Characterization of glucan-producing Leuconostoc strains isolated from sourdough

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by ¹H and ¹³C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1 → 2), α-(1 → 3) and α-(1 → 6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)₅ primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1 → 2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.     

Functional properties of Lactobacillus strains isolated from kimchi

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) from kimchi, a traditional Korean fermented vegetable product generally consumed raw as a side-dish with practically every meal.Twelve mild acid producing facultatively heterofermentative Lactobacillus strains were selected for their potential as starter cultures for fermentation of kimchi, and evaluated for their functional properties. Eleven strains were identified as Lactobacillus sakei and one as Lactobacillus plantarum. The strains identified as L. sakei differed in some physiological features; of particular interest was the fact that 9 of these strains produced L(+) lactic acid from glucose in presence of acetate.All strains were able to survive gastrointestinal conditions simulating stomach and duodenum passage. In addition, they showed higher adherence to HT-29 cells than Lactobacillus rhamnosus GG, a commercial probiotic strain used worldwide. These strains also showed antimicrobial activity against a number of food-borne pathogens. Their ability to lower cholesterol was demonstrated by BSH (bile salt hydrolytic) activity, and cholesterol assimilation tests in vitro. The results suggest the probiotic potential of these strains for use in kimchi fermentation.     

Identification of lactic acid bacteria isolated from kimchi and studies on their suitability for application as starter culture in the production of fermented sausages

The aim of the investigation was to identify strains of lactobacilli coming from kimchi with properties suitable for use as starter cultures in sausage fermentation. A total of 31 strains of lactobacilli were isolated from kimchi on the 4-6th day of fermentation at 20°C using MRS agar plates and identified on the basis of morphological, biochemical, and physiological characteristics. The isolates were identified as Leuconostoc mes.mes./dent (12.9%), Lactobacillus curvatus (9.7%), Lactobacillus brevis (35.5%), Lactobacillus sake (25.8%), and Lactobacillus plantarum (16.1%). Thus, 51.6% of the isolates were homo-fermentative or facultative hetero-fermentative bacteria and the rest (48.4%) were hetero-fermentative bacteria. Among them L. brevis, L. curvatus, L. plantarum, and L. sake were investigated for their growth profile and metabolism characteristics in the fluid (submerged) model-medium modified according to the special conditions of fermented sausages. Relatively good growth properties were found for L. brevis, L. plantarum, and L. sake with maximum numbers of 8.18, 8.51 and 8.17cfu/ml, respectively, whereas L. curvatus could not adapt to the special environmental conditions. Regarding souring properties, L. brevis showed little ability to decrease pH, whereas L. curvatus, L. plantarum, and L. sake showed relatively good acidifying properties. According to the results of glucose fermentation and its products, only L. plantarum exhibited homo-fermentative characteristics. As a result only L. plantarum among the isolates from kimchi had an ability to adapt to the complex environment of fermented sausage, which will thereby allow them to act as starter cultures and natural preservatives in sausage production.     

Tyramine production among lactic acid bacteria and other species isolated from kimchi

Lactic acid bacteria (LAB) are naturally found in fermented vegetable products. The ability of 230 kimchi bacterial isolates was investigated to produce tyramine by biochemical and genetic methods. The production of tyramine was determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The presence of the gene encoding the corresponding tyrosine decarboxylase was also determined by PCR assay. After the production of tyramine was confirmed by chromatographic and molecular methods, the bacterial isolates producing the amine were identified by 16S rRNA gene sequence and species-specific PCR analyses. Only a small proportion of the bacterial isolates (14/230 isolates) decarboxylated tyrosine in vitro. All of the 14 bacterial isolates that produced tyramine were shown to possess the tdc gene, indicating that a positive correlation existed between the production of tyramine and the presence of the corresponding decarboxylase gene. The 14 isolates included three LAB species and one other species: Lactobacillus brevis (six), Lactobacillus curvatus (four), Leuconostoc mesenteroides (two), and Staphylococcus hominis (two). This study demonstrated that only a small proportion of LAB and other microbiota growing in kimchi had the ability to produce tyramine.     

