Modeling bacterial survival in unfavorable environments

Summary The long-term survival of pathogenic microorganisms was evaluated and modeled in simulated fermented and dried, uncooked sausages, such as salami and pepperoni.Listeria monocytogenes andSalmonella were inoculated in BHI broths with added lactic acid or lactate (0–1.5%), NaCl (0–19%) and NaNO₂ (0–200 ppm) and then incubated at 4–42°C for up to 9 months. Enumerations of surviving cells showed several forms of declining curves, including classic first-order declines, shoulder or lag phases, and two-phase declines with shoulder. Two primary models were tested for their ability to depict the data. The effect of the environmental conditions on the parameters of the models were described with multiple regression equations (secondary models).Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

Predictive modelling of Lactobacillus casei KN291 survival in fermented soy beverage

The aim of the study was to construct and verify predictive growth and survival models of a potentially probiotic bacteria in fermented soy beverage. The research material included natural soy beverage (Polgrunt, Poland) and the strain of lactic acid bacteria (LAB) — Lactobacillus casei KN291. To construct predictive models for the growth and survival of L. casei KN291 bacteria in the fermented soy beverage we design an experiment which allowed the collection of CFU data. Fermented soy beverage samples were stored at various temperature conditions (5, 10, 15, and 20°C) for 28 days. On the basis of obtained data concerning the survival of L. casei KN291 bacteria in soy beverage at different temperature and time conditions, two non-linear models (r ²= 0.68–0.93) and two surface models (r ²=0.76–0.79) were constructed; these models described the behaviour of the bacteria in the product to a satisfactory extent. Verification of the surface models was carried out utilizing the validation data — at 7°C during 28 days. It was found that applied models were well fitted and charged with small systematic errors, which is evidenced by accuracy factor — Af, bias factor — Bf and mean squared error — MSE. The constructed microbiological growth and survival models of L. casei KN291 in fermented soy beverage enable the estimation of products shelf life period, which in this case is defined by the requirement for the level of the bacteria to be above 10⁶ CFU/cm³. The constructed models may be useful as a tool for the manufacture of probiotic foods to estimate of their shelf life period.     

Ensilage of tropical grasses mixed with legumes and molasses

The effects of adding two legumes, Gliricidia sepium and Leucaena leucocephala, cv. Cunningham, and molasses on the fermentation characteristics of silages made from two tropical grasses (Pangola grass, Digitaria decumbens, and Setaria sphacelata cv. Kazungula) were investigated. Pangola grass silages contained significantly higher contents of water-soluble carbohydrates and lactic acid than did setaria silages after 100 days fermentation, but there were no significant differences between the two silages in populations of lactic acid bacteria and contents of total N and NH₃–N. Addition of either species of legume had no significant effect on fermentation acids and NH₃–N contents, and numbers of lactic acid bacteria. Addition of both legumes reduced NH₃–N production in the silages by 59% after 5 days' fermentation. Numbers of lactic acid bacteria were not significantly affected by the different treatments. Enterococcus faecalis represented 60% of the lactic acid bacteria isolated from the treated herbages prior to ensiling. By 100 days of fermentation, only lactobacilli were isolated: 82% homo-fermenters and 18% hetero-fermenters. Lactobacillus mesenteroides subsp. dextranicum was found only in the silage supplemented with 33% (w/w) legume. It was concluded that the low quality of tropical grasses used as feeds for ruminants may be significantly improved by ensiling these grasses with small amounts of molasses and with high-protein tree leaves.M. Tjandraatmadja and B.W. Norton are with the Department of Agriculture. The University of Queensland, Queensland, 4072, Australia; I.C. Mac Rae is with the Department of Microbiology, The University of Queensland, Queensland, 4072, Australia.     

Modelling of growth conditions for cyanobacterium Spirulina platensis in microcosms

The influence of cultivation conditions on the growth of the cyanobacterium Spirulina platensis was investigated by using two types of photobioreactors. In a rotative photobioreactor the doubling time (t _d) was 3.54 days. The better value found for t _d in an aerated photobioreactor by changing the initial nitrogen concentration (NaNO₃) at 0.003, 0.015, 0.030 and 0.060M was 2.5 days. A factorial experimental design was performed in order to estimate the contributions of initial nitrogen concentration, inoculum and cultivation time as well as their interactions. All three factors and their interactions proved to be significant in influencing the cellular concentration of S. platensis.     

