Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti‐inflammatory cheese

Scope : Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti‐inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti‐inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. Methods and results : We developed a single‐strain Ld‐fermented milk and a two‐strain P. freudenreichii and Ld‐fermented experimental pressed cheese using strains previously selected for their anti‐inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. Conclusion : This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti‐inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.     

Screening selected strains of probiotic lactic acid bacteria for their ability to produce biogenic amines (histamine and tyramine)

The aim of this study was to investigate the production of biogenic amines (BA), histamine and tyramine by some probiotic lactic acid bacteria (LAB). Fifteen strains representing six LAB species were screened qualitatively by growing them in a decarboxylase medium. Quantitative analysis was carried out by HPLC analysis with direct derivatization of acid extracts. Lactobacillus casei (TISTR 389) and Lactobacillus delbrueckii subsp. bulgaricus (TISTR 895) were found to produce BA. The highest levels of histamine (1820.9 ± 3.5 mg L^?1) and tyramine (5486.99 ± 47.6 mg L^?1) formation were observed for the TISTR 389 strain, while TISTR 895 produced only histamine (459.1 ± 0.63 mg L^?1) in the decarboxylase broth. Biogenic amine potential was not observed for the Lactobacillus acidophilus, Lactobacillus lactis subsp. lactis, Lactococcus lactis subsp. lactis, and Lactobacillus plantarum strains studied. This study confirmed that BA formation is strain dependent and not related to the species. Therefore, careful screening for amino acid decarboxylase activity is recommended before selecting LAB as appropriate starter or probiotic strains in food and dairy industry.     

Bile salt deconjugation activity of Propionibacterium strains and their cholesterol co‐precipitation abilities

The present study investigated the deconjugation of bile salts and co‐precipitation of cholesterol with deconjugated bile salts by seven Propionibacterium spp. strains in vitro. Propionibacterium spp. could deconjugate sodium glycocholate (1.53–5.31 mM) and sodium taurocholate (0.08–0.25 mM) bile salts. The highest cholesterol precipitation (47.8 µg/mL) was determined with Propionibacterium freudenreichii subsp. freudenreichii SP3 strain in a 0.15% oxgall‐containing medium. Significant (P < 0.05) correlations were observed between cholesterol co‐precipitation and deconjugation of sodium glycocholate among the strains. In vitro bile salt deconjugation activity studies of P. freudenreichii subsp. freudenreichii SP3 strain revealed that this strain may have potential as a probiotic strain for deconjugation of bile salts in vivo studies.     

New insights into physiology and metabolism of Propionibacterium freudenreichii

Dairy propionibacteria are Actinobacteria, mainly isolated from dairy environments. Propionibacterium freudenreichii has been used for a long time as a ripening culture in Swiss-type cheese manufacture, and is more and more considered for its potent probiotic effects. This review summarises the knowledge on the main P. freudenreichii pathways and the main features explaining its hardiness, and focuses on recent advances concerning its applications as a cheese ripening agent and as a probiotic for human health. Propionibacteria have a peculiar metabolism, characterised by the formation of propionic acid as main fermentation end-product. They have few nutritional requirements and are able to use a variety of carbon substrates. From the sequence of P. freudenreichii CIRM-BIA1^T genome, many pathways were reconstituted, including the Wood-Werkman cycle, enzymes of the respiratory chain, synthesis pathways for all amino acids and many vitamins including vitamin B₁₂. P. freudenreichii displays features allowing its long-term survival. It accumulates inorganic polyphosphate (polyP) as energy reserve, carbon storage compounds (glycogen), and compatible solutes such as trehalose. In cheese, P. freudenreichii plays an essential role in the production of a variety of flavour compounds, including not only propionic acid, but also free fatty acids released via lipolysis of milk glycerides and methyl-butanoic acids resulting from amino acid degradation. P. freudenreichii can exert health-promoting activities, such as a bifidogenic effect in the human gut and promising immunomodulatory effects. Many P. freudenreichii properties involved in adaptation, cheese ripening, bio-preservation and probiotic effects are highly strain-dependent. The elucidation of the molecular mechanisms involved is now facilitated by the availability of genome sequence and molecular tools. It will help in the selection of the most appropriate strain for each application.     

