Development of a dairy-based exopolysaccharide bioingredient

An exopolysaccharide (EPS)-producing Streptococcus thermophilus was used to develop an “all-dairy” ingredient with increased EPS content and greater functionality for dairy applications such as Mozzarella cheese. In laboratory-scale trials, milk protein hydrolysate (MPH), whey protein isolate (WPI) and whey protein hydrolysate (WPH) in reconstituted low-heat skim milk powder (LHSMP) were examined as additional dairy protein sources to increase the production of EPS. WPH supplementation resulted in the greatest EPS production. In batch fermentation, a medium based on LHSMP and WPH, combined with optimum conditions of temperature and pH, was used to obtain maximum production of EPS from S. thermophilus. EPS production was growth associated. The fermented biomass was harvested at the end of the exponential phase and freeze-dried. The reduced viable cell count and the retention of ropiness of the powder from the drying process enabled a higher level of EPS inoculation in a preliminary Mozzarella cheese manufacturing trial.     

Exopolysaccharide-producing lactic acid bacteria strains from traditional Thai fermented foods: isolation, identification and exopolysaccharide characterization

Lactic Acid Bacteria (LAB) isolated from various traditional Thai fermented foods were screened for exopolysaccharides (EPS) production. From 104 isolates, two rod-shaped and five coccal-shaped LAB were able to produce EPS from sucrose on solid media. However, only the cocci were capable of producing EPS in liquid media and these were identified as Pediococcus pentosaceus. Pediococcus pentosaceus strains AP-1 and AP-3 produced EPS in high yield. In liquid media containing sucrose as carbon source, the amount of EPS produced by AP-1 and AP-3 strains was 6.0 and 2.5 g/L, respectively. The isolated and purified EPSs were chemically characterized. On the basis of sugar composition, methylation analysis and nuclear magnetic resonance spectroscopy, both the EPSs were shown to belong to the same dextran class. In particular, both EPSs differed from linear dextran by branching through 3,6-di-Osubstituted alpha-D-glucopyranosyl residues. The EPS from P. pentosaceus AP-3 was characterized by a relatively higher degree of branching and by a higher molecular weight than that from P. pentosaceus AP-1.     

Diversity and functional characteristics of lactic acid bacteria from traditional kefir grains

Traditional kefir grains were collected from distinct parts of Turkey, and their microbial profile was determined. A wide bacterial biota was observed formed by distinct lactic acid bacteria (LAB) in which Lactococcus lactis strains appeared to be dominant. Yeast species were also identified in kefir grains. Significant levels of antifungal and antibacterial activities were monitored in kefir isolates. All tested LAB produced an exopolysaccharide (EPS) containing glucose and galactose, and some strains formed a fructan‐type EPS. Importantly, low levels of antibiotic resistance were observed among the kefir isolates.     

Quality Control of Malt: Turbidity Problems of Standard Worts Given by the Presence of Microbial Cells

Since the main effect on beer colour originates from the chemical composition of malt, the measurement of the colour of wort obtained from malt is one of the most important quality parameters to be controlled. Malt colour determination is usually performed spectrophotometrically on EBC Congress worts, which are generally bright. The quality control on the malt harvested in the year 2007 highlighted a problem of turbid Congress wort, for a part of Northern Europe samples analyzed, and therefore the spectrophotometric determination of the colour of wort was not possible. Thus both analytical screening of the quality parameters of malt and the scanning electron microscope (SEM) were used to identify the nature of the turbidity in the worts. The results obtained suggested that the nature of the turbidity of Congress worts could have been due to microbial contamination of the barley, enhanced in particular by the rainy harvest and the consequent formation of exopolysaccharides (EPS) by stressed yeast and bacteria during malting.     

Binding of mutagens to exopolysaccharide produced by Lactobacillus plantarum mutant strain 301102S

Exopolysaccharide (EPS) was produced by Lactobacillus plantarum 301102 on exposure to the mutagenic action of acridine orange and novobiocin. The biological characteristics of this mutant strain 301102S were the same as those of the parent strain, but fermented milk prepared with the mutant strain showed antimutagenic activity on 3-amino-1,4-dimethyl-5H-pyrido indole. Only EPS-bound cells of strain 301102S showed binding ability to mutagens such as heterocyclic amines, and the mutagens were inactivated by binding to EPS. The binding ability was affected by pH; the greatest percentage binding was noted at pH 8.0. Addition of Mg²⁺ and sodium dodecyl sulfate, but not oxgall, inhibited the binding ability. Therefore, the binding mechanism of the EPS may consist of ion-exchange and hydrophobic bonds, and the EPS would bind mutagens in the intestine.     