ALGORITHM FOR APPLICATION OF FOURIER ANALYSIS FOR BIORHYTHMIC BASELINES OF PHARMACODYNAMIC INDIRECT RESPONSE MODELS

The change of an indirect pharmacological response R(t) can be described by a periodic time-dependent production rate k_in (t) and a first-order loss constant k_out. If k_in(t) follows some biological rhythm (e.g., circadian), then the response R(t) also displays a periodic behavior. A new approach for describing the input function in indirect response models with biorhythmic baselines of physiologic substances is introduced. The present approach uses the baseline (placebo) response R_b(t) to recover the equation for k_in(t). Fourier analysis provides an approximate equation for R_b(t) that consists of terms (usually two or three) of the Fourier series (harmonics) that contribute most to the overall sum. The model differential equation is solved backward for k_in(t), yielding the equation involving R_b(t). A computer program was developed to perform the square L²-norm approximation technique. Fourier analysis was also performed based on nonlinear regression. Cortisol suppression after inhalation of fluticasone propionate (FP) was modeled based on the inhibition of the secretion rate k_in(t) using ADAPT II. The pharmacodynamic parameters k_out and IC₅₀ were estimated from the model equation with k_in(t) derived by the new approach. The proposed method of describing the input function needs no assumption about the behavior of k_in(t), is as efficient as methods used previously, and is more flexible in describing the baseline data than the nonlinear regression method. (Chronobiology International, 17(1), 77–93, 2000)     

北疆传统发酵生奶酪中乳酸菌的耐受性及益生特性测定

【背景】北疆乳制品中生奶酪作为发酵乳制品,其中有多种微生物的参与,是优质食品级微生物的宝贵来源,然而其中蕴含的丰富的微生物资源也面临着流失。【目的】研究已筛选出的41株乳酸菌的生长曲线变化规律,并对其在低pH环境的耐酸特性及益生特性进行分析。【方法】通过人工模拟胃肠液和含0.3%、0.5%、1.0%浓度牛胆盐溶液培养,对从生奶酪中分离鉴定的41株乳酸菌菌株进行人工胃肠液、牛胆盐耐受性试验。【结果】41株乳酸菌中有6株乳酸菌(编号为QM-5、QM-27、UM-12、UM-18、NM-11和NM-14)均能在pH值分别为2.0、2.5、3.0、3.5、4.0时生长较好,在pH 2.0的环境下也保持一定程度的生长。菌株存活率大于50%,乳酸菌含量为108 CFU/mL以上。从生奶酪分离的乳酸菌有极强的耐酸、耐胆盐特性,可以预测在胃肠道环境生存。41株菌对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和枯草芽孢杆菌(Bacillus subtilis)均有抑制作用。【结论】通过比较耐酸性、胃肠道模拟、胆盐耐受性和抑菌特性等实验结果,初...     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

Antimicrobial activity of lactobacilli and streptococci

Of nine strains of lactic acid bacteria commonly used as starter cultures for the dalry industry and ensiling, six (Lactobacillus bulgaricus, L. casei, L. acidophilus CH=5, L. plantarum, Streptococcus latis and Strep. taecium) had antibiotic activity. Gram-positive bacteria were more sensitive than Gram-negative bacteria to the antibiotics. The most sensitive strain of Staphylococcus aureus was used as a target micro-organism for the characterization of the antimicrobial substance. The cultures of Streptococcus faecium and L. plantarum gave the most intense antimicrobial activity. Adding CaCO₃ to the medium (to bind accumulated lactic acid) increased the antibiotic activity of the lactic acid bacteria.The authors are with the Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 41000 Zagreb, Yugoslavia.     

