培养条件对乳酸菌发酵剂抗冷冻干燥性能影响的研究进展

乳酸菌发酵剂应具有高活力、耐贮藏性以及对不同环境的适应性,特别是在冷冻干燥过程中活性的保持。本文从乳酸菌发酵剂菌种的内在特性、培养时间、培养基成分、培养温度及pH值的变化等方面综述了不同培养条件对乳酸菌发酵剂抗冷冻干燥性能影响的研究进展。说明乳酸菌在冷冻干燥中的存活率依赖于它的内在基因,基因差别决定了不同的乳酸菌对冷冻干燥的抗性存在差异;同时与细胞膜成分以及菌株形态有一定的关联;通常生长在稳定期的乳酸菌其抗冷冻干燥能力较强;培养基成分对菌株的影响因菌株不同而异;较低的培养温度和pH值,有助于提高乳酸菌的抗冷冻干燥性能。本文为提高乳酸菌的冻干存活率提供一些参考。     

自然发酵肉制品中乳酸菌的分离及特性研究

以MRS和改良TJA(改良番茄汁琼脂培养基)培养基为基础培养基,从自然发酵肉品中分离得到8个菌株,其中4株为乳酸菌,3株为杆菌,1株为球状菌。进一步测试表明:4株乳酸菌在30℃培养时,生长最好;在pH3.5～4.0时生长良好;4株乳酸菌在10%NaCl的环境中均能生长,其中以2%～6%的NaCl浓度内生长最适宜;属兼性厌氧菌;对大肠杆菌产生一定的抑制作用。分离出的4株乳酸菌均适宜作为发酵肉制品的发酵剂。     

自然发酵肉制品中乳酸菌的筛选

为获得适合肉制品发酵的优良乳酸菌菌株,对来源于自然发酵肉制品中的25株乳酸菌进行筛选,其中19株在6 g/100 mL NaCl和150 mg/kg NaNO_2液体培养基中生长较好,结合其他发酵特性实验,13株乳酸菌符合肉制品发酵剂标准,挑选其中3株进行产酸能力、生长曲线及不同温度条件下生长情况的研究。结果表明:3株菌均具有较强的增殖及产酸能力;培养12 h时菌株即进入稳定生长期,培养24 h时培养基pH值降到4左右;在肉制品的发酵温度范围内能较好地生长。经形态学和16S rRNA序列分析,3株菌均为弯曲乳杆菌,它们均可作为潜在的开发肉制品发酵剂的出发菌株。     

分离自内蒙古传统肉肠乳酸菌的生长及产酸能力研究

对从内蒙古传统肉肠中分离的8株乳酸菌在不同温度、pH条件下的生长能力以及在不同培养基中的产酸能力进行研究。结果显示,8株菌株在不同温度及不同pH条件下均能够生长,在30℃及pH6.5的条件下生长能力最强。8株乳酸菌在MRS液体培养基、MRS+6%NaCl+150mg/kgNaNO2培养基及模拟肉汤培养基中培养1～13d的产酸能力基本一致,在不同培养基中均有很强的产酸能力,所有菌株在培养24h后pH均迅速下降至4.2以下,此后逐渐趋于平缓。     

发酵肉制品中乳酸菌计数培养基的优化

采用平板计数法比较了5种乳酸菌在4种培养基(MRS、SL、TJA与酪蛋白山梨酸培养基)上的生长情况,选择乳酸菌生长最好的培养基,对其pH值、添加山梨酸和纳他霉素进行改良,检测了5种乳酸菌及6种杂菌在此培养基上的生长情况,并用发酵肉制品样品进行验证.结果表明,采用改良TJA培养基对发酵肉制品中乳酸菌计数效果最好,在pH 5.5、添加山梨酸(1000mg/L)和纳他霉素(20mg/L)条件下能排除其他杂菌的干扰,提高乳酸菌计数的准确性.优化后的改良TJA培养基能较好地排除发酵肉制品中的杂菌对乳酸菌计数的影响,可应用于发酵肉制品中乳酸菌的计数.     