芽孢杆菌胞外多糖的结构初步分析

从筛选自新疆罗布泊沙漠的1株芽孢杆菌的发酵滤液中分离得到胞外多糖EPSⅠ和EPSⅡ,其中EPSⅠ为主要成分,占总糖的89.5%,EPSⅠ为糖蛋白,其分子质量为96.292ku,多糖含量为93.79%,蛋白含量为5.62%,不含糖醛酸,是由D-N-乙酰葡萄糖胺,D-木糖和D-甘露糖构成,它们的摩尔比例是2.36∶0.98∶1.75。EPSⅠ为β型吡喃糖,经高碘酸氧化和Smith降解,EPSⅠ中含有(1→),(1→3)和(1→4)糖苷键,且(1→)和(1→4)糖苷键的比例为1∶24.05,其中的糖肽是O-糖苷键。     

Presumptive Identification of Stock Cultures and Food Isolates As Salmonella, Shigella, or Yersinia with the Enteric Pathogen Screen

The enteric pathogen screen (EPS) card designed for use with the Vitek Auto Microbic System (AMS) was evaluated with stock cultures and fresh isolates from ground beef, pork sausage, chicken and turkey to determine its effectiveness as a screen for Salmonella, Yersinia enterocolitica and Shigella. Each isolate was concurrently tested with the Micro-ID test strip and the AMS Gram negative identification card to confirm its identification. The EPS accurately classified 115 of 125 (92%) of the stock cultures and 207 of 212 (97%) of the fresh food isolates. The EPS gives a rapid presumptive screen at a relatively low cost and as such is a viable alternative to conventional procedures.     

Antioxidant and antitumour activities of exopolysaccharide from liquid‐cultured Grifola frondosa by chemical modification

Grifola frondosa is a popular edible mushroom for its tasty flavour and can be used as a dietary supplement for its functional substances. In this study, an exopolysaccharide (EPS), purified from the submerged culture of G. frondosa, was chemically modified to obtain its carboxymethylated (CM‐EPS) and selenising derivatives (Se‐EPS). The chemical characteristics of these polysaccharides were determined based on high‐performance liquid chromatography (HPLC) and infrared analysis (IR). The antioxidant and antitumour activities of EPS and its derivatives were further investigated, respectively. The results showed that the antioxidant capacity of CM‐EPS and Se‐EPS were much better than that of EPS. Moreover, the Se‐EPS could significantly inhibit the proliferation of Hela cells compared with EPS. This study demonstrated that the carboxymethylation and selenylation played a great influence on enhancing the antioxidant and antitumour activities of exopolysaccharides from G. frondosa.     

Aggregation, dissolution, and stability of quantum dots in marine environments: importance of extracellular polymeric substances

There is an increasing concern that a considerable fraction of engineered nanoparticles (ENs), including quantum dots (QDs), will eventually find their way into the marine environment and have negative impacts on plankton. As ENs enter the ocean, they will encounter extracellular polymeric substances (EPS) from microbial sources before directly interacting with plankton cells. In this study, EPS harvested from four phytoplankton species, Amphora sp., Dunaliella tertiolecta, Phaeocystis globosa, and Thalassiosira pseudonana, were examined for potential interactions with CdSe nonfunctionalized and functionalized (carboxyl- and amine-) QDs in artificial seawater. Our results show that EPS do not reduce the solubility of QDs but rather decrease their stability. The degradation rate of QDs was positively correlated to the protein composition of EPS (defined by the ratio of protein/carbohydrate). Two approaches showed significant inhibition to the degradation of carboxyl-functionalized QDs: (1) the presence of an antioxidant, such as N-acetyl cysteine, and (2) absence of light. Owing to the complexity in evaluating integrated effects of QDs intrinsic properties and the external environmental factors that control the stability of QDs, conclusions must be based on a careful consideration of all these factors when attempting to evaluate the bioavailability of QDs and other ENs in the marine environments.     