MATING SYSTEM IN NATURAL POPULATIONS OF JEFFREY PINE

The mating system and allozyme variation at 20 loci in three Klamath Mountains and two Sierra Nevada populations of Jeffrey pine (Pinus jeffreyi Grev. & Balf.) were investigated. On average, multilocus estimates of the proportion of viable progeny due to outcrossing (t_m) were high in all populations (mean t_m = 0.935, range 0.881 to 0.971). Despite differences in stand structure, t_m did not differ (P > 0.05) between the Klamath (mean t_m = 0.933) and Sierra Nevada (mean t_m = 0.937) populations. At all but one locus in one population and at two in another, genotype frequencies fit (P > 0.05) Hardy-Weinberg expectations. Mean estimates of observed heterozygosity in Klamath (0.182) and Sierra Nevada (0.327) populations were comparable to values reported for other conifers.     

Effect of maize stunting mollicutes on survival and fecundity of Dalbulus leafhopper vectors

The effect of corn stunt spiroplasma (CSS) on survival and fecundity of three Dalbulus leafhopper species was determined. CSS significantly reduced the survival, as measured by the time to 50% (t₅₀) and 25% (t₂₅) survival, and by the scale parameter (b) of the Weibull model, for D. elimatus and D. gelbus. Fecundity of these two species, as measured by the net and gross reproductive rates, was also reduced by CSS. CSS did not significantly affect the corn leafhopper, D. maidis. In a separate experiment, maize bushy stunt mycoplasma (MBSM) reduced the survival and fecundity of D. maidis at temperatures from 20 to 29 °C. The effect of MBSM on D. maidis survival was less severe than CSS on D. elimatus and D. gelbus; t₂₅, but not t₅₀, was reduced by MBSM. Survival times and the cohort generation time generally declined with increasing temperature. Fecundity, however, generally increased with increasing temperature.     

Optimum conditions for the biological production of lactic acid by a newly isolated lactic acid bacterium,Lactobacillus sp. RKY2

Lactic acid is a green chemical that can be used as a raw material for biodegradable polymer. To produce lactic acid through microbial fermentation, we previously screened a novel lactic acid bacterium. In this work, we optimized lactic acid fermentation using a newly isolated and homofermentative lactic acid bacterium. The optimum medium components were found to be glucose, yeast extract, (NH₄)₂HPO₄, and MnSO₄. The optimum pH and temperature for a batch culture ofLactobacillus sp. RKY2 was found to be 6.0 and 36°C, respectively. Under the optimized culture conditions, the maximum lactic acid concentration (153.9 g/L) was obtained from 200 g/L of glucose and 15 g/L of yeast extract, and maximum lactic acid productivity (6.21 gL⁻¹h⁻¹) was obtained from 100 g/L of glucose and 20 g/L of yeast extract. In all cases, the lactic acid yields were found to be above 0.91 g/g. This article provides the optimized conditions for a batch culture ofLactobacillus sp. RKY2, which resulted in highest productivity of lactic acid.     

Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E?I complex and first-order rate constant for inactivation were determined by incubation (K_i* = 26.6?±?2.8 µM, k₂ = 12.2?±?0.6 min–1) or progress curve (K_i* = 6.5?±?1.5 µM, k₂ = 0.11?±?0.01 min–1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t_1/2 = 11.3?±?0.2?h), matrix-assisted laser desorption/ionization (MALDI, t_1/2 = 13.5?±?0.4?h), and electro-spray ionization (ESI, t_1/2 = 12.2?±?0.5?h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.     

Modeling of recycling oxic and anoxic treatment system for swine wastewater using neural networks

A recycling reactor system operated under sequential anoxic and oxic conditions for the treatment of swine wastewater has been developed, in which piggery slurry is fermentatively and aerobically treated and then part of the effluent is recycled to the pigsty. This system significantly removes offensive smells (at both the pigsty and the treatment plant), BOD and others, and may be cost effective for small-scale farms. The most dominant heterotrophic were, in order,Alcaligenes faecalis, Brevundimonas diminuta andStreptococcus sp., while lactic acid bacteria were dominantly observed in the anoxic tank. We propose a novel monitoring system for a recycling piggery slurry treatment system through the use of neural networks. In this study, we tried to model the treatment process for each tank in the system (influent, fermentation, aeration, first sedimentation and fourth sedimentation tanks) based upon the population densities of the heterotrophic and lactic acid bacteria. Principal component analysis (PCA) was first applied to identify a relationship between input and output. The input would be microbial densities and the treatment parameters, such as population densities of heterotrophic and lactic acid bacteria, suspended solids (SS), COD, NH₄ ⁺-N, or-tho-phosphorus (o-P), and total-phosphorus (T-P). Then multi-layer neural networks were employed to model the treatment process for each tank. PCA filtration of the input data as microbial densities was found to facilitate the modeling procedure for the system monitoring even with a relatively lower number of input. Neural networks independently trained for each treatment tank and their subsequent combined data analysis allowed a successful prediction of the treatment system for at least two days.     