降亚硝酸盐乳酸菌的鉴定及生长特性的研究

从传统发酵食品中分离乳酸菌,筛选鉴定降解亚硝酸盐能力较强的菌株,并研究其在白菜汁培养基中的生长及产酸情况,为制作泡菜发酵剂储备优良菌株。结果分离了144株乳酸菌,并获得5株亚硝酸盐降解率在99%以上的菌株,经鉴定菌株Mao21.1和Mao6.2为戊糖乳杆菌,Wang3.1和Mao20.1为植物乳杆菌,Lin2.4为戊糖片球菌。5株菌在白菜汁培养基中生长4h后,各菌株培养液的pH均快速下降;培养20h后,除菌株Mao21.1外,其他菌株培养液pH均降到3.5以下;对数生长末期菌株Mao6.2和Lin2.4活菌数达到108cfu/mL,该两株菌可作为制作泡菜发酵剂的储备菌株。     

乳酸菌耐酸机理的研究

为研究乳酸菌的耐酸机理,对4株乳酸菌株在人工胃液中的生存率及葡萄糖的影响作了分析,当葡萄糖存在时,耐酸菌株的生存率大大提高;培养24h后,耐酸菌株的生物量大于非耐酸菌株,菌液的pH值低,非耐性菌株在酸性培养基的生长情况较差;耐酸菌株能够消耗人工胃液中的葡萄糖并使悬浊液的pH值升高;耐酸菌株的ATP酶活性小于非耐酸菌株。以上的结果可以看出,耐酸菌株在酸性环境中可以较好的生长,并具有可以在强酸性条件下利用碳源(如葡萄糖)进行代谢的耐酸机理,而ATP酶活性不是影响耐酸性的主要因素。     

乳酸菌耐酸机理的研究

为研究乳酸菌的耐酸机理，对4株乳酸菌株在人工胃液中的生存率及葡萄糖的影响作了分析，当葡萄糖存在时，耐酸菌株的生存率大大提高；培养24h后，耐酸菌株的生物量大于非耐酸菌株，菌液的pH值低，非耐性菌株在酸性培养基的生长情况较差；耐酸菌株能够消耗人工胃液中的葡萄糖并使悬浊液的pH值升高；耐酸菌株的ATP酶活性小于非耐酸菌株。以上的结果可以看出，耐酸菌株在酸性环境中可以较好的生长，并具有可以在强酸性条件下利用碳源（如葡萄糖）进行代谢的耐酸机理，而ATP酶活性不是影响耐酸性的主要因素。     

分离自腊鱼的乳酸菌生长及产酸特性

采用Gompertz模型拟合从腊鱼中分离的5株乳酸菌的生长和产酸曲线,分析其生长和产酸特性。结果表明：Gompertz模型对乳酸菌的生长和产酸曲线的拟合精度很高;在生长模型和产酸模型中,5株乳酸菌生长的早期适应性相似,其中乳酸片球菌和食品乳杆菌的生长速率(0.32h-1)和产酸速率(0.32h-1)高于其他菌株;在生长模型中两者平衡菌数最高(分别为2.48和2.49);在产酸模型中两者平衡pH值最低(都为3.76);乳酸菌培养液的pH值与总菌数OD600nm值显著线性负相关。     

8株窖泥链霉菌对酸、乙醇的适应性研究

对分离自窖泥的8株放线菌进行鉴定,并考察其对窖泥环境中pH值、乙醇的耐受性.采用ISP2液体培养基在pH值2.0～3.5、乙醇浓度3%vol ～6%vol条件下培养链霉菌菌株,结果显示,分属于链霉菌属的8个种的8株放线菌均不能耐受pH值2.0及6%vol的乙醇浓度,均能耐受pH值3.5及3%vol的乙醇浓度,5株链霉菌在pH值3.0时可以生长,2株链霉菌可耐受5%vol乙醇,说明分离自窖泥的多数链霉菌对窖泥环境的适应能力较好,但不同种链霉菌的适应能力存在差异,窖泥环境在长期生产过程中对链霉菌属菌株具有一定的选择驯化作用.     

