Optimization of Medium Composition for Nisin Fermentation with Response Surface Methodology

ABSTRACT:  Nisin is an effective food biopreservative widely used in food industry. However, 1 problem of concern is limited production rate and final nisin concentration. A nisin-producing strain, L. lactis Lac2, a mutant strain with high yield of nisin, was obtained in our laboratory recently. In the present study, a fractional factorial design was applied to investigate the main factors that affect the yield of L. lactis Lac2. Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium. The results showed that the optimum medium for nisin production of L. lactis Lac2 was composed of 2.68% sucrose (w/v), 0.5% tryptone (w/v), 1% yeast extract (w/v), 0.3% Tween-80 (w/v), 0.02% MgSO₄·7H₂O (w/v), 0.81% NaCl (w/v), 1.91% K₂HPO₄ (w/v), 0.05% ascorbic acid (w/v), and 2% agar (w/v) (if necessary) at pH 6.5. When cultured in the optimum medium, the nisin yield is an average of 3381.81 IU/mL, which nearly doubled the yield when incubated in the initial medium. Also, the concentration of tryptone was decreased while that of the sucrose was increased when compared with CM broth, which means a reduction of the fermentation cost.     

The effect of redox potential,and its interaction with sodium chloride concentration,on the probability of growth of Clostridium botulinum type E from spore inocula

A most probable number (mpn) count has been used to assess the effect of redox potential and of redox potential combined with increasing concentrations of sodium chloride on the probability of growth of spores of C. botulinum type E strain Beluga. In the majority of cases the maximum count developed within five days. In medium at pH 6·8 – 7·0, containing 0·1% w/v NaCl, incubated at 20°C, the probability of growth of spores in five days was not significantly lower at an E_h of approx +60 mV, adjusted by the introduction of air, than in strictly anaerobic conditions at E_h ?400 mV. Increasing the E_h above +60 mV resulted in a decrease in the probability of growth of spores, and at a redox potential between approx +122 mV and +164 mV the probability of growth was decreased by a factor of over 10⁵ compared with that at the lowest E_h. The inclusion of 3·25% w/v and 4% w/v NaCl in medium at a redox potential of ?400 mV decreased the probability of growth by factors of 10² and 10⁴ respectively, while the combination of 3·25% w/v NaCl and an E_h between +62 mV and +122 mV decreased the probability of growth by a factor of 10⁶.     

Response surface optimization of lipid and protein extractions from <Emphasis Type="Italic">Spirulina platensis</Emphasis> using ultrasound assisted osmotic shock method

In this study, we optimized the process for extracting lipids and proteins from wet biomasses of Spirulina sp. using a 4-kHz ultrasonic osmotic shock method with ultrasound enhancement at a constant frequency of 40 kHz. Optimization was conducted using a response surface methodology (RSM) at an osmotic NaCl concentration of 10–30%, solvent:biomass ratio of 5–15 v/w, and extraction times of 20–50 min. The present osmotic shock method with ultrasound irradiation increased lipid yields to 6.65% in the presence of 11.9% NaCl, a solvent:biomass ratio of 12:1 v/w, and a 22-min extraction time, and protein yields to 43.96% with 15.12% NaCl, a solvent:biomass ratio of 10:1 v/w, and a 30-min extraction time.     

Expression of Lactobacillus pentosus B96 bacteriocin genes under saline stress

This research studied the influence of sodium chloride on bacteriocin activity of table olives’ strain Lactobacillus pentosus B96. The strain was cultured in MRS under different NaCl concentrations (0, 4, 6 and 8%, in w/v). In MRS, maximum bacteriocin activity was achieved 9 h later. A medium containing 4 or 6% NaCl (w/v) increased the total bioactivity of the strain and an 8% NaCl reduced it. Real-time PCR was used to monitor the genetic expression of the bacteriocin genes plnA, plnB, plnC, plnE/F, plnJ, plnK, plnN and plantaricin S. Cultured in MRS, plantaricin S reached its maximum expression during the lag phase while plnE/F expresses during the exponential phase. The presence of sodium chloride in the medium moved the maximum expression of plantaricin S to the stationary phase, independently of the concentration. 4% (w/v) of NaCl didn’t affect the expression pattern of plnE/F while promotes the expression of plnN during both the lag and the exponential phases. More sodium chloride, 6% (w/v) maintained the expression of plnN in the pag phase but not in the exponential and moved plnE/F expression to the stationary phase. Plantaricin S, plnE/F and plnN over-expressed during the stationary phase in the higher sodium chloride concentration assayed, 8% (w/v). The relative expression level of plsA was 1000-fold higher than that of the plnE/F and plnN genes and even the ldhD constitutive gene used. Under our conditions, expression of plnA, plnB, plnC, plnJ and plnK genes was not observed.     

