Enhancement of trehalose production in dairy propionibacteria through manipulation of environmental conditions

We have shown that the ability to produce trehalose is widespread within the genus Propionibacterium. Eighteen strains isolated from dairy sources were screened for trehalose synthesis; the effect of environmental conditions on trehalose production was evaluated in Propionibacterium freudenreichii ssp. shermanii NIZO B365, a strain that accumulated high amounts of this disaccharide. Lactose was the best carbohydrate source for trehalose production, whereas lactate, the substrate that led to the highest specific growth rate, was a poor precursor. Trehalose was consumed after exhaustion of the carbon source in the medium, suggesting its role as a reserve compound. The production of trehalose was not affected by lowering the growth temperature from 30 to 20 degrees C. On the other hand, the maximum trehalose accumulation increased from about 200 to 400 mg of trehalose/g of cell protein upon decreasing the pH from 7.0 to 4.7, by increasing the concentration of NaCl to 2% (w/v), or during growth under aerobic conditions (50% air saturation, 24 microM O(2), pH 7.0). In the absence of NaCl, trehalose accumulated concomitantly with growth, but an increase in salinity triggered a high trehalose production already in the early exponential growth phase. The data provide evidence for a dual function of trehalose as a reserve compound and as a stress-response metabolite. Moreover, P. freudenreichii ssp. shermanii NIZO B365 was able to produce high levels of trehalose in skim milk, which is promising for the implementation of fermented dairy products.     

Genetic manipulation of the peptidolytic system in lactic acid bacteria

Due to their presumed involvement in product flavour the peptidases of lactic acid bacteria have been subject to extensive research. A major breakthrough was the isolation and purification of the various enzymes to homogeniety. This allowed a reevaluation of the number of different enzymes in one species by careful investigation of their specificities. Moreover, the purified proteins offered novel ways to the genetic analysis of the peptidolytic system. N-terminal amino acid sequences of, and antibodies against, the purified peptidases were used to clone the majority of peptidases described for Lactococcus lactis. Some peptidase genes, especially those from a number of lactobacilli, were obtained by complementation of peptidase-negative cloning hosts. Thus, by deduction, the amino acid sequences of these peptidases is known and all belong to known families of peptidases. The recently developed chromosomal integration systems for LAB have been used to construct (multiple) peptidase-negative mutants in L. lactis. The growth characteristics of these strains are now under study. As a number of the mutants were made in a food grade way their performance in cheesemaking can be evaluated. The review presented will focus on the latest developments in the genetic analysis of peptidases in LAB and will evaluate our current understanding of peptidolysis by LAB and its importance in dairy fermentations.     

乳酸乳球菌中双乙酰代谢支路的调控

描述了从柠檬酸代谢、糖酵解来形成双乙酰的途径及其调节思路，并提出乳酸酸乳球菌的混合酸发酵及其调控机理，对双乙酰代谢支路的的遗传修饰方法进行了综述。结果表明，在特定条件下，有些乳酸菌从同型乳酸发酵转道成为异型乳酸发酵而产生其他代谢物(如甲酸或CO2，乙酸和乙醇)，这种变化涉及到丙酮酸代谢的遗传修饰：降低乳酸脱氢酶(LDH)活力或α-乙酰乳酸脱羧酶(ALD)活力、增加丙酮酸甲酸盐裂解酶(PFL)(厌氧条件)或α-乙酰合成酶(ALS)或丙酮酸脱氢酶(PDH)活力(好氧条件)，这些方法会有效地将糖转化成为双乙酰。对双乙酰代谢支路的调控主要集中在：超产αls或ileBN，失活ldh，pft，aldB或与超产NADH-氧化酶方法相结合使用。     

Quantitative measurement of tetrahydromenaquinone-9 in cheese fermented by propionibacteria