Growth characteristics of virulent Bacillus anthracis and potential surrogate strains

The objectives of this study were to compare generation and lag times of virulent Bacillus anthracis strains with those of other Bacillus strains, to identify possible surrogates for growth studies, and to determine if the B. cereus module of the U.S. Department of Agriculture Pathogen Modeling Program (PMP) had predictive value for B. anthracis. Growth characteristics of B. anthracis, B. cereus, B. mycoides, and B. subtilis strains in brain heart infusion broth at pH 6.5, 6.0, and 5.5 were determined by absorbance measurements. Growth curves of B. anthracis Sterne and B. cereus strains appeared similar, and the generation times for strain Sterne fell within the PMP's 95% confidence interval for B. cereus. However, the virulent B. anthracis strains Vollum and Pasteur had shorter generation times than the avirulent Sterne strain and most other surrogates and were lower than the PMP's 95% confidence interval for B. cereus. Growth curves of B. cereus ATCC 9818 and B. subtilis ATCC 6633 were more similar to those of virulent B. anthracis strains, but all potential surrogates had significantly different generation times and lag times under some conditions.     

Growth responses of Staphylococcus aureus and Streptococcus agalactiae to Corynebacterium bovis metabolites

Staphylococcus aureus and Streptococcus agalactiae growth responses to metabolites of Corynebacterium bovis cultured in media containing polyoxyethylenesorbitan monolaurate, monooleate, or trioleate and milk were determined. Filter sterilized metabolites of 48-h C. bovis cultures in synthetic media were added to cultures of Staph. aureus and Strep. agalactiae. Staphylococcus aureus and Strep. agalactiae were inoculated into 12-h C. bovis milk cultures. Growth responses of Staph. aureus and Strep. agalactiae were not affected by C. bovis metabolites of synthetic media. Staphylococcus aureus growth was inhibited during logarithmic and stationary phases in milk containing mixed cultures of C. bovis compared with growth in pure Staph. aureus cultures. Streptococcus agalactiae growth curves were similar in pure and C. bovis mixed cultures. Fatty acid compositions were not different in sterile milk and milk containing bacterial cultures. Growth responses of Staph. aureus and Strep. agalactiae were not related to concentration of C. bovis metabolites or fatty acid content of media in which C. bovis were cultured.     

Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production

Bifidobacterium adolescentis Int57 (Int57) and Propionibacterium freudenreichii subsp. shermanii ATCC 13673 (ATCC 13673) were grown either in coculture or as pure cultures in different media, such as cow's milk, soybean milk, and modified MRS medium. The viable cell counts of bacteria, changes in pH, concentrations of organic acids, and contents of various sugars were analyzed during incubation up to 7days. In soy milk, the survival of cocultured Int57 was six times higher than the monocultured cells, and ATCC 13673 cocultured with Int57 consumed 69.4% of lactic acid produced by Int57 at the end of fermentation. In cow's milk, coculture with ATCC 13673 increased the growth of Int57 from 24h until 120h by approximately tenfold and did not affect the survival of Int57 cells. After 96h of fermentation of modified MRS, the survival of ATCC 13673 cells cocultured with Int57 increased by 3.2- to 7.4-folds as compared with ATCC 13673 monoculture, whereas the growth of Int57 cells was unaffected. The growth and metabolic patterns of two strains during coculture showed noticeable differences between food grade media and laboratory media. The consumption of stachyose in soy milk during coculture of Int57 with ATCC 13673 was increased by more than twice compared with Int57 monoculture, and completed within 24h. The combinational use of Bifidobacterium and Propionibacterium could be applied to the development of fermented milk or soy milk products.     

OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF EICOSAPENTAENOIC ACID BY MORTIERELLA

This research characterized the optimum growth conditions of Mortierella for accumulation of eicosapentaenoic acid (EPA). Eight strains of Mortierella, NRRL 5513, 2761, 1757, 6337, 2493 and ATCC 24786, 16271, 16266, were evaluated for their ability to produce EPA. Temperature, temperature shifting, media, and media supplementation (carbon/nitrogen sources, growth factors and antibiotic supplementation) were studied to determine their effect on mycelial mass and EPA accumulation. M. elongata ATCC 16271 was the most effective strain for EPA production when grown at 18C, in yeast malt broth, pH 5. 1, and harvested after 7 days, yielding 66 mg EPA/g dry mycelial cells. In another experiment, temperature shifting (2 days at 24C, to 5 days at 12C) increased the yield of EPA about eight times to 32 mg EPA/g dry mycelial celis from 4 mg EPA/g dry mycelial cells for nontemperature shifted cultures. This research describes methodologies that resulted in EPA content higher than any previously reported.     