Influence of the Selected Antioxidants on the Stability of the Celsior Solution Used for Perfusion and Organ Preservation Purposes

The purpose of the following research was to improve the original Celsior solution in order to obtain a higher degree of stability and effectiveness. The solution was modified by the addition of selected antioxidants such as vitamin C, cysteine, and fumaric acid in the following concentrations: 0.1, 0.3, and 0.5 mmol/l. The solution’s stability was estimated using an accelerated stability test based on changes in histidine concentrations in the solution using Pauly’s method for determining concentrations. Elevated temperatures, the factor accelerating substances’ decomposition reaction rate, were used in the tests. The research was conducted at four temperatures at intervals of 10°C: 60 ± 0.2°C, 70 ± 0.2°C, 80 ± 0.2°C, and 90 ± 0.2°C. It was stated that the studied substances’ decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters (the rate constant of decomposition k, activation energy E _a, and frequency factor A). On the basis of these parameters, the stability of the modified solution was estimated at +5°C. The results obtained show that the proposed antioxidants have a significant effect on lengthening the Celsior solution’s stability. The best results were reached when combining two antioxidants: vitamin C and cysteine in 0.5 mmol/l concentrations. As a result, the Celsior solution’s stability was lengthened from 22 to 299 days, which is 13.5 times. Vitamin C at a concentration of 0.5 mmol/l increased the solution’s stability by 5.2 times (t ₉₀ = 115 days), cysteine at a concentration of 0.5 mmol/l caused a 4.4 times stability increase (t ₉₀ = 96 days), and fumaric acid at a concentration of 0.5 mmol/l extended the stability by 2.1 times (t ₉₀ = 48 days) in relation to the original solution.     

Transition temperature and enthalpy change dependence on stabilizing and destabilizing ions in the helix–coil transition in native tendon collagen

The transition temperatures t_t and enthalpy changes ΔH in the helix–coil transition of solid tendon collagen soaked in a solution containing one of the following stabilizing or destabilizing agents, HCHO, NaF, NaCl, NaI, NaBr, NaOH, NH₂CONH₂, CaCl₂, MgCl₂, were measured as a function of molar concentration by a calorimetric method. The temperature and the enthalpy changes accompanying the transition behaved in a similar manner: when the t_t was depressed by the presence of ions, similar behaviour was observed in ΔH. Both parameters (t_t and ΔH) increased for HCHO, and decreased for NaF and NaCl at concentrations lower than 0.2 M. Above 0.2 M they increased for NaF and NaCl, and decreased in the presence of the other reagents listed above. The average t_t and the ΔH observed in collagen soaked in water were 63.5°C and 12.3 cal/g, respectively. In addition to the parameters mentioned above, the molar effectiveness of the various reagents was obtained for the cases where there was a linear relationship between the t_t and molar concentration of the reagent in the solution. Since both the t_t and the ΔH were observed to vary, the entropy change (ΔS) accompanying the transition was calculated using thermodynamic relations. In order to explain the ΔS observed as a function of ionic concentration, the thermodynamic relationships have been obtained from a partition function under suitable assumptions. Since the partition function is dependent on the number of hydrogen bonds responsible for collagen stability, the result obtained has been compared with the values predicted by the two most quoted models for collagen. The present study is in accordance with the Ramachandran model for collagen structure, which predicts more than one hydrogen bond per three residues.     