Propionibacteria produce tetrahydromenaquinone-9 [MK-9 (4H)] as a major menaquinone (vitamin K₂). This study aimed to determine the MK-9 (4H) concentration in commercial propionibacteria-fermented cheese. The MK-9 (4H) concentration was quantified using an HPLC instrument with a fluorescence detector after postcolumn reduction. Among the various cheese samples, the MK-9 (4H) concentration was highest in Norwegian Jarlsberg cheese, followed by Swiss Emmental cheese. In contrast, the MK-9 (4H) concentrations in Appenzeller or Gruyère cheeses were extremely low or undetected. Likewise, the concentrations in Comte and Raclette cheeses were lower than those in Jarlsberg and Emmental cheeses. In the present study, the MK- 9 (4H) concentration in cheese showed a correlation with the viable propionibacterial cell count and propionate concentration. This implies that the increase in propionibacteria contributed to the generation of MK-9 (4H) in cheese. We presumed, based on these results, that Swiss Emmental and Norwegian Jarlsberg cheeses contain a meaningful amount of vitamin K because of their high MK-9 (4H) concentrations (200 to 650 ng/g).     

Inability of dairy propionibacteria to grow in milk from low inocula

Growth of propionibacteria in complex media was independent of the initial number of cells; in contrast, growth of propionibacteria in milk and whey did not occur if the initial level of cells was < 10(6) cfu/ml. Addition of vitamins, minerals or complex nitrogen sources to the milk or whey, or incubation under anaerobic conditions had no effect on the lack of growth. Addition of freeze-dried whey, prepared from skim milk reconstituted from powder, to a complex medium prevented growth from low inocula in the complex medium, demonstrating the presence of an inhibitor or inhibitors in the whey. The inhibitor(s) was heat stable, had a low molecular mass and retained its activity for at least 4 weeks at 20 degrees C. Pregrowth of some lactic acid bacteria, used as starter cultures in Swiss-type cheese manufacture, in milk for 2 weeks at 20 degrees C removed the inhibition, which explains how propionibacteria develop in Swiss-type cheese from low numbers even though they are inhibited in milk.     

Genetic manipulation of the IGF-I axis to regulate mammary gland development and function

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.     

Determination of menaquinone production by Lactococcus spp. and propionibacteria in cheese

Two genera of lactic acid bacteria are principally known to produce menaquinones in cheese: Lactococcus spp. in semi-hard cheese such as Vacherin Fribourgeois and Raclette, and propionibacteria in Swiss-type cheese such as Emmental. Menaquinone (MK) content of several cheese loaves was analysed to determine the impact of sampling site, ripening time and cultures used during cheese making. With Lactococcus spp., in Vacherin and in Raclette, the principal menaquinone was MK-9 (median = 149 μg kg⁻¹ and median = 167 μg kg⁻¹) followed by MK-8 (median = 70 μg kg⁻¹ and median = 66 μg kg⁻¹). In Emmental cheese, the principal menaquinone was MK-4 (median = 48 μg kg⁻¹) in young cheese and MK-9(4H) (median = 468 μg kg⁻¹) in cheese older than 90 days. The only difference in sampling site was found in Raclette for MK-9. In Vacherin, MK-8, MK-9 and total contents of menaquinones were significantly different according to the strain of Lactococcus lactis ssp. cremoris used.     

Adhesion of dairy propionibacteria to intestinal epithelial tissue in vitro and in vivo

