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.     

响应面法优化乳酸链球菌素发酵培养基成分

乳酸链球菌素由于对革兰氏阳性腐败和致病菌的厂。谱抗菌活性而作为天然防腐剂在食品工业中广泛使用。为提高乳酸链球菌素发酵水平,以乳酸乳球菌（Lactococcus lactis subsp．1actis）TCCC12001为生产菌株,采用响应面法对发酵培养基进行了优化,确定了该菌发酵的最佳培养基配方（g／L）：蔗糖20、酵母粉10、蛋白胨9、牛肉膏5、K2HP04·121-12010、NaCl2、MgS04·7H2O 0．2。最适发酵条件为：培养基初始pH值7．0,发酵温度30c,种龄10h,接种量3％,培养10h。在此条件下乳酸链球菌素的效价最高达1436IU／mL,比初始效价提高了69％。     

Optimization of the fermentation medium for alpha-galactosidase production from Aspergillus foetidus ZU-G1 using response surface methodology

ABSTRACT:  The optimization of fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was investigated in shaker flask fermentation. A one-factor-at-a-time experiment was used to screen the preferable nutriment (carbon sources, nitrogen sources, and essential elements) for alpha-galactosidase production. A fractional factorial design was used to screen the main 5 factors, soybean meal, wheat bran, KH₂PO₄, FeSO₄·7H₂O, and the medium initial pH, that affected the production of alpha-galactosidase. The central composite experimental design was further adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 3.2% soybean meal (w/v), 2% wheat bran (w/v), 0.1% KH₂PO₄ (w/v), and 0.05% FeSO₄·7H₂O (w/v); initial medium pH was 6.31. The results further predicted that alpha-galactosidase activity reached 64.75 U/mL after 96-h incubation in this medium, which was approximately 7 times higher than that incubated in the nonoptimized medium. The time course of alpha-galactosidase production in the optimized medium composition was also carried out to validate the model.     

Optimization of beta-carotene production by Rhodotorula glutinis using high hydrostatic pressure and response surface methodology

ABSTRACT:  Rhodotorula glutinis RG6 was treated by high hydrostatic pressure (HHP) of 300 MPa for 15 min for improving its ability of β-carotene production. After the treatments of 5 repeated cycles, the mutant strain RG6p was obtained, β-carotene production of which reached 10.01 mg/L, increased by 57.89% compared with 6.34 mg/L from parent strain RG6. To optimize the medium for β-carotene fermentation by mutant RG6p, a response surface methodology (RSM) approach was used in conjunction with a factorial design and a central composite design, and the maximum yield of β-carotene (13.43 mg/L), an increase of 34.17% compared to the control, was obtained at a pH 6.7 with an optimum medium (40 mL/250 mL) of yeast extract (4.23 g/L), glucose (12.11 g/L), inoculum (30 mL/L), tomato extract (2.5 mL/L), peanut oil (0.5 mL/L), and (NH₄)₂SO₄ (5 g/L).     

Isolation of Tomato Seed Meal Proteins with Salt Solutions

Salt solutions were used in isolating tomato seed meal proteins. Na₂SO₃ and NaCl solutions at different concentrations, and pH were included in a central composite design to find optimum conditions of protein isolation. The highest total protein yield was achieved with water extraction (no salt present). Salt extraction at pH 7.5 produced isolates with protein content of 93.4% (NaCl 5% w/v) and 77.1% (Na₂SO₃ 0.5% w/v). Observed values were in good agreement with predicted values. Isolates extracted with different salt solutions ranged from less soluble but very resistant to heat and Ca²⁺, to very surface active with functional properties comparable to commercial soy isolates.     

