Simultaneous production of nisin and lactic acid from cheese whey

A biorefinery process that utilizes cheese whey as substrate to simultaneously produce nisin, a natural food preservative, and lactic acid, a raw material for biopolymer production, was studied. The conditions for nisin biosynthesis and lactic acid coproduction by Lactococcus lactis subsp. lactis (ATCC 11454) in a whey-based medium were optimized using statistically based experimental designs. A Plackett-Burman design was applied to screen seven parameters for significant factors for the production of nisin and lactic acid. Nutrient supplements, including yeast extract, MgSO₄, and KH₂PO₄, were found to be the significant factors affecting nisin and lactic acid formation. As a follow-up, a central-composite design was applied to optimize these factors. Second-order polynomial models were developed to quantify the relationship between nisin and lactic acid production and the variables. The optimal values of these variables were also determined. Finally, a verification experiment was performed to confirm the optimal values that were predicted by the models. The experimented results agreed well with the model prediction, giving a similar production of 19.3 g/L of lactic acid and 92.9 mg/L of nisin.     

Stimulation of Nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.     

Stimulation of Nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.     

Value-Added Production of Nisin from Soy Whey

The objective of this study was to evaluate the potential of low/negative value soy whey (SW) as an alternative, inexpensive fermentation substrate to culture Lactococcus lactis subsp. lactis for nisin production. Initially, a microtiter plate assay using a Bioscreen C Microbiology Plate Reader was used for rapid optimization of culture conditions. Various treatments were examined in efforts to optimize nisin production from SW, including different methods for SW sterilization, ultrasonication of soy flake slurries for possible nutrient release, comparison of diluted and undiluted SW, and supplementation of SW with nutrients. In subsequent flask-based experiments, dry bacterial mass and nisin yields obtained from SW were 2.18 g/L and 619 mg/L, respectively, as compared to 2.17 g/L and 672 mg/L from a complex medium, de Man–Rogosa–Sharpe broth. Ultrasonication of soybean flake slurries (10% solid content) in water prior to production of SW resulted in ∼2% increase in biomass yields and ∼1% decrease in nisin yields. Nutrient supplementation to SW resulted in ∼3% and ∼7% increase in cell and nisin yields, respectively. This proof-of-concept study demonstrates the potential for use of a low/negative value liquid waste stream from soybean processing for production of a high-value fermentation end product.     

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.     

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.     

Utilization of condensed distillers solubles as nutrient supplement for production of nisin and lactic acid from whey

The major challenge associated with the rapid growth of the ethanol industry is the usage of the coproducts, i.e., condensed distillers solubles (CDS) and distillers dried grains, which are currently sold as animal feed supplements. As the growth of the livestock industries remains flat, alternative usage of these coproducts is urgently needed. CDS is obtained after the removal of ethanol by distillation from the yeast fermentation of a grain or a grain mixture by condensing the thin stillage fraction to semisolid. In this work, CDS was first characterized and yeast biomass was proven to be the major component of CDS. CDS contained 7.50% crude protein but with only 42% of that protein being water soluble. Then, CDS was applied as a nutrient supplement for simultaneous production of nisin and lactic acid by Lactococcus lactis subsp. lactis (ATCC 11454). Although CDS was able to support bacteria growth and nisin production, a strong inhibition was observed when CDS was overdosed. This may be caused by the existence of the major ethanol fermentation byproducts, especially lactate and acetate, in CDS. In the final step, the CDS based medium composition for nisin and lactic acid production was optimized using response surface methodology.     