Adhesion to the intestinal mucosa is a desirable property for probiotic microorganisms and has been related to many of their health benefits. In the present study, 24 dairy Propionibacterium strains were assessed with regard to their hydrophobic characteristics and their autoaggregation and hemagglutination abilities, since these traits have been shown to be indicative of adherence in other microorganisms. Six strains were further tested for their capacity to adhere to ileal epithelial cells in vitro and in vivo. The results of the study showed that propionibacteria were highly hydrophilic, and hemagglutination and autoaggregation were properties not commonly found among these microorganisms. No relationship was found between surface characteristics and adhesion ability, since hemagglutinating, autoaggregating, and nonautoaggregating bacteria were able to adhere to intestinal cells both in vitro and in vivo. Microscopic examination revealed that autoaggregating cells adhered in clusters, with adhesion being mediated by only a few bacteria, whereas the hemagglutinating and nonautoaggregating strains adhered individually or in small groups making contact with each epithelial cell with the entire bacterial surface. The in vitro assessment of adhesion was a good indication of the in vivo association of propionibacteria with the intestinal epithelium. Therefore, the in vitro method presented here should be valuable in screening routinely adhesive properties of propionibacteria for probiotic purposes. The adhesion ability of dairy propionibacteria would prolong their maintenance in the gut and increase the duration of their provision of beneficial effects in the host, supporting the potential of Propionibacterium in the development of new probiotic products.     

基于模糊控制的岩茶做青系统设计

为实现岩茶加工智能化控制,降低做青过程中劳动强度和人为因素的影响,提出了一种基于模糊控制的岩茶做青控制方案。系统地分析了做青工艺中温、湿度环境因子对岩茶品质的影响,设计了以做青温湿度偏差为输入参数、以专家数据库做青工艺参数变化值为输出参数的模糊控制器,模拟手工控制思路,调整做青过程各工艺时间,实现做青过程的智能控制。试验结果表明:做青工艺参数的控制与手工控制趋势基本一致。     

Effect of feeding propionibacteria on milk production by early lactation dairy cows

This experiment was conducted to determine the effect of a direct-fed microbial agent, Propionibacterium strain P169 (P169), on rumen fermentation, milk production, and health of periparturient and early-lactation dairy cows. Starting 2 wk before anticipated calving, cows were divided into 2 groups and fed a control diet or the control diet plus 6 × 10¹¹ cfu/d of P169. Cows were changed to a lactation diet at calving, and treatments continued until 119 d in milk. Rumen fluid samples were taken about 1 wk before calving, and at 1 and 14 wk after calving. Cows fed P169 had lower concentrations of acetate (mol/100 mol of total volatile fatty acids) at all time points, greater concentrations of propionate on the first and last sampling points, and greater concentrations of butyrate on the first 2 time points. Concentrations of glucose in plasma and milk and plasma concentrations of β-hydroxybutyrate were not affected by treatment. Cows fed P169 had greater concentrations of plasma nonesterified fatty acids on d 7 of lactation. The high nonesterified fatty acids at that time point was probably related to the high production of milk during that period by cows fed the additive. Cows fed P169 during the first 17 wk of lactation produced similar amounts of milk (44.9 vs. 45.3 kg/d, treatment vs. control) with similar composition as cows fed the control diet. Calculated net energy use for milk production, maintenance, and body weight change was similar between treatments, but cows fed the P169 consumed less dry matter (22.5 vs. 23.5 kg/d), which resulted in a 4.4% increase in energetic efficiency.     

The effect of NaCl and metabolic profile of propionibacteria on eye formation in experimental Swiss-type cheese

The influence of three brining times (0, 1, and 3 d) and two propionibacteria (PAB) cultures (Prop A and Prop B) on eye formation was investigated in experimental Swiss-type cheeses by comparing PAB counts, biochemical parameters, eye numbers, diameters and volumes. Prop A was strongly inhibited with increasing NaCl content. As a result, eye volume decreased considerably, as well as eye diameter especially towards the cheese surface. Prop B exclusively converted l-lactate and was much less affected by increasing NaCl content, hence, eye formation changed less. The salt sensitivity of the cultures also influenced the spatial distribution of the eyes. The width of the eyeless border zone was mainly affected by three factors: CO₂ diffusion to the outside, propionic acid fermentation, and body firmness. The study clearly showed that strain-specific salt sensitivity of PAB is an important feature that strongly influences eye formation in Swiss-type cheeses with different salt content.     