Antifungal activity of sourdough fermented wheat germ used as an ingredient for bread making

This study aimed at investigating the antifungal activity of sourdough fermented (Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5) wheat germ (SFWG). Preliminarily, methanol and water/salt-soluble extracts from SFWG were assayed by agar diffusion towards Penicillium roqueforti DPPMAF1. As shown by hyphal radial growth rate, the water/salt-soluble extract showed the inhibition of various fungi isolated from bakeries. The antifungal activity was attributed to a mixture of organic acids and peptides which were synthesized during fermentation. Formic (24.7 mM) acid showed the highest antifungal activity. Four peptides, having similarities with well known antifungal sequences, were identified and chemically synthesized. The minimal inhibitory concentration was 2.5–15.2 mg/ml. Slices of bread made by addition of 4% (wt/wt) of freeze dried SFWG were packed in polyethylene bags and stored at room temperature. Slices did not show contamination by fungi until at least 28 days of storage and behaved as the calcium propionate (0.3%, wt/wt).     

Effect of sourdough fermentation on stabilisation,and chemical and nutritional characteristics of wheat germ

Lactic acid bacteria strains were identified from wheat germ by 16S rRNA partial sequencing, subjected to RAPD-PCR typing and screened. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were used as starters to produce sourdough fermented wheat germ (SFWG). The chemical and nutritional characteristics of SFWG were compared to those of the raw wheat germ (RWG). Lipase activity in SFWG was ca. 2.6-fold lower than that found in RWG. As shown by SPME/GC/MS analysis, most of the volatile compounds derived from lipid oxidation during storage (40 days) were at markedly lower levels in SFWG compared to RWG. Fermentation of wheat germ increased of ca. 50% the concentration of free amino acids. Glu markedly decreased in SFWG, due to its conversion in GABA. The concentration of the anti-nutritional factor raffinose also decreased in SFWG. The in vitro protein digestibility, the concentration of total phenols, phytase and antioxidant activities were increased by fermentation.     

Use of selected sourdough lactic acid bacteria to hydrolyze wheat and rye proteins responsible for cereal allergy

This work was aimed at showing the capacity of selected sourdough lactic acid bacteria to hydrolyze wheat and rye allergens. Hydrolysis was investigated after wheat sourdough fermentation and after treatment of wheat and rye sourdough breads with pepsin, trypsin and pancreatin, which mimicked the digestive process. As shown by immunoblotting with sera from allergic patients, wheat sourdough fermentation caused the disappearance of some IgE-binding proteins (albumins/globulins and gliadins mainly) with respect to the chemically acidified dough used as the control. The IgE-binding protein profile of wheat and rye sourdough breads differed from those of baker's yeast breads. The signals of the IgE-binding proteins contained in the sourdough breads disappeared after in vitro digestion with pepsin, trypsin and pancreatin. The same effect by digestive enzymes was not found for baker's yeast breads which showed persistent IgE-binding proteins. As shown by ELISA inhibition assays, the presence of IgE-binding low molecular weight proteins/peptides in sourdough breads significantly decreased with respect to baker's yeast breads. Proteolytic activity by selected sourdough lactic acid bacteria may have an importance during food processing to produce pre-digested wheat and rye dough which contains IgE-binding proteins degradable by digestive enzymes.     