Utilization of Shochu distillery wastewater for production of polyunsaturated fatty acids and xanthophylls using thraustochytrid

The industrial production of barley shochu, a distilled alcoholic beverage, results in distillery waste that is currently incinerated or disposed of in landfills, causing environmental pollution. The supernatant of distillery waste contains organic matter such as proteins ( approximately 2.5%) and amino acids ( approximately 0.2%). This study demonstrates that the utilization of distillery wastewater as a sole nitrogen source enables a marine thraustochytrid, Schizochytrium sp. strain KH105, to propagate and accumulate valuable lipids including docosahexaenoic acid (DHA) and astaxanthin. Under optimized culture conditions, the highest DHA and astaxanthin yields were obtained at 3.4 g/l and 7.7 mg/l, respectively, after 4 or 5 d of cultivation in a 3-l jar fermentor. The chemical oxygen demand of the wastewater was reduced by 35%. About 67% of crude protein content and 85% of total free amino acid content also decreased in the culture supernatant. The thraustochytrid therefore serves to upgrade the distillery by-product to one containing value-added lipids for functional foods as well as to regulate the environmental contamination.     

Proximate composition and extraction of carotenoids and lipids from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis)

The chemical composition of redspotted shrimp (Penaeus paulensis) waste was investigated. The shrimp waste (freeze-dried heads, shells and tails) was found to have high protein (49% d.w.) and ash (27% d.w.) contents, but a low lipid content (4.9% d.w.) although the latter was higher than those found in other kinds of shrimp captured in Brazil. The fatty acid compositions showed that the lipids had a high content of unsaturated fatty acids, mainly EPA (C20:5; n-3) and DHA (C22:6; n-3). In order to establish an efficient and environmentally friendly recovery process for the astaxanthin (principal carotenoid and antioxidant present in the waste), the following processes were examined: traditional solvent extraction (TSE), super-critical fluid extraction (SC-CO₂) and super-critical fluid extraction with co-solvent (SC-CO₂ + ethanol). The temperature and pressure conditions for all the SC-CO₂ extractions were 50 °C and 30.0 MPa. The results showed that the mixture of 60% (v/v) n-hexane:isopropyl alcohol gave the highest (53 mg/kg waste) carotenoid extraction yield as compared to acetone, SC-CO₂ and SC-CO₂ + ethanol. The SC-CO₂ showed the lowest extraction yield of astaxanthin, but the addition of the entrainer (10% w/w) produced an important effect, increasing the astaxanthin extraction to values of 57.9%, similar to extraction with acetone (63.3%).     

Optimization of Medium Composition for Cultivating Clostridium butyricum with Response Surface Methodology

ABSTRACT: Probiotic bacteria, such as some strains of Clostridium butyricum , have been frequently used as the active ingredient in functional foods. However, slow growth of the probiotic bacteria has been one of the big concerns for their potential commercial application. In the present study, a fractional factorial design was applied to investigate the main factors, namely, the concentrations of the glucose, tryptone, yeast extract, beef extract, cys-teine, (NH₄) ₂SO₄, NaCl, K₂HPO₄, and the medium initial pH that affected the growth of 1 probiotic strain, C. butyricum ZJUCB, currently preserved in our laboratory. Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium. The experimental results showed that the optimum medium for incubating the C. butyricum ZJUCB was composed of 2.44% (w/v) glucose, 2.08% yeast extract (w/v), 1% tryptone (w/v), 0.1% (NH₄)₂SO₄ (w/v), 0.1% NaHCO₃ (w/v), 0.02% MnSO₄. H₂O (w/v), 0.02% MgSO₄. 7H₂O (w/v), 0.002% CaCl₂ (w/v), and 2% agar (w/v) (if necessary) at pH 8.55. After incubation for 24 h in the optimum medium, the populations of the viable organisms could reach 10⁹ colony-forming units (CFU)/mL, which was 100 times higher than that incubated in the initial medium.     