大豆锰超氧化物歧化酶基因在乳酸乳球菌中的分泌表达

为使大豆锰超氧化物歧化酶（Mn superoxide dismutase,MnSOD）在乳酸乳球菌中高效表达,将克隆的MnSOD基因开放阅读框序列分别重组到质粒pNZ8149和经改造的质粒pNZS上,表达载体pNZ-SOD和分泌表达载体pNZS-SOD,将两者分别以电穿孔法转化乳酸乳球菌L. lactis NZ3900,经Elliker琼脂板筛选、聚合酶链式反应（polymerase chain reaction,PCR）、酶切鉴定正确后,获得的两重组菌株加入Nisin进行诱导表达,对L. lactisNZ3900/pNZ-SOD和L. lactis NZ3900/pNZS-SOD的表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodiumdodecyl sulfate - polyacrylamide gel electrophoresis,SDS-PAGE）和酶活性对比分析。结果显示：L. lactis NZ3900/pNZS-SOD菌株表达SOD总活性约是L. lactis NZ3900/pNZ-SOD菌株的1.6 倍,是L. lactis NZ3900/pNZ8149的13.5倍,且可将表达的约2/3的SOD分泌到胞外,表明经改造的乳酸菌分泌表达系统L. lactis NZ3900/pNZS能够分泌表达SOD,并增强SOD的表达。     

腐乳源乳酸乳球菌17M1高密度培养条件研究

为了获得乳酸乳球菌(Lactococcus lactis)17M1的高密度培养方法,以菌体密度和活菌数为评价指标,以豆腐乳清为基础培养基,采用响应面法对该菌株的培养基组成进行优化,并研究其培养条件。结果表明,菌株17M1的最佳培养基组成为：在豆腐乳清中加入2.00%大豆蛋白胨、胰酪蛋白胨1.70%、柠檬酸铵2.30%。在此优化培养基中初始pH值6.15,于37℃培养15 h,乳酸乳球菌17M1的活菌数达到了1.17×10¹⁰CFU/mL,高于M17肉汤培养基活菌数(1.04×10⁹CFU/mL)。该研究结果为乳酸乳球菌17M1的工业化应用提供了技术支持。     

营养因素对乳酸乳球菌DL-203生长及其Nisin生物合成的影响

对Nisin高产菌株乳酸乳球菌DL—203的营养要求及其 对Nisin生物合成的影响进行了研究。结果表明,乳链菌 肽的生物合成与DL-203的生长呈极显著正相关,乳酸 乳球菌DL—203生长和合成Nisin的最佳碳源和氮源分 别为蔗糖和大豆蛋白胨,其适宜浓度均为15～20g/L。磷 酸盐对乳酸乳球菌DL—203生长及其Nisin生物合成具 有极大的促进作用,其中以Na_2HPO_4效果最好,其浓度 在224mmol/L以下时对菌体生长和Nisin生物合成具有 较大的促进作用。     

EMS诱变甘蔗愈伤组织的初步研究

为了探索EMS对甘蔗愈伤组织进行化学诱变的合适浓度和时间组合,以ROC22和粤糖93-159甘蔗愈伤组织为材料,研究了不同浓度(8、24、40μmol/L)、不同时间(2、4、6 h)诱变处理对愈伤组织的每克鲜重相对增重量和相对分化率的影响。并通过分化试验选择适合诱变后愈伤组织分化的培养基。结果表明,适合诱变处理后愈伤组织分化的培养基为:MS基本培养基+2 mg/L KT+2 mg/L NAA+3.0%蔗糖(W/V)+0.8%琼脂(W/V)。适合EMS诱变甘蔗的处理为:24μmol/L诱变处理2 h或8μmol/L诱变处理6 h。     

Effect of Manganese Ions on the Fermentative Activity of Frozen-Thawed Lactobacilli

The lactic acid fermentation of monoand disaccharides by frozenthawed Lactobacillus plantarum in modified All Purpose Tween (MAPT) broth was stimulated by manganese ions (Mn₂₊). Beaker sausage formulated with either dextrose or sucrose attained pH 5.0 faster with Mn₂₊. L. plantarum frozen stored for 2 wk was stimulated by Mn₂₊ more than the same culture frozen stored for 2 yr. The fermentation period of sucrose with Mn₂₊ was shorter than that of dextrose. CaC12 (as a substitute for NaCI) and Mn₂₊ reduced the fermentation period more than NaCI. The fermentation of dextrose by L. lactis was reduced the most by Mn₂₊, followed by L. casei subspecies casei, L. plantarum and L. casei subsp. alactosus. Calcium ions were not efficient in reducing the fermentation time of dextrose by lactobacilli in summer sausage.     