Evaluation of the yield,molar mass of exopolysaccharides,and rheological properties of gels formed during fermentation of milk by Streptococcus thermophilus strains St-143 and ST-10255y

The yield and chemical structures of exopolysaccharides (EPS) produced by many strains of Streptococcus thermophilus have been characterized. However, the kinetics (or production profile) for EPS during milk fermentation is not clear. In this study, we investigated whether any differences existed in the yield and molar mass of EPS when milk was fermented at the same acidification rate by 2 strains of S. thermophilus (St-143 and ST-10255y). The type of EPS produced by these 2 strains is different. Milk samples were analyzed for EPS concentration every 30 min during a fermentation period of 270 min (final pH 4.5) by using a modified quantification method, which was faster and validated for its recovery of added EPS. Rheological properties of milks during fermentation were also analyzed using small-strain dynamic oscillatory rheology. For the determination of molar mass, EPS extracts were isolated by ultrafiltration of whey obtained during fermentation of milk to pH values 5.2, 4.9, 4.7, and 4.5, and molar mass was analyzed using size-exclusion chromatography–multi-angle laser light scattering. During fermentation, both strains appeared to start producing significant amounts of EPS after about ～150 min, which corresponded to pH ～5.3, which was close to the point of gelation. During the remainder of the fermentation process (150–270 min), the EPS concentration from strains St-143 and ST-10255y significantly increased from 30 to 72 mg/L and from 26 to 56 mg/L, respectively. The quantity of EPS recovered by our modified method was estimated to represent ～60% of the total EPS added to milk. The molar mass of EPS produced by both strains appeared to slightly decrease during fermentation. At pH 5.2, EPS from St-143 and ST-10255y had molar masses of 2.9 × 10⁶ and 1.4 × 10⁶ g/mol, respectively, which decreased to 1.6 × 10⁶ and 0.8 × 10⁶ g/mol, respectively, when the pH of milk was 4.5. Distinct differences were apparent in the rheological properties of gels fermented by the 2 strains. At the end of fermentation, St-143 fermented milk had weaker gels with storage modulus (G′) value at pH 4.6 of 26 Pa, whereas gels made with ST-10255y were stiffer with a G′ value at pH 4.6 of 82 Pa. For St-143 gels, maximum loss tangent (LT_max) values were higher (0.50) and occurred earlier (at a higher pH value) than the LT_max values (0.46) for gels from ST-10255y strain. Because the fermentation conditions were identical for both strains, the observed changes in rheological properties could be due to the differences in chemical structures and molar mass of the EPS produced by these 2 S. thermophilus strains.     

Whey protein aggregates and their interaction with exo-polysaccharides

Summary We made a systematic investigation of the interaction of an exo-polysaccharide (EPS) with whey proteins. The EPS was produced by a lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40. The interaction of the EPS with milk components is of relevance to the consistency of fermented milk products. EPS and whey protein isolates (WPI) can be mixed in all proportions. Even at an EPS concentration of 10 g.L⁻¹ no instability is visible. In yoghurt milk the whey proteins are usually denatured. We prepared aggregated whey protein colloid particles (AWC particles) which had an average radius of 27 ± 3 nm, as derived from the radius of gyration. The interaction of AWC particles and EPS was studied at a neutral pH and an ionic strength of 0.1 m. At low EPS concentrations the mixture was stable. At higher concentrations the mixtures showed phase separation owing to depletion interactions. In the one-phase region the strength of the segregative interaction can be determined from turbidity, light scattering and neutron scattering measurements. Then, by applying statistical mechanical methods, the position of the phase boundary could be predicted. The viscosity of the mixtures was increased owing to the segregative interactions in the EPS/AWC mixtures. Within the two-phase region the mixtures showed so-called spinodal decomposition, during which different microstructures were formed. Understanding the formation of such structures is of interest for the development of new food structures.     

猪肉源大肠杆菌分离鉴定及其生物被膜形成

为研究猪肉来源大肠杆菌分离菌株的生物被膜形成及相关基因表达变化，首先从生猪屠宰线、猪胴体表面及生鲜猪肉中采用选择平板和特异性聚合酶链式反应（polymerase chain reaction，PCR）分离鉴定大肠杆菌，采用微孔板结晶紫染色法评价分离菌株15 ℃培养72 h时生物被膜形成能力，进而选取代表菌株研究生物被膜形成过程中被膜量、胞外聚合物（extracellular polymeric substances，EPS）组成，以及基于反转录荧光定量PCR的成膜基因表达的变化。结果表明：共分离到猪肉源大肠杆菌31 株，包括生猪屠宰线11 株、猪胴体表面4 株、生鲜猪肉16 株；微孔板结晶紫染色结果显示，被膜形成能力存在明显菌株差异，64.52%菌株成膜能力较弱，选取其中1 株（D4-18）进行成膜过程研究；微孔板中菌株D4-18 15 ℃培养168 h过程中被膜量持续增加，培养72 h和168 h时，菌株D4-18分泌EPS，培养72 h时，papC、fimH、csgA基因表达量分别为0.095、0.933、0.435 copies/cm2，随着培养时间延长，松散型EPS中蛋白质及多糖含量、紧密型EPS中蛋白质含量极显著增加（P＜0.01），培养168 h时，papC和fimH基因表达量增加，csgA基因表达量无显著变化，表明上述基因在大肠杆菌生物被膜形成过程中发挥了不同程度的调控作用。     