In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria

This study aimed to assess the transit tolerance of potential probiotic dairy propionibacteria strains in human upper gastrointestinal tract in vitro, and to evaluate the effect of food addition on viability of these strains in simulated pH 2.0 gastric juices. The transit tolerance of 13 dairy propionibacteria strains was determined at 37 degrees C by exposing washed cell suspensions to simulated gastric juices at pH values at 2.0, 3.0, and 4.0, and simulated small intestinal juices (pH 8.0) with or without 0.3% bile salts. The viability of dairy propionibacteria in pH 2.0 simulated gastric juice with So-Good original soymilk or Up & Go liquid breakfast was also determined. The simulated gastric transit tolerance of dairy propionibacteria was strain-dependent and pH-dependent. All tested strains were tolerant to simulated small intestinal transit. The addition of So-Good original soymilk or Up & Go liquid breakfast greatly enhanced the survival of dairy propionibacteria strains in pH 2.0 simulated gastric juices. Dairy propionibacteria strains demonstrate high tolerance to simulated human upper gastrointestinal tract conditions and offer a relatively overlooked, yet alternative source for novel probiotics besides Lactobacillus and Bifidobacterium.     

Production of acetic and propionic acids from labneh whey by fermentation with propionibacteria

Whey produced during the manufacture of labneh was supplemented with yeast extract (10 g/1), and then fortified with lactose, treated with β-galactosidase or fermented with Lactobacillus helveticus, prior to inoculation with free living cells of Propionibacterium freudenreichii ssp shermanii or Propionibacterium acidipropionici or cells immobilized in aliginate beads. Under anaerobic batch conditions, fermentation of the whey with Lb helveticus followed by P acidipropionici (free cell system) for 2.5 days at 32°C gave a broth with 5.9 g/l of propionic acid and 2.4 gll of acetic acid, while immobilized cells of the same organisms gave a broth with 11.0 gll propionic acid and 3.2 g/l acetic acid over 4 days. These latter values were the maximum levels recorded with any of the treatments, and it is suggested that such yields might make recovery economically feasible in certain countries.     

In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices

This study investigated Propionibacterium freudenreichii tolerance to stresses encountered during food technology or in the digestive tract. The impact of food matrices on stress survival was evaluated. The ability of P. freudenreichii strains SI 41 and CNRZ 81 to survive both acid and bile salts stresses was studied in vitro. Stress tolerance was examined after inclusion of bacteria in different food matrices (alginate beads, xanthan–gellan beads, fermented milk) and compared to the stress tolerance of the same strains in pure cultures (either in exponential growth phase, in stationary phase, or after freeze-drying). Fermented milk was determined as the best probiotic vector to protect propionibacteria from this trial. This in vitro investigation gave promising results. Indeed, some food matrices can significantly improve protection of bacterial cells from stress injury. These data, which should be confirmed in an in vivo study, will be taken into consideration for the improvement of technological processes and for the choice of an adequate probiotic vector.     

根瘤农杆菌介导的紫色红曲菌遗传转化体系的研究

通过构建根瘤农杆菌介导的红曲菌遗传转化体系,将含有桔霉素生物合成基因同源片段的T-DNA导入紫色红曲菌Monascus purpureus SM005的子囊孢子中,以期对产桔霉素的关键酶基因pksCT进行定向敲除.优化得到的遗传转化系统为进一步对红曲菌进行分子生物学操作提供了一条高效可行的途径.     