Production of Nisin Z by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Isolated from Dahi

Lactococcus lactis CM1, an isolate from homemade “Dahi,” a traditional fermented milk from India, used maltose as carbon source to produce a high level of bacteriocin. The bacterial cell mass and the bacteriocin production correlated with the initial pH of the medium and were highest when the initial pH was 11.0. The level of bacteriocin reached its peak at the late log phase with concomitant reduction of culture pH to 4.2, regardless of the initial pH of the medium. A combination of maltose and an initial medium pH of 11 resulted in the highest bacteriocin production. The antibacterial spectrum of the bacteriocin was closely similar to that of nisin and it inhibited a number of food spoilage and pathogenic bacteria. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the compound migrated close to the position of nisin (3.5 kDa). However, it had higher stability than nisin at a wide range of pH and temperature. PCR amplification using nisin gene-specific primers and sequencing of the amplified DNA revealed the structural gene for the bacteriocin to be identical to that of nisZ.     

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.     

Nutritional requirements of a strain of bacillus thuringiensis subsp. kurstaki and use of gruel hydrolysate for the formulation of a new medium for δ-endotoxin production

Nutritional requirements of a strain ofBacillus thuringiensis (Bt) subsp.kurstaki were elucidated for δ-endotoxin production. The effect of some principal nutrients was deeply investigated, showing several nutritional and metabolite limitations in Bt growth and δ-endotoxin synthesis. This led us to formulate a new medium based on the hydrolysate of gruel, a cheap and abundant byproduct of semolina factories, supporting growth and δ-endotoxin synthesis. After hydrolysis of gruel by α-amylase, followed by proteolysis using alcalase, the resultant soluble material substituted glucose very well for Bt δ-endotoxin production. Indeed, 15 g/L total sugars coming from that hydrolysate, supplemented by 5.4 g/L ammonium sulfate as nitrogen source and either 5 g/L yeast extract or 3 g/L peptone from casein or 3 g/L casaminoacids or 0.25 g/L cysteine or aspartic acid, were the principal components of this new medium in which almost 1 g/L of δ-endotoxin in 4.5 g/L total dry biomass was produced.     

Production of lactic acid from food wastes

Conversion of food wastes into lactic acid by simultaneous saccharification and fermentation (SSF) was investigated. The process involves saccharification of the starch component in food wastes by a commercial amylolytic enzyme preparation (a mixture of amyloglucosidase, α-amylase, and protease) and fermentation by Lactobacillus delbrueckii. The highest observed overall yield of lactic acid in the SSF was 91% of theoretical. Lactic acid concentration as high as 80 g/L was attainable in 48 h of the SSF. The optimum operating conditions for the maximum productivity were found to be 42°C and pH 6.0. Without supplementation of nitrogen-containing nutrients, the lactic acid yield in the SSF decreased to 60%: 27 g/L of lactic acid from 60 g/L of food waste. The overall performance of the SSF, however, was not significantly affected by the elimination of mineral supplements.     

Antimicrobial nanocomposite films made of poly(lactic acid)-cellulose nanocrystals (PLA-CNC) in food applications: part A—effect of nisin release on the inactivation of Listeria monocytogenes in ham

New bioactive nanocomposite films were prepared by compression molding method for food applications. Film matrix was composed of poly(lactic acid) containing cellulose nanocrystals (PLA-CNC). Nanocomposite films were converted to bioactive films using nisin as an antimicrobial agent by an adsorption coating method. Resulting antimicrobial films were then introduced in packages containing sliced cooked ham as a food model and stored for 14 days at 4 °C to determine their inhibiting capacity against Listeria monocytogenes and their physicochemical and structural properties. The study also focused on the nisin release from the films by using an agar diffusion bioassay. It was observed that mechanical properties such as tensile strength, tensile modulus, elongation at break and water vapor permeability values of the bioactive films were stable after 14 days of storage. Fourier transform infrared spectroscopy analysis allowed characterizing the adsorption of nisin onto PLA-CNC surface. Microbiological analysis of sliced cooked ham inoculated with L. monocytogenes (3 log CFU/g) allowed determining the potentiality of nisin as a strong antimicrobial agent in PLA-CNC-based films. Bioactive PLA-CNC-nisin films showed a significant reduction of L. monocytogenes in ham from day 1 and a total inhibition from day 3. The percentage of nisin release increased continuously from day 0 to day 14, up to 21 % at day 14. These results demonstrated the potential application of PLA-CNC-nisin films on controlling the growth of food pathogens in meat products.     