How to routinely assess transition,adhesion and survival of probiotics into the gut: a case study on propionibacteria

Two commercial promising probiotic strains (Propionibacterium jensenii and Propionibacterium freudenreichii) were studied to evaluate two basic probiotic requisites (adhesion and survival during the transit into the gut); two commercial starter cultures (Lactobacillus plantarum and Lactobacillus delbrueckii subsp. bulgaricus) were used as references. As an additional goal, the technological robustness was evaluated as growth as a function of pH, temperature and salt. Adhesion was studied as biofilm formation and hydrophobicity, whereas a simplified protocol was used to simulate the transit into the gut, along with the assessment of survival at pH 2.5 and in the presence of 0.3% bile salts. Propionibacteria were hydrophobic and form biofilm on glass slides, thus confirming the correlation of these properties and with the ability to adhere. None of the strains survived after the simulation of the transit into the gut, but this property could be induced in propionibacteria using a simple protocol of adaptive evolution. This paper suggests the possibility of using hydrophobicity as a screening tool to assess adhesion in propionibacteria and highlights the necessity of using a complete protocol of simulation of the gut to study the resistance to the conditions of stomach and intestine to avoid false‐positive results.     

Manufacture of Turkish Beyaz cheese added with probiotic strains

The survival of the probiotic strains Lactobacillus fermentum (AB5-18 and AK4-120) and Lactobacillus plantarum (AB16-65 and AC18-82), all derived from human faces, was investigated in Turkish Beyaz cheese production. Three batches of Turkish Beyaz cheese were produced: one with the test probiotic culture mix (P), another with a commercial starter culture mix including Lactoccocus lactis subsp. cremoris, Lactococcus lactis subsp. lactis (C) and the third with equal parts of the commercial starter culture mix and test probiotic culture mix (CP). The cheeses were ripened at 4 °C for 120 days and the viability of cultures was determined monthly. Cheese samples were analyzed for total solids, fat in solids, titratable acidity, pH, salt in total solids, proteolysis, sensory evaluation, aroma compounds and biogenic amines. While initial lactic acid bacteria load in P cheese was 2.7 × 10⁹ at the beginning, it was 7.42 × 10⁷ cfu/g at the end of 120 days of ripening. The results showed that test probiotic culture mix was successfully used in cheese production without adversely affecting the cheese quality during ripening. The chemical composition and sensory quality of P cheeses were also comparable with C cheeses. The present study indicates that probiotic cultures of human origin are feasible for Turkish Beyaz cheese production.     

Dried Tomato‐Flavored Probiotic Cream Cheese with Lactobacillus paracasei

Abstract: A dried tomato‐flavored probiotic cream cheese (P) containing Lactobacillus paracasei Lpc‐37 was developed for the purpose of this study. The same product, but without probiotic addition (C) was used as control. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were used as lactic starter cultures. Chemical composition analyses and sensory tests were performed on days 1 and 7, respectively. Titratable acidity, pH value and L. paracasei population were determined every 7 d during the refrigerated storage (21 d) of the cream cheeses. The experiment and analyses were performed in triplicate, using standard methods. Probiotic population remained greater than 10⁷ CFU/g throughout the storage period, thereby characterizing the product as potentially probiotic. Cream cheeses C and P did not differ on the sensory tests, both obtaining good overall acceptance by the consumers, of which 82.6% stated that they certainly or probably would buy the product. Practical Application: Lactobacillus paracasei Lpc‐37 is a probiotic bacterium and clinical studies have shown that this microorganism beneficially affects its host. In general, dried tomato‐flavored products and cream cheese are products with good acceptance by the consumers. Thus, regular consumption of the probiotic cream cheese developed in this study may have positive effects on health and well being of people if incorporated into their diet.     

Performance in Nondairy Drinks of Probiotic L. casei Strains Usually Employed in Dairy Products

The increase in vegetarianism as dietary habit and the increased allergy episodes against dairy proteins fuel the demand for probiotics in nondairy products. Lactose intolerance and the cholesterol content of dairy products can also be considered two additional reasons why some consumers are looking for probiotics in other foods. We aimed at determining cell viability in nondairy drinks and resistance to simulated gastric digestion of commercial probiotic lactobacilli commonly used in dairy products. Lactobacillus casei LC‐01 and L. casei BGP 93 were added to different commercial nondairy drinks and viability and resistance to simulated gastric digestion (pH 2.5, 90 min, 37 °C) were monitored along storage (5 and 20 °C). For both strains, at least one nondairy drink was found to offer cell counts around 7 log orders until the end of the storage period. Changes in resistance to simulated gastric digestion were observed as well. Commercial probiotic cultures of L. casei can be added to commercial fruit juices after a carefull selection of the product that warrants cell viability. The resistance to simulated gastric digestion is an easy‐to‐apply in vitro tool that may contribute to product characterization and may help in the choice of the food matrix when no changes in cell viability are observed along storage. Sensorial evaluation is mandatory before marketing since the product type and storage conditions might influence the sensorial properties of the product due to the possibility of growth and lactic acid production by probiotic bacteria.     

