Production of nisin by Lactococcus lactis in media with skimmed milk

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30°C for 36 h) in both M17 and MRS standard broth media (pH 6.0–7.0) supplemented with sucrose (1.0–12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30°C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of “standard nisin” (Nisaplin®, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.     

Environmental Effects on Transglutaminase Production and Cell Sporulation in Submerged Cultivation of <Emphasis Type="Italic">Bacillus circulans</Emphasis>

In this research, the effects of pH, temperature, and oxygen on growth kinetics of a newly isolated strain of Bacillus circulans from the Amazon and their correlations with transglutaminase (TGase) production and cell sporulation were investigated. Statistical experimental methods were used to optimize these parameters, while induction of sporulation was achieved by oxygen culture control. Full factorial composite experimental design and response surface methodology were experimentally tested. The model showed that temperature has a positive and significant effect on TGase production (P < 0.05) while pH and temperature, associated with anoxic conditions, have a marked effect on cell sporulation which is consistently linked with TGase production. The contour plot of results showed that the best culture conditions for TGase production of B. circulans were 30°C, initial pH 8.5, and the highest production was obtained in late-stationary culture phase with maximal specific enzyme activity of 655 U g⁻¹ of cells (0.37 U/mL). A correlation between enzyme production and cell sporulation, as mediated by oxygen culture conditions, was also demonstrated and, although demonstrated only for B. subtilis, it corroborates the molecular mechanisms involved in this process. It can be suggested that B. circulans BL32 is a strong biological system for the industrial production of TGases.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii

The effects of environmental conditions, namely initial pH (2.5–7.0) and temperature (25 and 35°C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Y_P/s and Q _p were 0.75 g/g and 0.95 g/[L·h], respectively, both attained at pH 6.0, 35°C), the highest xylose reductase activities (nearly 900 1U/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35°C (577 1U/mg of protein).     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Constitutive Expression and Optimization of Nutrients for Streptokinase Production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> Using Statistical Methods

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett–Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Optimization,Purification, and Characterization of Extracellular Mesophilic Alkaline Cellulase from Sponge-Associated <Emphasis Type="Italic">Marinobacter</Emphasis> sp. MSI032

Marinobacter sp. (MSI032) isolated from the marine sponge Dendrilla nigra was optimized for the production of extracellular cellulolytic enzyme (CMCase) by submerged fermentation. Initial experiments showed that the culture medium containing 1% maltose as carbon source and 1% peptone and casein as nitrogen source supported maximal enzyme production at 27 °C and at a pH of 9.0. Further optimization carried out showed the maximal enzyme production was supported by the presence of 2% NaCl and 10 mM Zn²⁺ ions in the production media. The production of enzyme cellulase occurred at 48 h of incubation which proved the importance of this strain for cellulase production in large scale. Further, the enzyme was purified to 12.5-fold with a 37% yield and a specific activity of 2,548.75 U/mg. The purified enzyme displayed maximum activity at mesophilic temperature (27–35 °C) and at a broad pH range with optimal activity at pH 9.0. The purified enzyme was stable even at a higher alkaline pH of 12.0 which is greater than the pH stability that has not been reported in any of the cellulolytic isolates studied so far. Thus, from the present study, it is crucial that, instead of exploring the thermophilic resource that is limited in natural environments, the mesophilic bacteria that occurs commonly in nature can be added up to the database of cellulolytic bacteria. Thus, it is possible that a wide diversity of mesophilic bacteria associated with marine sponges opens up a new doorstep for the degradation of cellulosic waste material for the production of liquid fuels. This is the first report elucidating the prospects of sponge-associated marine bacterium for the production of extracellular alkaline cellulase.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Production of γ-Decalactone by a Psychrophilic and a Mesophilic Strain of the Yeast <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis>

Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic strain Rhodotorula aurantiaca A19 was selected for its ability of growth and γ-decalactone production at low temperatures. The effects of temperature, initial pH, and castor oil concentration on the growth and γ-decalactone production by a psychrophilic and a mesophilic strain of R. aurantiaca were investigated. The highest γ-decalactone production in flasks (5.8 g/l) was obtained with the strain A19 at 14 °C and initial pH 7.0 in medium containing 20 g/l castor oil. On the other hand, these factors did not affect the production of γ-decalactone by the mesophilic strain. In fermentor, a γ-decalactone concentration of 6.6 g/l was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the mesophilic strain. Our results suggest that the ability to synthesize γ-decalactone is a particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of γ-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. Moreover, the amount of γ-decalactone obtained in fermentor with the strain 19 was on the order of concentrations usually described in patents.     