Modelling Growth and Bacteriocin Production by <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> PA003 as a Function of Temperature and pH Value

To investigate the effect of pH and temperature on the cell growth and bacteriocin production of Pediococcus acidilactici PA003, a lactic acid bacterium isolated from traditionally fermented cabbage, the kinetic behaviour of P. acidilactici PA003 was simulated in vitro during laboratory fermentations by making use of MRS broth. Firstly, primary models were developed for cell growth, glucose consumption, lactic acid and bacteriocin production for a given set of environmental conditions. Based on primary models, further study was undertaken to fit secondary models to describe the influence of temperature and pH on microbial behaviour. The models were validated successfully for all components. The results from the cell yield coefficient for lactic acid production reflected the homofermentative nature of P. acidilactici PA003. Both cell growth and bacteriocin production were very much influenced by changes in temperature and pH. The optimal condition for specific growth rate and biomass concentration was almost the same at pH 6.5 and 35 °C. At 35 °C and pH 6.1, the maximal bacteriocin activity was also achieved. The kinetic models provide useful tools for elucidating the mechanisms of temperature and pH on the kinetic behaviour of P. acidilactici PA003. The information obtained in this paper may be very useful for the selection of suitable starter cultures for a particular fermentation process and is a first step in the optimization of food fermentation processes and technology as well.     

Conversion of food industrial wastes into bioplastics

The usage of plastics in packaging and disposable products, and the generation of plastic waste, have been increasing drastically. Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. In the authors’ laboratories, various carbohydrates in the growth media, including sucrose, lactic acid, butyric acid, valeric acid, and various combinations of butyric and valeric acids, were utilized as the carbon (c) sources for the production of bioplastics byAlcaligenes eutrophus. As the first step in pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesize bioplastics, the authors investigated the usage of malt wastes from a beer brewery plant as the C sources for the production of bioplastics by microorganisms. Specific polymer production yield by A. Latus DSM 1124 increased to 70% polymer/cell (g/g) and 32g/L cell dry wt, using malt wastes as the C source. The results of these experiments indicated that, with the use of different types of food wastes as the C source, different polyhydroxyal-kanoate copolymers could be produced with distinct polymer properties.     

Optimization of l -(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure l-(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties

Background: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. Methods: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. Results: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. Conclusions: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.     

High-yield fermentation of pentoses into lactic acid

Lactobacillus species capable of fermenting glucose are generally incapable of utilizing xylose for growth or fermentation. In this study, a novel aspect of a well-known Lactobacillus strain, L. casei subsp. rhamnous (ATCC 10863), was uncovered: it can ferment xylose as efficiently as glucose. This strain is a registered organism, extremely stable on long-term operation. Fermentation by this strain is characterized by an initial lag phase lasting 24–72 h before xylose consumption takes place. The yield (grams/gram) of lactic acid from xylose is in excess of 80% with initial volumetric productivity of 0.38 g/(L-h). Acetic acid is the primary byproduct formed at the level of about 10% of the lactic acid. In addition to xylose, it can ferment all other minor sugars in hemicellulose except arabinose. Subjected to mixed sugar fermentation, this strain consumes glucose first, then mannose, followed by almost simultaneous utilization of xylose and galactose. It shows high tolerance for lactic acid as well as extraneous toxins. It can ferment the mixed sugars present in acid-treated hydrolysate of softwood, giving yields similar to that of pure sugar but at a slower rate. Author to whom all correspondence and reprint requests should be addressed.     