Production of probiotic Feta cheese using Propionibacterium freudenreichii subsp. shermanii as adjunct

Probiotic Feta cheese was produced using Propionibacterium freudenreichii subsp. shermanii as an adjunct since it has been shown to induce apoptosis of colon cancer cells in vitro and in vivo through the production of propionate and acetate. Microbiological and physicochemical analysis of the cheese was performed through the 60 d period of ripening. Counts of propionibacteria increased until day 7 and then remained constant until the end of ripening (approximately 9 log cfu g⁻¹). Moreover, throughout ripening the presence of P. freudenreichii subsp. shermanii LMG 16424^T was confirmed by 16S rRNA gene sequence analysis, while propionic acid was first detected on day 7 and reached a concentration of 52.1 mm on day 60. The ripened cheese containing P. freudenreichii subsp. shermanii was very well accepted by the sensory evaluation panellists. This is the first time that P. freudenreichii subsp. shermanii was studied as a probiotic adjunct in a white-brined cheese.     

Effects of tropical fruit pulps and partially hydrolysed galactomannan from Caesalpinia pulcherrima seeds on the dietary fibre content,probiotic viability,texture and sensory features of goat dairy beverages

The use of cheese whey and probiotic cultures in the production of dairy beverages has been highly attractive; nonetheless, whey-based goat beverages tend to be poor and watery when compared to fermented milks. The addition of fruits and fibre ingredients might improve texture and mouthfeel of this kind of product. Fermented whey-based goat beverages prepared using Streptococcus thermophilus TA-40 as starter culture, with added guava or soursop pulps, and with or without addition of partially hydrolysed galactomannan from Caesalpinia pulcherrima seeds (PHGM), showed to be good vehicles for Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus Lr-32, maintaining their viability above 7 log CFU/ml during 21 days. PHGM increased the dietary fibre content and enhanced the instrumental texture and sensory features of both guava and soursop dairy beverages, especially texture, appearance, and overall acceptability. The PHGM might be recommended to improve nutritional and sensory quality of fermented probiotic beverages produced with goat milk and cheese whey.     

Production of propionic acid in a fermented dairy beverage

A dairy beverage containing propionic acid was produced using the adjunct starter cultures with Lactobacillus acidophilus and Propionibacterium freudenreichii ssp. shermanii. The impacts of temperature (30, 35 and 40 °C) and inoculation ratio (1:2, 1:4 and 1:8) on propionic acid production and viability of micro‐organisms were studied. The incubation temperature had a significant adverse (P < 0.05) effect on the viability of P. freudenreichii and propionic acid production. Maximum amount of produced propionic acid (in 1:4–30 °C treatment) was 0.77%w/w. P. freudenreichii and L. acidophilus counts remained high (9 × 10⁶ and 1.5 × 10⁶ cfu/mL, respectively) after cold storage.     

The first dairy product exclusively fermented by Propionibacterium freudenreichii: A new vector to study probiotic potentialities in vivo

Dairy propionibacteria display probiotic properties which require high populations of live and metabolically active propionibacteria in the colon. In this context, the probiotic vector determines probiotic efficiency. Fermented dairy products protect propionibacteria against digestive stresses and generally contain a complex mixture of lactic and propionic acid bacteria. This does not allow the identification of dairy propionibacteria specific beneficial effects. The aim of this study was to develop a dairy product exclusively fermented by dairy propionibacteria. As they grow poorly in milk, we determined their nutritional requirements concerning carbon and nitrogen by supplementing milk ultrafiltrate (UF) with different concentrations of lactate and casein hydrolysate. Milk or UF supplemented with 50 mM lactate and 5 g L⁻¹ casein hydrolysate allowed growth of all dairy propionibacteria studied. In these new fermented dairy products, dairy propionibacteria remained viable and stress-tolerant in vitro during minimum 15 days at 4 °C. The efficiency of milk fermented by the most tolerant Propionibacterium freudenreichii strain was evaluated in piglets. Viability and SCFA content in the colon evidenced survival and metabolic activity of P. freudenreichii. This work results in the design of a new food grade vector, which will allow preclinical and clinical trials.     

