Final ReportSummary - QUALVIVO (The Development of a Next Generation Probiotic Supplement for Treatment and Prevention of Antibiotic Associated Diarrhoea)
Market needs:
The effects of antibiotic associated diarrhoea have a major detrimental socio-economic impact on the European Union (EU) due to additional treatment costs, increased length of stay in hospital and the use of vancomycin, which is traditionally regarded as an antibiotic associated diarrhoea treatment of last resort.
The rising occurrence and severity of antibiotic associated diarrhoea poses an increased risk for the European Community. According to the United Kingdom (UK)'s Department of Health there were 52 000 antibiotic associated aiarrhoea cases in 2006 in the UK.
Each patient suffering from antibiotic associated diarrhoea (Clostridium Difficile) requires on average an additional 21 days in hospital, taking up hospital beds and requiring nursing care. The cost of using these resources and treating antibiotic associated diarrhoea has been estimated at EUR 5920 per patient.
QUALVIVO will develop a next generation of synbiotic products that can be consumed not only when a person is already infected with antibiotic associated diarrhoea but also as prophylaxes (preventive measures).
Activities during the two year project duration:
Analysed the market situation - the lack of such a type of product currently on the market that will have no side effects is believed by both the consortium partners and the European Commission (EC) evaluators to have the potential to create excellent business opportunities by taking a niche in the market that is still unexplored.
Screened lactic acid bacteria and bifidobacteria - a very large part of the first year's work has been the screening of about 100 existing and new lactic acid bacteria (LAB) and bifidobacteria. This has enabled the project to establish a short list to be used.
Selected the bacteria with the desired properties together with complementary ingredients - the selected bacteria with desired antimicrobial activities (AMA) were verified in-vitro during the first year. Complementary ingredients have been selected for inclusion in the final composition.
Animal models for safety and proof of concept - safety is a key component for the project and animal models have been prepared for safety and proof of concept trials.
Work package (WP)4 outlines development of the optimum isolation and packaging conditions for the final symbiotic product. It is envisaged that a freeze drying technique will be employed for isolation.
WP5 brought together all the outputs from WP1 - WP4 for a prototype evaluation. The culmination of the extensive and sometimes complex scientific and technology development in the preceding WPs leading to a relatively simple prototype manufacturing process.
The QUALVIVO research project is being part financed by the EC under the Seventh Framework Programme (FP7) for Research for the benefit of SMEs (Contract No. 232087).
A consortium of 6 organisations comprising companies and research partners has developed the technology and will disseminate the results.
The technical work programme over the period 1 July 2009 to 31 June 2011 has involved the following:
WP1: Scientific basis for 'tailor made' pro and prebiotics
WP2: Technology development - probiotic methodology
WP3: Technology development - prebiotic methodology
WP4: Technology development - isolation
WP5: Technology integration
WP6: Technology validation.
Other, management and research and technology development (RTD0 work programmes over the same period has been as follows:
WP7: Innovation related activities
WP8: Consortium management
WP9: Project management.
The activities in the QUALVIVO project have been conducted by a consortium consisting of six organisations from four different European countries. The project has been coordinated by Probiotics International Ltd.
Project context and objectives:
The main objective of the QUALVIVO project is the development of a next generation probiotic supplement for treatment and prevention of antibiotic associated diarrhoea. Antibiotic associated diarrhoea and clostridium difficile associated disease can be hazardous for the aging population and immuno-compromised patients. Due to severe dehydration, antibiotic associated diarrhoea can be especially hazardous, leading in some cases to fatalities.
The increase and spread of antimicrobial resistance is a rapidly growing global problem following the antibiotic medication and over-consumption of antibiotics. At the European Union (EU) - United States of America (USA) summit on 3 November 2009, the EU and USA decided to establish a joint task force to combat antimicrobial resistance - 'The EU and the USA will, along with the World Health Organization (WHO), take the lead in dealing with the disastrous situation we find ourselves in.' One in every 250 death certificates now cites clostridium difficile as a contributory or main factor.