高铁营养酵母发酵培养及营养评价

采用生长周期短，易于工业化生产酵母为菌株，以糖蜜为主要培养基，通过现代发酵工程技术，在3m^3发酵罐上进行中间试验，在12m^3发酵罐进行试生产，发酵产品高铁营养酵母铁的含量1061mg／kg，高铁营养酵母的产率2．54％(w／v)，蛋白质的含量51．90％(w／v)，为工业化生产提供科学依据，并探讨作为食品添加剂的营养价值。     

Resistance of Escherichia coli and Salmonella against nisin and curvacin A.

We have determined the effects of the following factors on the resistance of Gram-negative bacteria against nisin and curvacin A: (i) chemotype of the lipopolysaccharide (LPS), (ii) addition of agents permeabilizing the outer membrane, (iii) the fatty acid supply of the growth medium, and (iv) the adaptation to acid and salt stress. Bacteriocin activity was determined against growing and resting cells as well as protoplasts. All smooth strains of Escherichia coli and Salmonella enterica serovar Typhimurium were highly resistant towards the bacteriocins, whereas mutants that possess the core of the LPS, but not the O antigen, as well as deep rough LPS mutants were sensitive. Antibiotics with outer membrane permeabilizing activity, polymyxin B and polymyxin B nonapeptide, increased the sensitivity of smooth E. coli towards nisin, but not that of deep rough mutants. Incorporation of 1 g l(-1) of either oleic acid or linoleic acid to the growth media greatly increased the susceptibility of E. coli LTH1600 and LTH4346 towards bacteriocins. Both strains of E. coli were sensitive to nisin and curvacin A at a pH of less than 5.5 and more than 3% (w/v) NaCl. Adaptation to sublethal pH or higher NaCl concentrations (pH 4.54 and 5.35 or 4.5% (w/v) NaCl) provided only limited protection against the bacteriocidal activity of nisin and curvacin A. Adaptation to 4.5% (w/v) NaCl did not result in cross protection to bacteriocin activity at pH 4.4, but rendered the cells more sensitive towards bacteriocins.     

Changing in processing yield and physical properties of frozen white shrimp (Penaeus vannamei) treated with lysine and sodium bicarbonate

The objective of this research was to investigate the phosphate alternative use of natural compound, lysine with sodium bicarbonate (NaHCO₃), at low concentration for freezing of white shrimp (Penaeus vannamei). Shrimp were treated with lysine, NaHCO₃ and lysine with NaHCO₃ at various concentrations and frozen in an air‐blast freezer. Thawing yield, cooking yield, colour, texture and nanostructure of the sample were studied compared to the control (nontreated sample) and sodium tri‐polyphosphate (STPP) treated one. Use of lysine/NaHCO₃ each at 1% (w/v) could improve water holding capacity (WHC) of the frozen shrimp, increasing cooking yield to 100.45% (w/w), comparable to the 101.73% (w/w) of STPP‐treated sample. The colour of the noncook‐thawed shrimp was also improved. Microstructure and lipid oxidation of the treated samples were also studied. The combination of lysine and NaHCO₃ indicated high potential use as phosphate alternative for frozen white shrimps.     