An Influence of Fructan Containing Concentrate from Jerusalem Artichoke Tubers on the Development of Probiotic Dairy Starters on Milk and Oat-based Substrates

Supplementation of milk and oat hydrolysate containing medium with Jerusalem artichoke concentrate (JAC) and subsequent fermentation with probiotic dairy starters resulted in substantial stimulation of probiotics Bifidobacterium lactis and Lactobacillus acidophilus as well as yogurt starter culture Lactobacillus bulgaricus development and acidification rate. The strain-specific responses of the general yogurt cultures, as well as probiotics to the addition of JAC, should be considered to achieve optimal composition of probiotic strains and conformable fermentation conditions. JAC is suggested to be perspective prebiotic additive for fermented synbiotic milks or oat-hydrolysate-based products.     

Development of new probiotics by strain combinations: is it possible to improve the adhesion to intestinal mucus?

We evaluated the ability of commercial probiotic strains (Lactobacillus rhamnosus GG, L. rhamnosus LC705, Bifidobacterium breve 99, and Propionibacterium freudenreichii ssp. shermanii JS) to adhere alone or in different combinations to immobilized mucus. Probiotic combinations were clearly able to enhance the adhesion of L. rhamnosus GG, L. rhamnosus LC705, and P. freudenreichii ssp. shermanii JS. For L. rhamnosus GG and P. freudenreichii JS, all the combinations significantly improved adhesion to intestinal mucus, from 29.7 to 34.9% and from 1.9 to 2.3%, respectively. The adhesion of L. rhamnosus LC705 was improved from 0 to 46.4%. The adhesion of B. breve 99 was improved only in combination with L. rhamnosus GG and P. freudenreichii JS. Our results suggest that probiotic combinations could increase the beneficial health effects as compared with individual strains. Combinations of probiotic strains may therefore have synergistic adhesion effects, and such combinations also should be assessed in clinical studies.     

Probiotic Bacillus spp. in Soy‐Curd: Nutritional,Rheological, Sensory,and Antioxidant Properties

The focus of this study was to coculture probiotic Bacillus spp. with dairy starter cultures namely, Streptococcus thermophilus and Lactobacillus bulgaricus for enhanced nutritional properties of soy‐curd. Subsequently, rheological, sensory, and antioxidant properties of soy‐curd along with mineral as well as fatty acid composition were analyzed. Data revealed an increase in the cell viability of probiotic Bacillus spp. on coculturing rather than as mono‐culture. Proximate analysis showed higher nutritional value along with increased trace elements. UFA/SFA ratio, rheology, and sensory properties of probiotic soy‐curd were in the acceptable range. Probiotic soy‐curd showed higher antioxidant activity as measured by the ability to scavenge free radicals. No significant difference in the overall quality within the probiotic products was observed. However, B. flexus MCC2427 cocultured product displayed slightly better attributes than other samples. In general, the results suggest that soy‐curd can be a suitable carrier for probiotic Bacillus spp. and the enhanced nutritional and antioxidant properties could be of additional advantage to combat malnutrition problem.     

Effect of different matrices on probiotic resistance to in vitro simulated gastrointestinal conditions

In this study, we evaluated the effect of different matrices (MRS, milk and milk with inulin) on the tolerance of probiotic strains (Lactobacillus acidophilus La‐5 and Bifidobacterium animalis subsp. lactis BB‐12) to simulated conditions similar to those found in the gastrointestinal tract. Both probiotic strains demonstrated a significantly lower viability after exposure to in vitro gastric and intestinal conditions, and B. animalis subsp. lactis BB‐12 showed higher survival during the test compared to L. acidophilus La‐5 in all tested matrices. Milk and inulin protected probiotics from in vitro gastrointestinal stress. These results suggest that it is critical to formulate the food matrix to be used as probiotic carrier.     

Persistence of probiotic strains in the gastrointestinal tract when administered as capsules, yoghurt, or cheese

Most clinical studies of probiotics use freeze-dried, powdered bacteria or bacteria packed in capsules. However, probiotics are commercially available in various food matrices, which may affect their persistence in the gastrointestinal tract. The objective of the study was to compare oral and faecal recovery during and after administration of a combination of Lactobacillus rhamnosus GG and LC705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium animalis subsp. lactis Bb12 as capsules, yoghurt, or cheese. This randomized, parallel-group, open-label trial (n = 36) included a 4-week run-in, 2-week intervention, and 3-week follow-up period. Participants consumed 10¹⁰ cfu/day of probiotic combination and provided saliva and faecal samples before, during, and after the intervention. Strain-specific real-time PCR was used to quantify the strains.L. rhamnosus GG was the only probiotic strain regularly recovered in saliva samples. During the intervention period it was recovered in the saliva of 88% of the volunteers at least once. No difference was found between the yoghurt and cheese groups. At the end of the intervention, L. rhamnosus GG and LC705 counts were high in faecal samples of all product groups (8.08 and 8.67 log₁₀ genome copies/g, respectively). There was no matrix effect on strain quantity in faeces or the recovery time after ceasing the intervention. For P. freudenreichii subsp. shermanii JS and B. animalis subsp. lactis Bb12, a matrix effect was found at the end of the intervention (P < 0.01 and P < 0.001, respectively) and in the recovery time during follow-up (P < 0.05 for both). Yoghurt yielded the highest faecal quantity of JS and Bb12 strains (8.01 and 9.89 log₁₀ genome copies/g, respectively). The results showed that the administration matrix did not influence the faecal quantity of lactobacilli, but affected faecal counts of propionibacteria and bifidobacteria that were lower when consumed in cheese. Thus, the consumption of probiotics in yoghurt matrix is highly suitable for studying potential health benefits and capsules provide a comparable means of administration when the viability of the strain in the capsule product is confirmed.     