Processing properties of yogurt as affected by the EPS produced by Leuconostoc mesenteroides XR1

The aim of this study was to examine the fermentation characteristics of a novel Leuconostoc mesenteroides strain (XR1) and to investigate the effect of XR1 exopolysaccharides (EPS) on the microstructure, texture and rheological properties of yoghurt. The novel Leuconostoc mesenteroides strain (XR1) was isolated from wild kefir grains obtained from Xinjiang Province. The results showed that XR1 had good EPS production capacity and favourable fermentation performance. The addition of XR1 EPS to fermented milk effectively improved the water-holding capacity (WHC) of the fermented milk and changed its microstructure. In subsequent fermentation studies, the XR1 strain was found to be a compatible strain, and the steric barrier formed between XR1 EPS and the fermented milk proteins enhanced its viscoelastic properties. Rheology studies indicated that fermented milk supplemented with EPS possessed a higher modulus (G’, G’’ ) and complex viscosity. Thus, these results indicate that XR1 EPS are useful as a natural organic additive that can replace chemical additives in dairy products.     

Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations

To increase the exopolysaccharide (EPS) yields from Streptococcus thermophilus LY03 and to unravel the nature of the EPS degradation process, fermentation experiments were carried out with this strain in a customized MRS medium, using different additional carbohydrates or amino acids possibly related to growth and EPS production. No significant increase of the EPS yields or activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase that are correlated with EPS production, or of the activity of dTDP-glucose pyrophosphorylase involved in the rhamnose synthetic branch of EPS biosynthesis, was observed. The EPS monomer composition remained unchanged for all experiments. Fermentations with a sudden temperature increase or lowered pH were carried out as well to try to avoid EPS degradation upon prolonged fermentation. It was demonstrated that EPS degradation took place enzymatically. Incubations of purified high-molecular-mass EPS with cell-free culture supernatant or cell extracts showed its degradation by enzymes with an endo-activity. This glycohydrolytic activity probably encompasses several enzymes having a molecular mass lower than 50,000 and 10,000 Da, and seems to be rather stable at high temperature and low pH. These results contribute to a better understanding of the physiological and chemical factors influencing EPS production and degradation.     

Streptococcus thermophilus strains of plant origin as dairy starters: Isolation and characterisation

Streptococcus thermophilus strains have been isolated mainly from dairy environments. To prospect for new strains of S. thermophilus, isolation was made from different plant sources. In this study, 74 plant isolates were characterised as S. thermophilus by a polyphasic approach and by 16S rRNA sequencing. The isolates were further evaluated for their physiological and biochemical properties. Plant isolates exhibited good acid production and varying proteolytic activity. All the isolates showed acetaldehyde and capsular exopolysaccharide (EPS) production and few revealed diacetyl production. In contrast to industrial strains, six isolates were able to ferment galactose and 25 were found to have no urease activity. Both the plant isolates and reference dairy cultures were found to possess similar physiological and biochemical properties and can be considered for developing new starters.     

Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt

Lactic acid bacteria (LAB) were isolated from traditional yogurt samples and genotypic characterization of these isolates revealed the presence of 21 distinct LAB strains belonging to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, and Lactobacillus plantarum as new LAB strains. Determination of the exopolysaccharide (EPS) production characteristics of the selected strains of each species revealed that all strains possessed at least one gene required for both homopolymeric‐ and heteropolymeric‐type EPS production. Structural analysis of the EPSs showed that L. delbrueckii subsp. bulgaricus Y39 and S. thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuc. mesenteroides Y35 and L. plantarum Y36 produced homopolymeric glucan‐type EPS. The level of EPS production in these strains was found to be in a similar range. These strains with EPS production characteristics are good candidates for future studies as new LAB for yogurt production.