Growth kinetics and cell morphology of Listeria monocytogenes Scott A as affected by temperature,NaCl, and EDTA

Growth kinetics and morphological characteristics of Listeria monocytogenes Scott A grown under stress conditions induced by increasing levels of NaCl and EDTA were studied as a function of temperature. L. monocytogenes Scott A was inoculated into brain heart infusion broth (pH 6) at 19, 28, 37, and 42 degrees C. Test cultures contained NaCl (at concentrations of 4.5, 6.0, and 7.5%) or EDTA (at concentrations of 0.1, 0.2, and 0.3 mM); control cultures contained 0.5% NaCl. Growth curves were fitted from plate count data by the Gompertz equation, and growth kinetics parameters were derived. Stationary-phase cells were examined by scanning and transmission electron microscopy. Generation times (GTs) and lag phase duration times (LPDs) increased as additive levels were increased. The bacterium grew at all NaCl levels. At 37 and 42 degrees C, growth was slow in media containing 7.5% NaCl, and no growth occurred in media containing 0.3 mM EDTA. Temperature was a major factor in certain stress conditions that led to cell elongation and loss of flagella. Cells in control media at 28 degrees C grew as short rods (0.5 by 1.0 to 2.0 microm), while at 42 degrees C most cells were 4 to 10 times as long. Higher levels of NaCl at higher temperatures resulted in longer and thicker cells. At 28 degrees C, 0.1 mM EDTA had little effect on growth kinetics and morphology; however, 0.3 mM EDTA caused a sixfold increase in GT and LPD and loss of flagellae, with most cells being two to six times as long as normal. Cell length did not correlate with growth kinetics. The results of this study suggest that the effect of altered morphological characteristics of L. monocytogenes cells grown under stress on the virulence and subsequent survival of these cells should be investigated.     

Influence of Potassium Sorbate and pH on Ten Strains of Type A and B Clostridium botulinum

Growth of each of ten strains of C. botulinum was measured as the average A_630nm of five replicate peptone-yeast extract-glucose (PYEG) broth cultures at 37°C. Media ranged in pH from 7.10–5.10 with potassium sorbate concentrations of 0–0.26%. Growth Ratios [GR = treatment/control] based on time to reach A₆₃₀=0.35 were calculated. The linear regressions of log [GR] vs log [undissociated sorbic acid in mg/L] or [1/GR] vs [pH] were used to predict GR's at 250 mg/L undissociated sorbic acid (pH 5.65 and 0.26% sorbate) and at pH 5.65 (0% sorbate) respectively. Among the ten strains examined, statistically significant differences (P<0.05) in resistance to pH and sorbate among strains and for the same strain from different spore suspensions were discovered. Resistance to pH and sorbate appeared to be correlated.     

壳聚糖复合生物防腐剂的抑菌效果研究

本实验研究了以壳聚糖及其金属锌螯合物为主复配的五个生物防腐剂配方以及配方中的主要组分在不同pH条件下的抑菌效果。研究选定的五种指示菌是大肠杆菌(Escherichia coli)8099、金黄色葡萄球菌(Staphylococcus aureus)ATCC6538、枯草杆菌黑色变种(Bacillus subtilis)ATCC9372、白色念珠菌(Candida albicans)ATCC10231和黑曲霉(Aspergillus niger ATCC16404),采用平板培养计数法检测了从加入不同种类的菌悬液至含抑菌剂和培养基的试管中培养0、4、24、48、72和96h的活菌残留数,以活菌残留数的对数值随时间变化的趋势表示抑菌效果。结果表明:配方A、B、D、E在p H6.0以下可以抑制五种指示菌。配方A～E和单组份壳聚糖金属锌螯合物对大肠杆菌、金黄色葡萄球菌和枯草芽胞杆菌的抑制效果好,而对羟基苯甲酸乙酯主要对枯草芽胞杆菌和黑曲霉的抑制较好,对其余三种菌的抑制效果较差,所有配方对白色念珠菌的抑菌效果较其它四种指示菌的抑制效果差,这是由于实验的起始菌悬液浓度较高造成的,同一配方在起始菌悬液浓度和作用pH值均低的情况下抑菌效果好。     