高产DHA裂壶藻突变株的选育

利用60Co-γ射线辐照诱变裂壶藻（Schizochytrium limacinum）ATCC20888,以平板生长情况、生物量、含油率和二十二碳六稀酸（DHA）产量为综合指标,最终选育出一株高产突变株,命名为1.6-7-1,其摇瓶发酵生物量为47.37 g/L,油脂含量为31.41%,DHA产量为5.65 g/L,且DHA产量较出发菌株提高了约40%,经连续5代发酵培养,其遗传性能稳定。     

Determination of total mercury in seafood by cold vapor-atomic absorption spectroscopy (CVAAS) after microwave decomposition

A method was developed for determination of total Hg in seafood using 10% w/v SnCl₂ · 2H₂O and continuous flow CVAAS after microwave decomposition in closed vessels. Seafoods were decomposed with 5 mL HNO₃ and 1 mL 1% w/v NaCl at 200 °C, transferred to polypropylene tubes containing 3.5 mL HCl, and diluted to 50 mL with H₂O. Standards were prepared in diluent containing 10% v/v HNO₃, 7% v/v HCl, and 0.02% w/v NaCl. Potential interference by 22 elements was evaluated. Interference by Se and Au was observed. Stability of standard solutions in HNO₃, HCl and NaCl was evaluated. Stabilizing effect of chloride was demonstrated. Results for six reference materials containing 0.0371–1.59 mg/kg Hg were 86–106% of certificate values. Concentrations in 11 varieties of seafood were 0.015–1.78 mg/kg. Average recoveries of inorganic and organic Hg added to seafood were 102% and 99%, respectively; average recoveries from fortified method blanks were 100% and 97%, respectively. The limits of quantitation were 0.0022 and 0.011 mg/kg in seafoods and reference materials, respectively.     

Degradation of curdlan using hydrogen peroxide

Curdlan, a linear glucan interconnected by β-(1 → 3) linkages, is soluble in alkaline solutions but not in water, which limits its wide application, particularly in the food industry. In this study, curdlan was subjected to oxidative degradation using hydrogen peroxide (H₂O₂). The optimal hydrolysis conditions were determined, and the results were as follows: reaction time, 40 min; temperature, 60 °C; H₂O₂ concentration, 1.5% (v/v); and NaOH concentration, 2.5 M. Under these optimised conditions, the maximum dextrose equivalent value (13.49%) was obtained. The composition and the structure of the hydrolysates were characterised by high-performance liquid chromatography and Fourier-transform infra-red spectroscopy, respectively. The hydrolysates were filtered, neutralised with HCl, concentrated to ∼12% (w/v), desalted, and freeze dried to yield a water-soluble, white powder. The (1 → 3)-β-d-glucan oligosaccharide content of the product was 98.6% and the yield was 91.4% (w/w).     

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations

The highly NaCl-tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra- and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl-tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species.     

Strategies for reducing supplemental medium cost in bioethanol production from waste house wood hydrolysate by ethanologenic Escherichia coli: inoculum size increase and coculture with Saccharomyces cerevisiae

In this paper, we report a simultaneous realization of both efficient ethanol production and saving medium nutrient (corn steep liquor [CSL]) during bioethanol fermentation of overliming-treated hydrolysate of waste house wood (WHW) using ethanologenic Escherichia coli KO11. In cultivation using WHW hydrolysate supplemented with 4% (v/v) CSL and 0.2 g-dry cell weight (DCW)/l E. coli KO11 cells, the overall ethanol yield reached 84% of the theoretical value at 61 h. When we conducted the cultivation with 1% CSL to reduce the supplemental medium cost, the overall ethanol yield remained in the range of 66-72% even at 90 h. We proposed two alternative methods for increasing the overall yield even with 1% CSL. The first method involved increasing the inoculum size of E. coli KO11 up to 0.8 g-DCW/l, where 83% of the overall yield was attained at 60 h of cultivation. The second method involved the coculture of 0.2 g-DCW/l E. coli KO11 together with 0.02 g-DCW/l of Saccharomyces cerevisiae TJ1, and the overall yield reached 81% at 47 h of cultivation.     