Influence of immobilization parameters on growth and lactic acid production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> co-immobilized in calcium alginate gel beads

Streptococcus thermophilusand Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1–1.5M) and alginate (1–2<><>, w/v) concentrations. Highest lactic acid production was 35 g l₁ when both bacteria were in high viscosity beads (1<><>, w/v alginate) hardened in 0.1 M CaCl₂ .The gel bead composition affected size and distribution of entrapped lactic acid bacteria.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Modeling the inactivation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spores by ethylene oxide processing

Ethylene oxide is currently a dominant agent in medical device sterilization. This work intends to study the main effects and interactions of temperature, ethylene oxide concentration, and relative humidity on commercial spore strips of Bacillus subtilis, var. niger (ATCC 9372) inactivation, the most common microorganism used in controlling the efficacy of the process. Experiments were carried out using a full factorial experimental design at two levels (2³ factorial design). Limit target exposure conditions for ethylene oxide concentration, temperature, and relative humidity were 250–1,000 mg EO/l, 40–60°C, and 50–90%, respectively. Adopting a different approach from the first-order kinetics, a Gompertz model was successfully applied in data fitting of the inactivation curves. Bacillus subtilis kinetic behavior presented a sigmoidal inactivation with an initial shoulder (λ), followed by a maximum inactivation rate (k_max), these being model parameters. It was concluded that temperature and ethylene oxide concentration were the most significant factors and consequently, additional experiments were carried out aiming at describing the parameters' dependence on these process factors. Mathematical relations describing such dependences were successfully developed and included in the Gompertz kinetic model. The predictive ability of this integrated model was assessed, and its adequacy in predicting B. subtilis inactivation was proven.     

两种方法形成菌丝球固定2-氯酚降解菌的对比

【目的】系统研究吸附法和同时培养法对所形成混合菌丝球的外观形态、内部结构及其去除2-氯酚效果的影响。【方法】采用吸附法和同时培养法将可降解2-氯酚的光合细菌PSB-1D固定在黄孢原毛平革菌(Phanerochaete chrysosporium)DH-1发酵而成的菌丝球上,形成混合菌丝球。以单一菌丝球为对照,利用光学显微镜、扫描电镜等仪器观察混合菌丝球的外观形态和内部结构,考察2种方法对混合菌丝球成球效果的影响;以无菌培养液为空白对照,考察游离光合细菌、单一菌丝球、2种方法形成混合菌丝球对2-氯酚的降解效能。【结果】在吸附法形成的混合菌丝球上,光合细菌主要集中在过渡区;而同时培养法将光合细菌牢固地包埋在菌丝球内核区,并大量簇状附着生长在菌丝交联的空隙处和每根菌丝上。在接种等量孢子和光合细菌的前提下,同时培养法较吸附法操作时间更短,成球数量更多,形成菌丝球干湿比更大,单位菌丝干重上固定的细菌数量更多。菌丝球降解体系和游离光合细菌对2-氯酚的降解均符合一级动力学特征。同时培养法形成的混合菌丝球降解效果最好,7 d内对初始浓度为50 mg/L的2-氯酚降解率可达89%以上,降解速率常数为0.3286 mg/(L·d),2-氯酚半衰期t1/2为2.8 d。【结论】首次报道黄孢原毛平革菌包埋固定化光合细菌形成混合菌丝球。该研究为生物质固定化材料的实际应用提供理论依据。     

Lactic acid production with Lactobacillus casei ssp. rhamnosus NBIMCC 1013: Modeling and optimization of the nutrient medium

The medium needed to perform a fermentation process with viable cells of Lactobacillus casei ssp. rhamnosus NBIMCC 1013 for the production of lactic acid was modeled and optimized. On the basis of single‐factor experiments and statistical analysis, the significant factors affecting the fermentation process, i.e. the concentration of carbon source, concentrations of both yeast and meat extracts, and the range of variability of these components were determined. Modeling and optimization of the medium contents were performed using central composite design. The composition of the medium used for the production of lactic acid (g/L) was as follows: glucose 69.8, meat extract 17.07, yeast extract 10.9, CH₃COONa 10, K₂HPO₄ 0.25, KH₂PO₄ 0.25, MgSO₄·7H₂O 0.05, and FeSO₄ 0.05. The maximum specific growth rate of the lactic acid bacteria (μ=0.51 h⁻¹) and other kinetic parameters were determined during cultivation in a laboratory bioreactor using the logistic equation and the Luedeking–Piret model. The obtained medium allows the production of lactic acid under optimum conditions, at high specific sugar assimilation rates and high lactic acid accumulation rates. The positive results of the paper are the new nutrient medium for lactic acid production and the process kinetic model, enabling scaling up and switching to a continuous process.