Production of Lactic Acid from Raw Sweet Potato Powders by Rhizopus Oryzae Immobilized in Sodium Alginate Capsules

Rhizopus oryzae immobilized in calcium alginate was applied in lactic acid fermentation with unhydrolyzed raw sweet potato powders as the sole carbon source. The effects of sodium alginate concentration, calcium chloride concentration, and the immobilized bead diameter on lactic acid production were investigated. Increase in sodium alginate concentration during the gelation process would harden the immobilized capsule, which led to a decrease in lactic acid production. The increase in calcium chloride would increase the thickness of the immobilized capsule, which would increase the mass transfer resistance. Nevertheless, while the calcium chloride was lower than 15%, it would not have obvious effects on lactic acid production. A larger bead could have more space for cell growth, which led to the maximum lactic acid production observed at the 5-mm bead diameter. Moreover, results of repeated-batch operation suggested that immobilized cells could have higher stability in lactic acid production than free cells. The total cumulative lactic acid in immobilized-cell operation could increase by 55% as compared with free-cell operation after 216 h (seven repeated-batches), and no loss of amylolytic activity was observed. The results indicated that immobilized R. oryzae by Ca-alginate could be suitable for lactic acid production from unhydrolyzed raw potato powders.     

Fungal Pretreatment by Phanerochaete chrysosporium for Enhancement of Biogas Production from Corn Stover Silage

Corn stover silage (CSS) was pretreated by Phanerochaete chrysosporium in solid-state fermentation (SSF), to enhance methane production via subsequent anaerobic digestion (AD). Effects of washing of corn stover silage (WCSS) on the lignocellulosic biodegradability in the fungal pretreatment step and on methane production in the AD step were investigated with comparison to the CSS. It was found that P. chrysosporium had the degradation of cellulose, hemicellulose, and lignin of CSS up to 19.9, 32.4, and 22.6 %, respectively. Consequently, CSS pretreated by 25 days achieved the highest methane yield of 265.1 mL/g volatile solid (VS), which was 23.0 % higher than the untreated CSS. However, the degradation of cellulose, hemicellulose, and lignin in WCSS after 30 days of SSF increased to 45.9, 48.4, and 39.0 %, respectively. Surface morphology and Fourier-transform infrared spectroscopy analyses also demonstrated that the WCSS improved degradation of cell wall components during SSF. Correspondingly, the pretreatment of WCSS improved methane production by 19.6 to 32.6 %, as compared with untreated CSS. Hence, washing and reducing organic acids (such as lactic acid, acetic acid, propionic acid, and butyric acid) present in CSS has been proven to further improve biodegradability in SSF and methane production in the AD step.     

Application of a pH Feedback-Controlled Substrate Feeding Method in Lactic Acid Production

Substrate concentration in lactic acid fermentation broth could not be controlled well by traditional feeding methods, including constant, intermittent, and exponential feeding methods, in fed-batch experiments. A simple feedback feeding method based on pH was proposed to control pH and substrate concentration synchronously to enhance lactic acid production in fed-batch culture. As the linear relationship between the consumption amounts of alkali and that of substrate was concluded during lactic acid fermentation, the alkali and substrate in the feeding broth were mixed together proportionally. Thus, the concentration of substrate could be controlled through the adjustment of pH automatically. In the fed-batch lactic acid fermentation with Lactobacillus lactis-11 by this method, the residual glucose concentration in fermentation broth was controlled between 4.1 and 4.9 g L⁻¹, and the highest concentration of lactic acid, maximum cell dry weight, volumetric productivity of lactic acid, and yield were 96.3 g L⁻¹, 4.7 g L⁻¹, 1.9 g L⁻¹ h⁻¹, and 0.99 g lactic acid per gram of glucose, respectively, compared to 82.7 g L⁻¹, 3.31 g L⁻¹, 1.7 g L⁻¹ h⁻¹, and 0.92 g lactic acid per gram of glucose in batch culture. This feeding method was simple and easily operated and could be feasible for industrial lactic acid production in the future.