Practical applications

Recent trends in yogurt production technology have led to an increased use of ropy starter cultures in yogurt production due to the technological roles of exopolysacharides (EPS) produced by these cultures. The main role of EPS in yogurt production is to improve the textural properties of yogurt as an in situ produced natural polymer. In addition to the yogurt starter cultures, use of adjunct cultures during production of yogurt is also of special interest to enhance the technological and nutritional characteristics of yogurt. Therefore, in this study, potential yogurt starter and adjunct cultures from traditional yogurt samples with EPS production characteristics were isolated. From these isolates, Lactobacillus delbrueckii subsp. bulgaricus Y39 and Streptococcus thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuconostoc mesenteroides Y35 and Lactobacillus plantarum Y36 produced homopolymeric glucan.     

Effect of intense pulsed light on Lactobacillus bulgaricus exopolysaccharide yield,chemical structure and antioxidant activity

In this work, IPL technology was used for improving the production of exopolysaccharide (EPS) in Lactobacillus bulgaricus. The effects of fluence per pulse, pulse number and pulse distance were investigated. Through GC–MS, Fourier transform infrared (FT-IR), ¹H nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) study the influence on EPS primary chemical structure and surface morphology. The antioxidant activity of EPS was evaluated by DPPH, Fe²⁺ chelating and reducing power assays. The findings demonstrate that IPL could be a novel environment protection method for improving the production of EPS. The research results provide a theoretical basis for whether IPL is a new type of environmental protection method and can be used to improve EPS production.     

Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir – Part II

ZW3 is a newly discovered exopolysaccharide (EPS) produced by Lactobacillus kefiranofaciens ZW3, isolated from Tibet kefir. Some of its properties have been characterized in our previous paper. Present research demonstrates some other important aspects of this EPS. The molecular weight obtained by gel permeation HPLC was 5.5 × 10⁴ Da. Solubility, water holding and oil binding capacity of ZW3 EPS were 14.2%, 496.0%, and 884.74% respectively. Scanning electron microscopy (SEM) of ZW3 EPS demonstrated a smooth surface with compact structures. A topographical examination of EPS by atomic force microscopy (AFM) revealed that ZW3 EPS is composed of almost uniform net of molecules. Rheological study indicated that common salt did not affect the viscous behavior of ZW3 EPS and acidic pH may enhance its viscosity. Exopolymer showed a melting point of 93.38 °C. A degradation temperature (Td) of 299.62 °C was observed from the TGA curve for the polysaccharide ZW3.     

产功能性胞外多糖及DL-乳酸的植物乳杆菌的高效发酵和应用

植物乳杆菌(Lactobacillus plantarum)是一种多功能乳酸菌,所产胞外多糖(exopolysaccharides, EPS)具有很多优良功能特性。但发酵生产EPS时,原料和操作成本高,EPS产量低限制了其工业化应用。该研究在5 L罐中,使用常规流加发酵、生物催化、生物催化结合液液萃取和基于pH-Stat自动流加葡萄糖法的反复生物催化等策略发酵,旨在提高EPS产量、降低原料和操作成本。其中,生物催化法仅使用葡萄糖即可将EPS质量浓度提升至3.34 g/L,较摇瓶发酵水平提高110%。使用基于pH-Stat自动流加葡萄糖法的反复生物催化策略,可以连续回用各反复发酵中的残存细胞,第2次反复发酵批次的EPS质量浓度达到3.33 g/L。由于在收集处理细胞时,细胞总量下降,导致EPS产量下降,但EPS/细胞量不变,细胞活性稳定。可以通过加大首批次发酵的装液量、提高细胞总量,解决EPS产量不断下降的问题。用基于pH-Stat法的反复生物催化策略进行EPS发酵,提升了EPS产量,省去了昂贵MRS培养基的使用,降低了原料成本,实现了自动化控制。该发酵策略还可联产具有一定价值的副产物4...     

Statistical optimisation of the exopolysaccharide production by Lactobacillus fermentum Lf2 and analysis of its chemical composition

Lactobacillus fermentum Lf2 produces high amounts of exopolysaccharides (EPS) (~1 g/L) with demonstrated functional and technological roles when applied as a food ingredient in dairy matrices, properties that made these EPS interesting in comparison with other similar molecules from lactic acid bacteria (LAB). Those characteristics encouraged us to optimise the production. The EPS extract is composed of a high molecular mass β‐glucan and a medium molecular mass heteroglycan. In the present work, the optimal conditions that doubled the EPS yield using a semidefined medium (SDM, 0.63% yeast nitrogen base, 0.53% bacto casitone, 0.53% ammonium citrate, 6.25% sucrose, pH 6.5) were found by means of response surface methodology (RSM). The chemical characterisation indicated that under optimised conditions the synthesis of the heteroglycan was favoured compared with that of the β‐glucan.