Alginate- and gellan-based edible films for probiotic coatings on fresh-cut fruits

ABSTRACT:  Alginate- (2% w/v) or gellan-based (0.5%) edible films, containing glycerol (0.6% to 2.0%), N-acetylcysteine (1%), and/or ascorbic acid (1%) and citric acid (1%), were formulated and used to coat fresh-cut apple and papaya cylinders. Water vapor permeability (WVP) was significantly higher ( P < 0.05) in alginate films (0.30 to 0.31 × 10⁻⁹g m/Pa s m²) than in the gellan ones (0.26 to 0.27 × 10⁻⁹g m/Pa s m²). Addition of 0.025% (w/v) sunflower oil decreased WVP of gellan films (0.20 to 0.22 × 10⁻⁹ g m/Pa s m²). Water solubility of gellan and alginate films at 25 °C (0.47 to 0.59 and 0.74 to 0.79, respectively) and their swelling ratios (2.3 to 2.6 and 1.6 to 2.0, respectively) indicate their potential for coating high moisture fresh-cut fruits. Fresh-cut apple and papaya cylinders were successfully coated with 2% (w/v) alginate or gellan film-forming solutions containing viable bifidobacteria. WVP in alginate (6.31 and 5.52 × 10⁻⁹g m/Pa s m²) or gellan (3.65 and 4.89 × 10⁻⁹ g m/Pa s m²) probiotic coatings of papaya and apple, respectively, were higher than in the corresponding cast films. The gellan coatings and films exhibited better water vapor properties in comparison with the alginate coatings. Values > 10⁶ CFU/g B. lactis Bb -12 were maintained for 10 d during refrigerated storage of fresh-cut fruits, demonstrating the feasibility of alginate- and gellan-based edible coatings to carry and support viable probiotics on fresh-cut fruit.     

产叶酸菌株的筛选及其发酵条件的优化

从土壤样本中筛选分离得到的菌株R-1和S-1具有产叶酸的能力,进行单因素实验和正交分析,优化碳源、氮源、初始pH、发酵时间等条件,叶酸产量提高约为10%;加入前体物质对氨基苯甲酸(PABA),菌株R-1和S-1产叶酸的能力显著提高,分别提高了2倍和1.8倍;最后进行正交分析得出各菌种的最佳发酵产叶酸的因素选择。结果显示,菌株R-1的最佳产叶酸条件是葡萄糖25g/L,柠檬酸铵4g/L,PABA0.3g/L,接种量2%,培养时间24h,初始pH6.5,叶酸产量达到了1.92μg/mL;菌株S-1最佳产叶酸条件是蔗糖20g/L,柠檬酸铵2g/L,PABA0.3g/L,接种量3%,培养时间48h,初始pH5.5,叶酸产量为1.31μg/mL。     

味精粗料作为聚谷氨酸合成前体的培养条件优化

为降低聚谷氨酸生产成本,采用本实验室筛选得到的枯草芽孢杆菌,编号为CGMCC No.1250。考察了不同的谷氨酸钠替代品,味精粗料最佳,并考察了味精粗料的含量、碳源和氮源及其浓度、NaCl浓度、装液量以及温度等对-γPGA产量的影响。实验结果表明,对于该菌株,最适碳源和氮源分别是蔗糖和蛋白胨;在含有70 g/L蔗糖5、0 g/L蛋白胨、30 g/L NaCl,pH7.0,含味精粗料体积分数为26.6%(含谷氨酸120 g/L)的发酵液中,37℃,220 r/min培养24 h,-γPGA的产量达到41.7g/L。     