Succinic Acid Production from Cheese Whey using Actinobacillus succinogenes 130 Z

Actinobacillus succinogenes 130 Z was used to produce succinic acid from cheese whey in this study. At the presence of external CO₂ supply, the effects of initial cheese whey concentration, pH, and inoculum size on the succinic acid production were studied. The by-product formation during the fermentation process was also analyzed. The highest succinic acid yield of 0.57 was obtained at initial cheese whey concentration of 50 g/L, while the highest succinic acid productivity of 0.58 g h⁻¹ L⁻¹ was obtained at initial cheese whey concentration of 100 g/L. Increase in pH and inoculum size caused higher succinic acid yield and productivity. At the preferred fermentation condition of pH 6.8, inoculum size of 5% and initial cheese whey concentration of 50 g/L, succinic acid yield of 0.57, and productivity of 0.44 g h⁻¹ L⁻¹ were obtained. Acetic acid and formic acid were the main by-products throughout the fermentation run of 48 h. It is feasible to produce succinic acid using lactose from cheese whey as carbon resource by A. succinogenes 130 Z.     

Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation

Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase, and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.     

Effects of Carbon and Nitrogen Sources and Oxygenation on the Production of Inulinase by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Cultivations of Kluyveromyces marxianus var. bulgaricus ATCC 16045 were performed on both minimal and complex media using different carbon and nitrogen sources either in the presence or absence of aeration. The results collected were worked out and compared so as to provide a useful contribution to the optimization of inulinase production. Kinetics of extracellular inulinase release were similar on glucose, fructose, and sucrose. Inulinase was detected at basal level since the beginning of batch runs on these three carbon sources and overproduced after their depletion. The highest inulinase activity in minimal medium containing 10 g/l sucrose (6.4 IU/ml) was obtained at an initial (NH₄)₂SO₄ concentration of 5 g/l, whereas it was reduced to about one fourth of this value and detected only at the beginning under nitrogen-limited conditions. The best sucrose concentrations for the enzyme production were 30 and 20 g/l in minimal and complex media, yielding 15.4 and 208 IU/ml, respectively. In general, the enzyme activity was much higher in complex than in minimal medium under all conditions. O₂-enriched air neither improved inulinase production nor prevented ethanol formation.     

High-Level Expression and Efficient Purification of Bioactive Swollenin in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The bioactivity of swollenin is beneficial to cellulose decomposition by cellulase despite the lack of hydrolytic activity itself. In order to improve the productivity of swollenin, the effects of culture conditions on the expression level in recombinant Aspergillus oryzae were investigated systematically. With regard to the bioactivity of swollenin, glycerin and peanut meal were the optimal carbon or nitrogen source, respectively. The highest level production of swollenin (50 mg L⁻¹) was attained after 88 h cultivation with the initial pH of 5.6 in the culture medium. Then the soluble swollenin was effectively purified from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography with recovery yield of 53.2%. The purified swollenin was fully bioactive due to its strong synergistic activity with cellulose.     

β-Glucosidase production by Trichoderma reesei

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies: (1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, (2) the pH was adjusted to 6.0 daily, and (3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.     

Response Surface Optimization of the Critical Medium Components for Pullulan Production by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> FB-1

Culture conditions for pullulan production by Aureobasidium pullulans were optimized using response surface methodology at shake flask level without pH control. In the present investigation, a five-level with five-factor central composite rotatable design of experiments was employed to optimize the levels of five factors significantly affecting the pullulan production, biomass production, and sugar utilization in submerged cultivation. The selected factors included concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride. Using this methodology, the optimal values for concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride were 5.31%, 0.11%, 0.07%, 0.05%, and 0.15% (w/v), respectively. This optimized medium has projected a theoretically production of pullulan of 4.44%, biomass yield of 1.03%, and sugar utilization of 97.12%. The multiple correlation coefficient ‘R’ was 0.9976, 0.9761 and 0.9919 for pullulan production, biomass production, and sugar utilization, respectively. The value of R being very close to one justifies an excellent correlation between the predicted and the experimental data.     

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Grampositive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-α expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin^®, 25 mg of pure nisin with an activity of 1×10⁶ AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1∶1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.     

Phytase Production by a Marine Yeast <Emphasis Type="Italic">Kodamea ohmeri</Emphasis> BG3

The marine yeast strain Kodamea ohmeri BG3 isolated from the gut of a marine fish (Hexagrammes otakii) was found to secrete a large amount of phytase into the medium. The crude phytase produced by this marine yeast showed the highest activity at pH 5.0 and 65 °C. The optimal medium for phytase production contained oat 10.0 g/l, ammonium sulfate 15.0 g/l, glucose 30 g/l, and NaCl 20.0 g/l, while the optimal cultivation conditions for phytase production were pH 5.0, a temperature of 28 °C, and a shaking speed of 170 rpm. Under the optimal conditions, over 557.9 mU/ml of phytase activity was produced within 72 h of fermentation at the shake flask level. This is a very high level of phytase activity produced by yeasts. We think that the medium and process for phytase production by the marine yeast strain were very simple, and such marine yeast from the gut of natural marine fish may have a potential application in the maricultural industry and marine environmental protection. The results demonstrate that phytate was actively degraded by the crude phytase within a short period.