The QUALVIVO product will be a highly sophisticated synbiotic product, it will through dosage control and a target delivery mechanism deliver medicinal effects.
Project results:
Work progress and achievements during the period
WP1 - Scientific basis for 'tailor made' pro and prebiotics: New scientific understanding
1. Thew following section describes the mouse and artificial gut models that are used in QUALVIVO project for in vivo studies in order to validate the use of preferred candidate probiotic strains and prebiotics, selected in the first WP. The entire study was approved by the ethical committee (months 16 - 10). C57BL/6 mice were segregated into four groups. Mice that received the antibiotic mixture for four days were given autoclaved water for one day. Then all mice including the control group were given a single dose of clindamycin intraperitoneally one day before challenging with106 cfu/ml of either CD 2167 and CD 027 by gastric gavage.
From all the groups, fecal samples were collected before the antibiotic administration, after antibiotic administration, after two day of C. difficile infection, after one day of synbiotic administration and at the time of sacrifice. The animals were sacrificed by carbon dioxide asphyxiation and the proximal and rectal part of the colons were excised and stored in histopathological studies.
The faecal samples were suspended and cultured anaerobically on selective cycloserine-cefoxitin-fructose agar plate for C. difficile counts. LAB counts were determined on MRS agar, total aerobes and anaerobes on blood agar and total bifidobacteria on Beeren's agar. PCR, strain specific PCR, qPCR were used to follow the changes in faecal microbial populations.
Laborious screening process lead to the identification of probiotic strains with:
(i) good cell surface hydrophobicity;
(ii) degradation of at least one prebiotic;
(iii) antimicrobial activity against CD, ESBL E. Coli, Salmonella, Campylobacter;
(iv) growth in presence of mucin, bile, tolerance to acid;
(v) mucin binding ability; and
(vi) certain strains showed production of antioxidants compounds and cytokines.
Based on this, the candidate pro/prebiotics combinations will be tested in artificial gut system (GITS), animal model and clinical trials.
WP2 - Technology development: Probiotic methodology
Closstridium difficile (CD) causes CD associated diarrhoea (CDAD) and pseudomembranous colitis in hospitalised patients when administered with proton pump inhibitors or antibiotics such as clindamycin, cephalosporins, fluoroquinolones and ampicillin. These drugs destroy the indigenous intestinal microbiota, thus leading to gut overgrowth of CD. Elderly populations (age = 65) and patients on prolonged antibiotic treatment are most susceptible for infection. Thus, CD is posing a challenge for the management in health care units. Colitis is mainly caused by two large protein toxins: TcdA (308 kDa, Toxin A) and TcdB (260 kDa, Toxin B). In addition, emerging hypervirulent CD strains like B1/NAP1 (Warny et. al., 2005) secrete a toxin that causes actin aggregates, that enhances the effects of Toxin A and B and destroys the cytoskeleton. Emerging hypervirulent strains and increasing relapses of severe CDAD demand the search for new alternatives. One approach could be the use of probiotics with prebiotics, a combination referred to as 'synbiotics'.
Prevention study
Administration of a synbiotic RSIG mix containing multi-strain probiotics and prebiotics conferred protective effect in the antibiotic treated mice against CD infection. The exact mechanism(s) of action is unknown but a possible modulation by the probiotic bacteria with AMA against CD 027 as well as inhibited toxin production might be one explanation.
Treatment study
In conclusion, the present study shows that treatment with only synbiotic IG successfully treated and reduced the CD 027 load and showed no toxin production and neat histopathology of the caecum samples. Synbiotic RSIG, probiotic and prebiotic treated groups showed reduced histopathological score but toxin titres and CD 027 colony counts were high. This implies that the treatment model requires further optimisation in a mouse model.
WP3 - Technology development: Prebiotic methodology
A. The main aim of this study is to identify starch degrading bifidobacteria and to select a suitable starch preparation for designing a synbiotic mix with starch as one of the ingredients.
B. To develop an in vitro co-culture method for the selection of an optimal synbiotic mix using a combination of probiotics and prebiotics.
