymatic production (966 and 1,117 S.U.). The results suggested that increase in glucose concentration affected the enzyme synthesis, and casein was the prime factor in the enzyme synthesis induction. SSF showed to be a good system for rennin production.
This paper presents a review of the microbial enzymes used in dairy applications; primarily, milk-clotting enzymes or rennets, recombinant fungal and bacterial rennets for cheese manufacture, and fungal lactases for the manufacture of some milk products with reduced content of lactose. Other dairy enzymes include proteinases for accelerated cheese ripening for good flavour and textural development, proteases to reduce allergic properties of cow milk products for infants, and lipases for the development of lipolytic flavours in speciality cheeses.
Batch shake flask experiments were carried out with a fungal strain of Mucor miehei ATCC 3420 to study the effects of five fermentation parameters; initial D ‐glucose concentration, temperature, initial pH, agitation rate and inoculum ratio, on the production of rennin in a chemically defined medium. A statistical design was used to assign the experimental levels of five fermentation parameters for the determination of optimum conditions. The highest concentration of milk‐clotting activity (MCA) obtained was 2286 SU cm ⁻³ (without concentration of the broth) at 29.6 g dm ⁻³ initial D ‐glucose concentration, 6.8 initial pH, 37.6 °C temperature, 81 stroke per min agitation rate and 5.2% (v/v) inoculum ratio. Although enzyme secretion was a function of all the operational parameters investigated, the pH range (4.6–5.2) reached at the time of enzyme production had a profound effect on milk‐clotting activity levels. In the fermentation sample at maximum milk‐clotting activity the R factor, the ratio of milk‐clotting activity to proteolytic activity, was determined as 1 SU PU ⁻¹ , denoting similar characteristics to a commercial animal rennet.
© 2001 Society of Chemical Industry
Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.
Fungus Mucor miehei NCAIM 5238 during its growth in complex nutritive media effectively produces proteolytic enzymes, which besides hydrolitic also exhibit coagulation effects. Therefore, it is applied in industrial production of complex enzymatic preparation (renin), which is used in cheese production. The kinetics of microorganism growth and renin production were studied in a pilot-plant bioreactor of 1200 L working volume. During the batch culture the microorganism was growing in filaments and pellets, the later becoming predominant at the end of batch culture. The present work is based on experimental evidence, which indicates that the process kinetics in general could be roughly described by applying a simple mathematical model based on the three dimensional growth concept. The applicability of the model with respect to biomass growth, substrate uptake, oxygen transfer and uptake as well as the product formation kinetics was investigated. The batch culture range after 10 hours of the apparent lag phase was subjected to the study with respect to the mathematical model application. Application of the minimum variance method yielded kinetic parameters which gave a close fit between experimental and simulated data (coefficients of determination are R2x = 0.985; R2s = 0.995; R2DO = 0.981; R2P = 0.975). Computer simulation of the combination of batch and fed batch operations indicated a possible increase in renin production up to 30%.
Ten articles provide an overview of the recently flowering research into the genetics and molecular biology of fungus species that are economically significant in industry. The contributors, from a variety of disciplines, report on work in such areas as industrial enzymes, antibiotics, toxins, heter
Proteolytic activity of some milk-clotting enzymes [calf rennet (chymosin), Mucor miehei rennet, and Cryphonectria parasitica rennet] on κ-casein was determined with reverse-phase HPLC. All enzymes were standardized to the same milk-clotting activity and incubated with κ-casein at 37°C for 1, 5, 15, 30, and 60 min. Protein hydrolysis was stopped by addition of 10–5M pepstatin in 8 M urea. Chromatograms of hydrolysis products revealed that the enzymes hydrolyzed κ-casein differently. Chymosin hydrolysis was limited to formation of κ-macropeptide and para-κ-casein; the microbial rennets, particularly that from M. miehei, effected extensive nonspecific hydrolysis of both κ-casein and para-κ-casein.
Accurate estimates of differences in yield of cheese are important in experiments comparing treatments. Small differences (.l%) are economically important. Estimates of least significant difference are appropriate for evaluating those differences; expression as a percentage of yield is preferred to expression as weight. A low least significant difference is essential to prevent a Type II error in statistical inference, viz., concluding no difference when an economically important difference exists. The results of an experiment showing no effect of a treatment, but with a high LSD, are misleading. A least significant difference of ± .l% seems to be a practical lower limit, although lower values would be desirable. Important factors in accurate yield estimates are: accuracies of estimating quantities of milk and cheese; accuracy of sampling and analysis; and experimental design, replication of trials, and statistical analysis. Adjustments of yields to constant moisture (whey) and to constant fat losses in whey are useful. The significance of yield experiments should include both statistical and economical inferences; the least significant difference should be low to conclude no effect of a treatment on yield.
Fractionation on a DEAE-Sephadex A-50 column was used to study the degradation of microbial rennets derived from Mucor miehei. A comparison was made between the crude culture filtrate, derived from the growth of the microorganism in submerged culture condition on wheat bran medium, and commercial preparation Marzyme and Rennilase. The elution protein profiles lacked some of the peaks associated with the crude culture filtrate however, the milk-coagulating activity eluted out at approximately the same NaCl molarity (.45 M). Each brand name product had a characteristic elution pattern, which did not vary between their thermostable and thermolabile enzyme preparations. With storage at 4°C the elution protein profiles of the thermolabile enzyme preparations changed in a distinctive manner characteristic of the brand name. With the Marzyme series, the milk-coagulating activity eluted out at the same NaCl molarity, whereas other established peaks increased in area. The milk-coagulating activity, from Rennilase, eluted from the column at a lower NaCl concentration (.35 M). The changes were evidence of enzyme degradation, which was manifested by a general decrease in specific enzyme activity. By using column chromatography it was possible to monitor the stages of degradation with time.