Effect of probiotic cultures on the stability of anthocyanins in blueberry yoghurts

Dependent on anthocyanin pigments, colour is one of the main quality factors of berry products. In this paper we assess the influence of probiotic bacteria on the degradation rate of anthocyanins in yoghurts with a highbush blueberry preparation during storage. Four types of yoghurts were prepared: first with the yoghurt starter culture YC-X16 only (Steptococcus thermophilus and Lactobacilllus delbrueckii subsp. bulgaricus) and three more, each with the starter culture YC-X16 and one of the three probiotic cultures: Bb-12, La-5 and LCP, respectively (Bif. animalis subsp. lactis – Bb-12; Lactobacillus. acidophilus – La-5 and Lactobacillus. paracasei subsp. paracasei – LCP).Derivatives of malvidin were the predominant anthocyanins in the tested yoghurts. Degradation of pigments occurred in accordance with the first-order reaction and its half-life time depended on the bacterial cultures. Anthocyanins in the probiotic yoghurt made with LCP culture were characterized by lower stability than those made with the other bacterial cultures. The impact of lactic bacteria or their metabolic products on the stability of anthocyanins in berry yoghurts shows that an appropriate selection of culture for production of yoghurt is recommended.     

Evaluation of novel lactose-positive and exopolysaccharide-producing strain of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> for fermented foods

A novel strain of lactic acid bacteria Pediococcus pentosaceus P 773 was isolated from spoiled beer and identified by means of 16S rDNA sequence analysis. The ability to assimilate lactose as a sole carbon source as a specific feature for this strain was detected and confirmed on dairy substrates. In the presence of sucrose containing substrates (sucrose, raffinose) this P. pentosaceus P 773 lactose-positive strain produced a complex of extracellular polysaccharides (Qp = 0.08 g/l/h) with a molecular mass about 2,000 kDa composed by glucose and fructose residues at a ratio 3:1, respectively. These exopolysaccharides were capable to stimulate the growth rate and biomass productivity of common constituent cultures of probiotic dairy starters (Bifidobacterium lactis, Lactobacillus acidophilus, Streptococcus thermophilus) as well as were assimilated as a sole carbon source by these strains. The present study confirmed the presence of lactose-positive and exopolysaccharide-producing strain of P. pentosaceus in natural environment which could be used as a starter culture to impart more functional attributes to fermented food.     

Development of novel species‐specific primers for species identification of the Lactobacillus casei group based on RAPD fingerprints

BACKGROUND: It is difficult to clearly distinguish and identify specific species of the Lactobacillus casei group using phenotypic and genotypic (16S ribosomal DNA sequence analysis) techniques alone. Some species of this group are probiotic and are widely used in the food and feed industries. The objective of this study was to develop species‐specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting for species identification within the closely related L. casei group of bacteria. RESULTS: Three random primers termed OPT‐14, OPA‐11 and OPT‐16 were developed for analysis. The primer pairs each produced a species‐specific band found only in the tested Lactobacillus rhamnosus, Lactobacillus paracasei subsp. tolerans and Lactobacillus zeae isolates respectively. These specific fragments were then sequenced for further analysis. The species‐specific primers were designed according to cloned sequencing, which was employed for polymerase chain reaction (PCR) with the template DNA of Lactobacillus strains. Single 102, 179 and 451 bp species‐specific bands were found only in L. rhamnosus, L. paracasei subsp. tolerans and L. zeae respectively. CONCLUSION: Using PCR, the novel species‐specific primers have been shown to rapidly, accurately and effectively identify species of L. rhamnosus, L. paracasei subsp. tolerans and L. zeae from within the L. casei group of probiotic bacteria. Copyright © 2009 Society of Chemical Industry