FED-BATCH FERMENTATION FOR RAPID PRODUCTION OF XANTHAN BY XANTHOMONAS CAMPESTRIS

The object of the present work was to optimise inoculum size and nitrogen source for rapid production of xanthan by Xanthomonas campestris by fed-batch fermentation. The fermentation period for production of xanthan by X. campestris decreased from 72 h to 48 h by using 30% (v/v) inoculum instead of the usual 10% level. Among various nitrogenous substances tested in the medium having the C/N ratio of 28, the best concentration of 1.9% (w/v) of xanthan with a viscosity of 10,000 cP was obtained for KNO₃ medium. Intermittent harvests operated for three cycles of 48 h each, using 30% inoculum resulted in xanthan broth of high viscosity (9000 cP) with a concentration of 1.9% and a yield of 68% (w/w). Potassium nitrate was a better suited nitrogen source for the medium used for rapid production of xanthan.     

绿色高效连续分离技术制备蛋黄中活性蛋白质和磷脂

为了实现蛋黄功能性成分的充分利用，采用绿色溶剂从新鲜蛋黄中高效连续分离具有生物活性的卵黄免疫球蛋白（immunoglobulin Y, IgY）、卵黄高磷蛋白（phosvitin, PV）和磷脂（phospholipids, PL）。首先采用水稀释法（稀释倍数为1:10）分离水溶性组分与脂蛋白，通过35%饱和(NH₄)₂SO₄和0.5% NaCl（w/v）对上清液进行盐析并用8% PEG 6000纯化IgY。脂质组分中的PV在pH5.0，90 ℃条件下的NaCl溶液（w/v）中进一步纯化。采用乙醇/乙酸乙酯比例为1:2（v/v）提取蛋黄中全部脂类，进一步采用乙酸乙酯在0 ℃下提纯蛋黄总脂中的PL。经脱盐、冷冻干燥后，IgY、PV和PL的纯度分别为97.38%、78.63%和85.94%。IgY和PV的得率分别为6.15、10.15 mg/mL。蛋黄总脂中PL的含量为35.18%。因其良好的多不饱和脂肪酸/饱和脂肪酸比率（0.70）和相对较低的n-6/n-3比率（5.37），该PL产品可应用于食品加工中。本方法简便、环保并且高效，可从蛋黄中依次分离出高纯度IgY、PV和PL，并为蛋黄的综合利用提供技术支持。     

PREDICTIVE MODEL FOR THE COMBINED EFFECT OF TEMPERATURE,pH, SODIUM CHLORIDE,AND SODIUM PYROPHOSPHATE ON THE HEAT RESISTANCE OF ESCHERICHIA COLI O157:H71

The effects and interactions of heating temperature (5–62.5C), pH (4 – 8), NaCl (0 – 6%, w/v), and sodium pyrophosphate (0 – 0.3%, w/v) on the heat resistance of a four strain mixture of Escherichia coli O157:H7 in beef gravy were examined. Thermal death times were determined using a submerged coil heating apparatus. The recovery medium was plate count agar supplemented with 1% sodium pyruvate. Decimal reduction times (D-values) were calculated by fitting a survival model to the data with a curve fitting program. The D-values were analyzed by second order response surface regression for temperature, pH, NaCl and sodium pyrophosphate levels. The four variables interacted to affect the inactivation of the pathogen. Thermal resistance of E. coli O157:H7 can be lowered by combining these intrinsic factors. A mathematical model describing the combined effect of temperature, pH, NaCl and sodium pyrophosphate levels on the thermal inactivation of E. coli O157:H7 was developed. The model can predict D-values for any combinations of temperature, pH, NaCl and sodium pyrophosphate that are within the range of those tested.     

重组猕猴桃果胶甲酯酶抑制剂发酵条件优化

在获得含高拷贝猕猴桃果胶甲酯酶抑制剂基因的重组菌株GS115基础上,通过分析甘油加入量、甲醇添加量、山梨醇与甲醇添加比例、诱导初始p H、诱导时间、培养基类型对重组猕猴桃果胶甲酯酶抑制剂(kw PMEI1)表达量的影响,优化了摇瓶发酵条件。结果表明:甘油加入量3%(v/v)、甲醇添加量1%(v/v)、山梨醇溶液(100%,w/v)和甲醇(分析纯)以体积比为1∶1的比例添加、p H5.5、在BMMY培养基中诱导表达5 d为最佳条件。该重组蛋白kw PMEI1的表达量最高可达700.302 mg/L。