The effects of temperature, pH, ionic strength, and ionic species on the stability of rennin were studied. Stability was determined by measuring decreases in clotting activity during 96 hr of incubation. Above pH 6.0, activity losses increased with pH, and were highly temperature dependent. They were accompanied by a precipitation of protein and appeared to be the result of pH denaturation. Maximum stability was maintained from pH 5.0 to 6.0. A region of instability was observed between pH 3.0 and 4.9 in buffers of 0.03 ionic strength. An instability maximum at pH 3.8 resulted in a loss of 35% of initial activity at 30 C over 96 hr. When the ionic strength was increased from 0.03 to 1.0 with NaCl, with other conditions constant, activity losses increased from 35 to 70%. Under the same conditions of pH, ionic strength, and temperature, rennin was more stable in sodium citrate, sodium lactate, and ammonium sulfate than in sodium chloride or potassium chloride. Below pH 4.5, activity losses were accompanied by an increase in the amount of Ruhemann's purple formed by the action of ninhydrin on the rennin mixture. This suggested that activity losses were due in part to self-digestion.
A milk--clotting protesse has been sep- arated in crystalline form from filtrates of Mucor michel. It is the major pro- teolytic component produced by the micro- organism, and has a molecular weight of 34,000 to 39,000. Determination of amino acid composition indicates: Asp4~, Thrls, Set25, Glu15, Ala2e, ¥a11~, l~(Cys)4, Mets, Ileu11, LeUl4, Tyr13, Phen14, Ornl, Lyss, His2, Arg4, Trya (NH3)39. The pH optima for proteolysis of hemoglobin, casein and bovine serum albumin classify the enzyme as an acid protease. The enzyme is most stable at acid pH. The effects of inhibitors indicate that this clotting enzyme is not metal dependent and does not have serine or SIt active groups. Aluminum sulfate has a peculiar depressing action on clotting activity, and study of the mechanism has pointed out similarities be- tween this M. miehei enzyme and calf rennin.
Recombinant calf prochymosin synthesized in E. coli was shown to accumulate in the form of insoluble inclusion bodies. Isolation of this aggregated material, combined with specific washing procedures, was the most significant stage of the purification protocol. Disruption of proteins in the inclusion bodies necessitated denaturation and renaturation. The method described can completely solubilize prochymosin. At this stage acidification produced active chymosin. Subsequently, ion-exchange chromatography produced highly purified prochymosin which, after acidification, yielded chymosin >99% pure.
An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.
In this study, microbial production of rennin, a milk-clotting enzyme, from a commercial strain of Mucor miehei NRRL 3420 has been investigated in a continuously fed fermenter for prolonged times. The spherical film-type growth of the culture has been accomplished in the fermenter and the effects of medium pH, mixing and dilution rates, and feed d-glucose concentration on milk-clotting activity have been elaborated. In the fermenter, optimum operational parameters have been determined as 400 rpm, 0.125 day−1, and 7.5 g l−1 for mixing rate, dilution rate, and feed d-glucose concentration, respectively. Under these conditions, the fermenter operated 575 h continuously producing 1.24 IU ml−1 maximum milk-clotting activity without concentration. In the fermenter sample at maximum milk clotting activity, the R factor and specific milk-clotting activity were determined as 1.55 × 10−3 IU PU−1 and 5.28 IU mg−1 medium protein, respectively, denoting competitive characteristics of a commercial rennet after concentration.
Production of microbial rennet, a milk clotting enzyme, from a commercial strain of Mucor miehei (NRRL 3420) was investigated in a continuously fed fermenter system for prolonged periods. The spherical film-wise growth of the culture has been accomplished and the effects of medium pH, mixing and dilution rates, and feed of D-glucose concentration on milk clotting activity was investigated. In model simulation studies, maximum milk clotting activity was generated from a multiple linear function. This was expressed in terms of fermentation medium pH, D-glucose, dissolved oxygen concentrations and dilution rate at the time of maximum milk clotting activity.
Different experiments using Mucor miehei CBS 370.65 were carried out to study the effect of agitation speed on the production of the mold acid protease. The experiments were conducted in shake flasks at a fixed substrate concentration of 58 g l−1 of total carbohydrates and at shaker speeds from 80 to 380 rev min−1. Enzyme production was found to be directly proportional to the shaker speeds, with the highest concentration of enzyme of 1,400 Soxhlet Rennet units (SU) ml−1 obtained at 380 rev min−1. The yield of product to substrate at 380 rev min−1 was determined to be 27,081.0 SU g−1 substrate and the productivity of the process was 221 SU g−1 h−1. Enzyme production was partially growth associated, and glucose supported both cell growth and enzyme production. Product formation and cell concentration were directly related to the rate of substrate consumption. The rate of product formation decreased when product started to accumulate, suggesting that the process was affected by feedback repression.
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