Lactobacillus reuteri CRL 1100 as starter culture for wheat dough fermentation

The effect of sucrose on the fermentation balance of Lactobacillus reuteri CRL 1100 and the invertase activity of this strain in wheat dough and culture medium (MRSs) was evaluated. The enzyme activity was dependent on the environmental pH releasing glucose and fructose from sucrose hydrolysis. Glucose was used as carbon source, while fructose was mainly used as electron acceptor to produce mannitol up to 10h of fermentation. Thereafter, fructose seemed to be metabolized by the heterofermentative pathway, which determined an increase in the concentration of acetate (6 mmol l(-1)), lactate (2 mmol l(-1)) and ethanol (1 mmol l(-1)) and the lack of mannitol formation after glucose depletion. The fermentation balance of Lb. reuteri CRL 1100 during the dough fermentation resulted in lower (63%) ethanol, higher (75%) acetate production and soluble carbohydrates concentrations, like MRSs cultures. This fermentation profile would be important to obtain an optimal growth of yeast and the optimal bread flavor and taste.     

Probiotic potential of Lactobacillus strains with anti-allergic effects from kimchi for yogurt starters

Lactobacillus strains were isolated from kimchi and investigated for their potential use for probiotic yogurt starters. Two of the isolated strains have over 90% survival rate to artificial gastric acid (pH 2.5, 1% pepsin) and artificial bile acid (0.3% oxgall). These strains were identified as Lactobacillus plantarum by 16S rRNA sequencing, and named as L. plantarum SY11 and L. plantarum SY12. The known carcinogenic enzyme, β-glucuronidase was not produced by L. plantarum SY11 and L. plantarum SY12. These 2 strains were found to be resistant to several antibiotics and strongly adhered to intestinal cells. Antiallergic effect of L. plantarum SY11 and L. plantarum SY12 was demonstrated using nitric oxide (NO) and cytokine production. L. plantarum SY11 and L. plantarum SY12 were capable of significantly decreasing NO production, and reduced T helper 2-associated cytokines, cyclooxygenase-2, tumor necrosis factor-α, and inducible nitric oxide synthase compared to control. Yogurt samples produced using L. plantarum SY11 and L. plantarum SY12 did not show significant differences in microbiological, physicochemical, and sensory properties compared with yogurt sample produced using commercial strain. Therefore, these two strains could be used as probiotic yogurt starters with antiallergic effects.     

Extraction of water-soluble xylan from wheat bran and utilization of enzymatically produced xylooligosaccharides by Lactobacillus, Bifidobacterium and Weissella spp.

Xylan was extracted from wheat bran after heat pretreatment in water using either an autoclave or a microwave oven. Xylooligosaccharides (XOS) were produced from the xylan using the thermostable xylanase RmXyn10A and the potential prebiotic properties of XOS were studied in vitro with different human gut bacteria: Lactobacillus brevis (DSMZ 1269), Bifidobacterium adolescentis (ATCC 15703) and two strains of recently isolated lactic acid bacteria from the species pair Weissella cibaria/confusa. The highest yield of (arabino)xylan with the heat pretreatment was obtained at 185 °C for 10 min. Higher temperature led to fewer arabinose substitutions present on the backbone which in turn resulted in a slightly more efficient enzymatic hydrolysis by RmXyn10A.     

Study of the behaviour of Lactobacillus plantarum and Leuconostoc starters during a complete wheat sourdough breadmaking process

The acidification properties, metabolic activity and technological performance of four individual Lactobacillus plantarum or Leuconostoc freeze-dried starters were investigated during a complete wheat sourdough breadmaking process including 0.2 g/100 g baker's yeast. Microbiological contents (lactic acid bacteria and yeasts), acidification characteristics (pH and total titratable acidity), soluble carbohydrates (maltose, glucose and fructose) and fermentative end-products (lactic and acetic acids, ethanol) contents were evaluated during both sourdough and corresponding bread dough fermentation. Biochemical and technological analysis of the resulting bread products are also presented. Some differences among strains in acidification properties and soluble carbohydrates availability were outlined both in sourdough and bread dough. Each individual Leuconostoc or Lb. plantarum starter was able to produce a characteristic fermentation and was found to ensure the production of breads with overall satisfactory acceptance.     

Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations

Presumptive lactic acid bacterial cocci were found in six sourdoughs (out of 20) from the Abruzzo region (central Italy) and subjected to phenotypic and genotypic characterization. A total of 21 isolates, recognized as seven strains by randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing, were identified by a polyphasic approach, consisting of 16S rRNA gene sequencing, multiplex PCR assays and physiological features, as Enterococcus faecium and Pediococcus pentosaceus. Four strains belonging to those species and previously isolated from wheat kernels were inoculated in sterile flour to verify their capacity to grow in sourdough environment. Doughs with several dual bacterial combinations, including Lactobacillus sanfranciscensis, were propagated for 11 days and pH measurements and bacterial counts were carried out.     

Effects of Leuconostoc mesenteroides starter cultures on microbial communities and metabolites during kimchi fermentation

Kimchi fermentation usually relies upon the growth of naturally-occurring various heterofermentative lactic acid bacteria (LAB). This sometimes makes it difficult to produce kimchi with uniform quality. The use of Leuconostoc mesenteroides as a starter has been considered to produce commercial fermented kimchi with uniform and good quality in Korea. In this study, a combination of a barcoded pyrosequencing strategy and a ¹H NMR technique was used to investigate the effects of Leu. mesenteroides strain B1 as a starter culture for kimchi fermentation. Baechu (Chinese cabbage) and Chonggak (radish) kimchi with and without Leu. mesenteroides inoculation were prepared, respectively and their characteristics that included pH, cell number, bacterial community, and metabolites were monitored periodically for 40 days. Barcoded pyrosequencing analysis showed that the numbers of bacterial operational taxonomic units (OTU) in starter kimchi decreased more quickly than that in non-starter kimchi. Members of the genera Leuconostoc, Lactobacillus, and Weissella were dominant LAB regardless of the kimchi type or starter inoculation. Among the three genera, Leuconostoc was the most abundant, followed by Lactobacillus and Weissella. The use of Leu. mesenteroides as a starter increased the Leuconostoc proportions and decreased the Lactobacillus proportions in both type of kimchi during kimchi fermentation. However, interestingly, the use of the kimchi starter more highly maintained the Weissella proportions of starter kimchi compared to that in the non-starter kimchi until fermentation was complete. Metabolite analysis using the ¹H NMR technique showed that both Baechu and Chonggak kimchi with the starter culture began to consume free sugars earlier and produced a little greater amounts of lactic and acetic acids and mannitol. Metabolite analysis demonstrated that kimchi fermentation using Leu. mesenteroides as a starter was completed earlier with more production of kimchi metabolites compared to that not using a starter, which coincided with the decreases in pH and the increases in bacterial cell number. The PCA strategy using all kimchi components including carbohydrates, amino acids, organic acids, and others also showed that starter kimchi fermented faster with more organic acid and mannitol production. In conclusion, the combination of the barcoded pyrosequencing strategy and the ¹H NMR technique was used to effectively monitor microbial succession and metabolite production and allowed for a greater understanding of the relationships between the microbial community and metabolite production in kimchi fermentation.     

Immobilization of kefir and Lactobacillus casei on brewery spent grains for use in sourdough wheat bread making

New types of bread were produced employing baker’s yeast, kefir or Lactobacillus casei immobilized on brewer’s spent grains. Bread was produced either by the straight-dough or the sourdough method. All the studied biocatalysts and their corresponding sourdoughs were found efficient for breadmaking. Good rising was achieved and the produced breads had good overall quality and remained fresher for longer, compared to commercial type baker’s yeast bread. The best results were obtained for sourdough breads, with higher moisture retention during baking, lower rates of water evaporation and staling, and maintenance of freshness for longer (4–5 days). Consumer evaluation showed bigger preference for the sourdough breads as far as aroma, taste and overall quality were concerned, justified by the GC-MS analysis of volatiles that revealed a different aroma profile, with more compounds identified than in breads produced by the straight-dough method, obviously due to variations in microbial populations.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Diversity and technological potential of lactic acid bacteria of wheat flours