Effect of dimorphic regulation on heterologous glucose oxidase production by Mucor circinelloides

The production of Aspergillus niger glucose oxidase (GOX) and native amylase by the recombinant M. circinelloides KFA199 strain under conditions of dimorphic growth was investigated. The recombinant KFA199 strain was compared to its parental ATCC 1216b strain and a wild‐type CBS 232.29 strain under similar morphology‐controlled conditions. Cultivation in Vogel's medium supplemented with ergosterol/Tween‐80 and sparged with nitrogen gas was most suitable for yeast‐like biomass production under anaerobic conditions. Anaerobic growth was characterized by high levels of ethanol formation and linear growth rates of 0.24–0.05/h, indicating metabolic stress. Subsequent to anaerobic growth, cultures were shifted to aerobic conditions to induce aerobic mycelial growth. GOX produced by the recombinant KFA199 after the shift to aerobic conditions was poorly secreted and accumulated intracellularly to 0.56 U/ml_culture. Amylase production by the KFA199, ATCC12b and CBS 232.29 strains was determined during growth on starch after the shift to aerobic culture. Growth‐associated amylase production by the ATCC 1216b (0.63 U/ml_culture) and wild‐type CBS 232.29 (0.33 U/ml_culture) strains was substantially higher than by the recombinant KFA199 strain (0.07 U/ml_culture), which may be related to the leucine auxotrophy of the transformation host, or genetic changes induced during transformation of the KFA199 strain. Copyright © 2010 John Wiley & Sons, Ltd.     

Absence of association of autoinducer-2-based quorum sensing with heat and acid resistance of Salmonella

This study used various approaches to investigate the potential association of autoinducer-2 (AI-2) presence with thermal and acid resistance of Salmonella cultures. Salmonella Thompson strains RM1987N (luxS-positive; AI-2 positive) and RM1987NLUX (luxS-negative; AI-2 negative) were exposed to 55 °C (6 h) in Luria-Bertani (LB) broth, while the luxS-negative S. Thompson strain and a Salmonella Typhimurium luxS-positive strain were exposed to 55 °C in AI-2-positive or -negative preconditioned (PC) media derived from S. Thompson and Escherichia coli O157:H7 luxS-positive and -negative strains. In addition, the luxS-negative S. Thompson strain was subjected to pH 3.5 PC media (35 °C, 6 h) with or without AI-2 activity, and acid-adapted or nonadapted S. Thompson strains were exposed to pH 3.0 LB broth (35 °C, 6 h). Surviving bacterial populations during exposure to 55 °C LB were not different between luxS-negative and -positive S. Thompson strains. In addition, heating at 55 °C of the luxS-negative S. Thompson strain in AI-2-positive and -negative PC media resulted in similar (P ≥ 0.05) survivor counts. Furthermore, surviving cell counts of S. Typhimurium (luxS-positive) in 55 °C AI-2-positive PC media were not different (P ≥ 0.05) than those in AI-2 negative PC media. No differences in surviving cell counts of the luxS-negative S. Thompson strain was found when exposed to pH 3.5 AI-2-positive and -negative PC media. Also, survivors of acid-adapted or nonadapted cells of luxS-negative and -positive S. Thompson strains were not different following exposure to pH 3.0 LB. The results indicated that, under the conditions of this study, AI-2-based quorum sensing did not appear to be associated with heat and acid resistance of Salmonella.     

Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores

Spores of Bacillus cereus strains ATCC 7004, ATCC 4342 and ATCC 9818 were produced in four sporulation media (Nutrient Agar supplemented with 1 ppm Mn²⁺, Fortified Nutrient Agar, Angelotti Medium and Milk Agar) and their percentages of sporulation and heat resistance parameters obtained in a wide temperature range were compared. In all conditions studied, high rates of sporulation were obtained. Clear differences among D-values for spores produced in the four media were observed. the medium which yielded the most resistant spores and the magnitude with which the sporulation medium affected D-values was different for each strain. z-Values of the three strains were not influenced by the medium used to obtain spores.