一株仅产L-乳酸的乳酸乳球菌发酵培养基的优化

为了研究一株分离自新疆牧民家庭自制酸马奶中的乳酸乳球菌KLDS 4.0325产L-乳酸的能力,以L-/D-乳酸试剂盒验证该菌种在发酵过程中所产L-乳酸的光学纯度为100%。利用Plackett-Burman设计法对影响该菌株发酵的培养基主要组分进行筛选,确定影响L-乳酸产量的主要因素为蔗糖、酵母粉、K2HPO4。在此基础上,采用响应面法优化发酵培养基的组成,结果表明：当蔗糖添加量为102.9 g/L、酵母粉添加量为2.5 g/L、K2HPO4添加量为7.9 g/L时,L-乳酸产量最大,可达86.6 g/L,在最优发酵条件下获得的实测值与模型预测值（86.3 g/L）吻合,说明所建立的模型是切实可行的。     

Optimization of Nisin Production by Lactococcus lactis UQ2 Using Supplemented Whey as Alternative Culture Medium

Abstract: Lactococcus lactis UQ2 is a nisin A-producing native strain. In the present study, the production of nisin by L. lactis UQ2 in a bioreactor using supplemented sweet whey (SW) was optimized by a statistical design of experiments and response surface methodology (RSM). In a 1st approach, a fractional factorial design (FFD) of the order 2^5-1 with 3 central points was used. The effect on nisin production of air flow, SW, soybean peptone (SP), MgSO₄/MnSO₄ mixture, and Tween 80 was evaluated. From FFD, the most significant factors affecting nisin production were SP (P = 0.011), and SW (P = 0.037). To find optimum conditions, a central composite design (CCD) with 2 central points was used. Three factors were considered, SW (7 to 10 g/L), SP (7 to10 g/L), and small amounts of added nisin as self-inducer (NI 34.4 to 74.4 IU/L). Nisin production was expressed as international units (IU). From RSM, an optimum nisin activity of 180 IU/mL was predicted at 74.4 IU/L NI, 13.8 g/L SP, and 14.9 or 5.11 g/L SW, while confirmatory experiments showed a maximum activity of 178 ± 5.2 IU/mL, verifying the validity of the model. The 2nd-order model showed a coefficient of determination (R²) of 0.828. Optimized conditions were used for constant pH fermentations, where a maximum activity of 575 ± 17 IU/mL was achieved at pH 6.5 after 12 h. The adsorption-desorption technique was used to partially purify nisin, followed by drying. The resulting powder showed an activity of 102150 IU/g. Practical Application : Nisin production was optimized using supplemented whey as alternative culture medium, using a native L. lactis UQ2 strain. Soybean peptone, SW, and subinhibitory amounts of nisin were successfully employed to optimize nisin production by L. lactis UQ2. Dried semipurified nisin showed an activity of 102150 IU/g.     

Incidence of nisin Z production in Lactococcus lactis subsp. lactis TFF 221 isolated from Thai fermented foods

Lactic acid bacteria isolated from various Thai fermented foods were screened for the presence of nisin gene by using PCR with primers specific to nisin A structural gene. Only one strain, Lactococcus lactis subsp. lactis TFF 221, isolated from kung jom, a traditional shrimp paste, was found to carry a nisin gene. The TFF 221 nisin had antimicrobial activity against not only closely related lactic acid bacteria but also some foodborne pathogens. It was heat stable and inactivated by alpha-chymotrypsin and proteinase K. Some characteristics of TFF 221 nisin were found to be very similar to those of nisin A produced by Lactococcus lactis subsp. lactis NCDO 2111. Both of them had the same antimicrobial spectrum and MICs against all indicator bacteria. However, when assayed with indicator organisms, in all cases the TFF 221 nisin produced larger zones of inhibition in agar diffusion assays than the nisin A did. Sequencing of the TFF 221 nisin gene showed that it was the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The nisin determinant in strain TFF 221 was found to be located on a conjugative transposon residing in the chromosome. The ability of the nisin produced by L. lactis subsp. lactis TFF 221 to inhibit a wide range of foodborne pathogens may be useful in improving the food safety of the fermented product, especially in the Thai environment, which suffers from perennial problems of poor food hygiene.     