Lactic acid bacteria (LAB) were analysed from wheat flours used in traditional bread making throughout Sicily (southern Italy). Plate counts, carried out in three different media commonly used to detect food and sourdough LAB, revealed a maximal LAB concentration of approximately 4.75 Log CFU g⁻¹. Colonies representing various morphological appearances were isolated and differentiated based on phenotypic characteristics and genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR. Fifty unique strains were identified. Analysis by 16S rRNA gene sequencing grouped the strains into 11 LAB species, which belonged to six genera: Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Weissella. Weissella cibaria, Lactobacillus plantarum, Leuconostoc pseudomesenteroides and Leuconostoc citreum were the most prevalent species. The strains were not geographically related. Denaturing gradient gel electrophoresis (DGGE) analysis of total DNA of flour was used to provide a more complete understanding of the LAB population; it confirmed the presence of species identified with the culture-dependent approach, but did not reveal the presence of any additional LAB species. Finally, the technological characteristics (acidifying capacity, antimicrobial production, proteolytic activity, organic acid, and volatile organic compound generation) of the 50 LAB strains were investigated. Eleven strains were selected for future in situ applications.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

Microbiological profile of maize and rye flours, and sourdough used for the manufacture of traditional Portuguese bread

A thorough microbiological study of maize and rye flours, and sourdoughs obtained therefrom for eventual manufacture of broa – a dark sour bread typical in Northern Portugal, following artisanal practices, was carried out. Towards this purpose, samples were supplied by 14 artisanal producers, selected from 4 sub-regions, during two periods of the year. Total viable counts, as well as viable mesophilic and thermophilic microorganisms, yeasts and molds, Gram⁻ rods, endospore-forming and nonsporing Gram⁺ rods, and catalase⁺ and catalase⁻ Gram⁺ cocci were assayed for. The comprehensive experimental dataset unfolded a unique and rather complex wild microflora in flours and sourdoughs throughout the whole region, which did not discriminate among sub-regions or seasons, or flour source for that matter. However, fermentation played a major role upon the numbers of the various microbial groups: the viable counts of yeasts, lactobacilli, streptococci, lactococci, enterococci and leuconostocs increased, whereas those of molds, Enterobacteriaceae, Pseudomonadaceae, staphylococci and micrococci decreased.     

Diversity of lactic acid bacteria from Hussuwa, a traditional African fermented sorghum food

The diversity of lactic acid bacteria associated with Hussuwa fermentation, a Sudanese fermented sorghum food, was studied using a polyphasic taxonomical approach. Predominant strains could be well characterised based on a combination of phenotypic tests and genotypic methods such as ARDRA, rep-PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Thus, the majority (128 of 220, 58.3%) of strains exhibited phenotypic properties typical of heterofermentative lactobacilli and of these, 100 strains were characterised more closely using the genotyping methods. The majority (97/100) strains could be characterised as Lactobacillus fermentum strains. Seventy-two of 220 strains (32.7%) showed phenotypic properties that are characteristic of pediococci. Of 41 selected strains investigated by genotyping techniques, 38 (92.7%) could be characterised as Pediococcus acidilactici strains, while three (7.3%) could be characterised as Pediococcus pentosaceus strains. The Hussuwa fermentation thus appears to be dominated by L. fermentum strains and P. acidilactici strains. For this reason, we selected representative and predominant strains as potential starter cultures for Hussuwa fermentation. These strains, L. fermentum strains BFE 2442 and BFE 2282 and P. acidilactici strain BFE 2300, were shown on the basis of RAPD-PCR fingerprinting to predominate in a model fermentation when used as starter cultures inoculated at 1 × 10⁶ CFU/g and to lower the pH of the fermentation to below pH 4.0 within 48 h. These cultures should be studied for further development as starter preparations in pilot scale studies in actual field fermentations.