Aggregation and hydrophobicity properties of 6 dairy propionibacteria strains isolated from homemade Turkish cheeses

In the present study, 6 dairy Propionibacterium strains were assessed with regard to their hydrophobic characteristics and their autoaggregation and coaggregation abilities since these traits have been shown to be indicative of adherence in other microorganisms. Aggregation assays and bacterial adhesion to hydrocarbons demonstrated significant differences in cell surface properties among the tested propionibacteria strains. Almost all strains appeared relatively hydrophilic, which showed low affinity for p-xylene. Four of the tested strains showed the strong adhesion to ethyl acetate, a basic solvent, in comparison with microbial adhesion to chloroform, an acidic solvent, which demonstrated the particularity of propionibacteria to have an important electron donor and acidic character. Also, these strains simultaneously showed affinity to 3 hydrocarbons, suggesting a high complexity of the cell surface. All propionibacteria strains tested showed autoaggregation and coaggregation ability with the Escherichia coli ATTC 11229, but the results were strain-specific and dependent on incubation conditions. Anaerobic incubation conditions were determined as the best condition for aggregation abilities of propionibacteria strains. A relationship was obtained between aggregation abilities (auto- and coaggregation) and a correlation between adhesion to hydrocarbon (chloroform) and autoaggregation was possible. Our results indicate that the ability to autoaggregation together with cell surface hydrophobicity and coaggregation abilities with E. coli strain can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use. PRACTICAL APPLICATION: Autoaggregation, cell surface hydrophobicity, and coaggregation abilities with E. coli of the selected dairy propionibacteria strains could be used as probiotic in foods after in vivo studies.     

Autolysis of propionibacteria: detection of autolytic enzymes by renaturing SDS-PAGE and additional buffer studies

Five strains of propionibacteria with 70-90% autolysis in sodium lactate broth (SLB) were studied by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Several lytic bands ranging in size between 25 and 143 kDa were detected by using propionibacteria cells or cell walls as substrate in the gel. Four Propionibacterium freudenreichii strains showed similar autolytic-enzyme profiles, consisting of two autolytic bands, one with molecular mass 162 kDa and one in the range 123-143 kDa. However, the Propionibacterium acidipropionici strain showed a completely different profile, consisting of 8 autolytic bands with molecular masses of 122, 97, 71, 55, 43, 39, 31, and 25 kDa. Lytic enzymes from P. freudenreichii INF-alpha, P. freudenreichii ISU P-59, P. freudenreichii ISU P-24, and P. freudenreichii ISU P-50 showed lytic activity against cells from all these four strains, but not against P. acidipropionici ATCC 4965. However, P. acidipropionici ATCC 4965 autolysed only its own cells. Effects of pH, temperature, and ions on autolytic activity were tested by renaturing SDS-PAGE and in buffer systems. Results from the SDS-PAGE electrophoresis showed optimal autolytic activity of P. acidipropionici ATCC 4965 at 37 degrees C and in the pH range 7 to 8.5 and of P. freudenreichii ISU P-59 at 20 degrees C and in the pH range 5 to 7. The autolytic activity of P. acidipropionici ATCC 4965 was extremely heat stable (100 degrees C, 2 h), in contrast to the lytic activity of P. freudenreichii ISU P-59, which was heat labile. The autolytic activities of P. acidipropionici ATCC 4965 were inhibited by divalent cations, however, the lytic activities of P. freudenreichii ISU P-59 were activated by Mn(2+), Ca(2+), and Co(2+). In buffer, optimum autolysis of P. acidipropionici ATCC 4965 was observed at pH 8.5 and at 40 degrees C. P. freudenreichii ISU P-59 showed optimum autolysis in buffer at pH 7.5 and at 30 degrees C.     

假交替单胞菌遗传操作系统的研究进展

假交替单胞菌能产生多种胞外活性物质,赋予其重要的基础研究和应用研究价值.目前已有多株假交替单胞菌完成基因组测序.基因组序列分析揭示不同的假交替单胞菌具有产生不同活性代谢产物的能力.假交替单胞菌遗传操作体系欠成熟,这极大地限制了假交替单胞菌的研究与应用.主要对近十多年来假交替单胞菌遗传操作系统研究进展进行综述,包括感受态制备,穿梭载体、自杀敲除载体及筛选标记的成功应用,以期为假交替单胞菌遗传操作系统的发展提供参考.