响应面优化侧孢短芽孢杆菌产短杆菌素发酵培养基

对侧孢短芽孢杆菌fmb70-24产短杆菌素的发酵培养基进行优化.在单因素实验的基础上利用Plackett-Burman设计对影响短杆菌素含量的6个主要因素进行评价,筛选出具有显著效应的因素为镁离子、牛肉浸膏和蔗糖,并利用爬坡试验确定响应面试验的最佳区域,设计三因素三水平的Box-Behnken试验得到侧孢短芽孢杆菌产短...     

培养基与分割发酵优化促进Nisin生物合成

为了提高乳酸乳球菌（Lactococcus lactis）细胞生长和乳酸链球菌素（Nisin）的合成效率,以正交优化法研究培养基分批发酵和分割发酵方式对Nisin生物合成效率的影响。结果表明,优化发酵培养基配方为：5%蛋白胨,3%蔗糖,2%玉米浆,1%酵母浸粉。在此优化条件下,摇瓶培养（11 h）峰值生物量为4.9×109 CFU/mL,较对照提高43%;10 L发酵罐分批发酵峰值生物量、Nisin效价及Nisin合成速率■q分别为7.75×109 CFU/mL、2 573 IU/mL、151.4 IU/（mL·h）,较优化前分别提升38.0%、56.6%、38.2%。分批发酵培养18 h Nisin达到峰值后[■q为147 IU/（mL·h）],以不同比例分割发酵并继续培养7 h,第二次分割（25%）为Nisin合成最适发酵方式,其Nisin平均合成速率达到294 IU/（mL·h）,较分批发酵提高了100%。     

Antimicrobial activity of nisin incorporated in pectin and polylactic acid composite films against Listeria monocytogenes

An extruded composite food packaging film containing pectin, polylactic acids (PLAs) and nisin was developed to inhibit Listeria monocytogenes . The mechanical properties and surface structure of the film were also examined. Cells of L. monocytogenes were reduced by 2.1, 4.5 and 3.7 log units mL⁻¹ by the pectin plus PLA (pectin/PLA) film containing nisin (1000 IU mL⁻¹ of tested liquid) in Brain Heart Infusion (BHI) broth, liquid egg white and orange juice, respectively, after 48 h at 24 °C. Pectin played an important roll in embedding nisin into the film. The pectin/PLA film had a similar stiffness but lower tensile strength, elongation and fracture energy than the pure PLA film. These data suggested that nisin incorporated into the pectin/PLA film was an effective approach to reducing L. monocytogenes in a typical growth medium (e.g. BHI broth) as well as in foods (e.g. orange juice and liquid egg).     

Nisin的生产、提纯和检测

Nisin是一种由乳酸乳球菌产生的羊毛硫氨酸类细菌素 ,在许多国家被许可作为生物防腐剂。Nisin的产量受许多因素的制约 ,如产生菌性能、培养基组成 (碳源、氮源、磷源和阳离子 )、发酵条件 (pH、温度、搅拌、通风 )、发酵类型 (分批发酵、连续发酵、自由细胞、固定化细胞 )等 ;大规模回收和纯化Nisin主要采用一些基于吸附-解析或者相分配原理的方法 ;最常用的定量检测Nisin的方法主要有生物分析法和免疫检测法 ,采用各种特定nisin抗体的免疫检测方法具有迅速、灵敏、准确等特性并能实现Nisin的在线检测。