C. To investigate the effect of in vitro screened combination of prebiotic mixes for their growth promoting effects of beneficial microbes in an in vivo murine model to be able to make prioritisation of prebiotics
Bifidobacteria are one of the prevalent culturable, first to colonise, anaerobic bacterial groups of the human and animal gastrointestinal tract (GIT). They exert health- promoting effects, such as antimicrobial activity against the pathogenic microorganisms (e.g. bacteriocins) and/or block the adhesion of pathogens, or the immuno modulation response. Growth of the probiotic bacteria including bifidobacteria can be stimulated by non-dietary carbohydrate oligosaccharides (NDO) known as prebiotics. 8 % of the genes in the whole genome of bifidobacteria code for carbohydrate metabolising enzymes and transport proteins. Starch, a storage polysaccharide of plants and is the energy source for humans. Undigested starch reaching the colon, referred to as resistant starch (RS), is further degraded by various colonic bacteria) to volatile acids of which butyric acid plays an important role in intestinal mucosal health. Epidemiological studies have shown a correlation between a high intake of dietary starch, e.g. up to 400 g per day for countries with high starch intakes and a lower incidence of colorectal cancer. Bifidobacteria is reported to degrade starch, amylopectin, amylose and pullalan by secreting amylolytic and amylopullalonolytic enzymes. In the present study, we have investigated the ability of bifidobacteria to degrade starches originated from different plant sources and also chemically modified starches.
WP4 - Technology development: Isolation
There is a growing interest for food products containing probiotics due to their beneficial effects on humans upon consumption like alleviation of antibiotic associated diarrhoea. Consequently, this has lead to focus on probiotic bacteria for formulation of efficient functional food products. Probiotics by defination are 'live microorganisms which upon ingestion in adequate amount provides health benefits to the host'. Bifidobacteria is one group of probiotic bacteria, branched or pleomorphic rods and present as a single chains or clumps. Bifidobacteria are Gram-positive, non-spore forming, non-motile and non-filamentous anaerobic bacteria.
The bifidobacteria Bif LU 30 and Bif LU 10 strains are two top strains well characterised within this project (deliverables 1.3 1. 4 and 1.5). The Bif LU 10 is an auto aggregating (AA) strain. They colonise the intestine of mouse, exhibits high antimicrobial activity (AMA) against CD, degrade the prebiotics galacto-oligosaccharide (GOS), isomalto-oligosaccharide (IMOS), lactulose and fructo-oligosaccharide (FOS) and resistant starch (corn) .They multiply well in a mouse gut model when fed as probiotic strains in a synbiotic mix. The Bif LU 30 strain exhibits high AMA against CD, is able to degrade prebiotics namely xylo-oligosaccharide (XOS), GOS, IMOS, Lactulose and FOS, and express high cell surface hydrophobicity during stress conditions. This strain multiplied well in a mouse model when fed as one of the strains in a probiotic and synbiotic mix as reported in deliverable 3.2.
The Bif LU 10 and Bif LU 30 production was successfully upscaled and a stable biomass was produced under pH controlled fermentation conditions as well as the freeze drying with 1 % (w/v) glycine as the cryoprotectant. The freeze dried powder obtained was stable when stored at + 4 oC over a period of 30 days.
WP5 - Technology integration
The work done throughout the QUALVIVO project has involved the identification of viable strains of probiotic bacteria able to help in the treatment of antibiotic associated diarrhoea. Two strains of bifidobacteria (Bif LU 30 and Bif LU 10) have shown excellent resistance to conditions similar to the ones in the gastrointestinal tract and thus have been considered viable for the purpose of this project. Two strains of Lactobacilli (LU28 and LU33) have also been identified to be suitable for the project. The following sections describe the pilot plant manufacturing processes and capability for both sets of bacteria.
The process starts with the selected strains which have previously been isolated and stored at -20 oC as glycerol stock cultures. A single colony is then transferred to a cultivation medium to prepare the 'seed culture' under controlled conditions. A fermenter containing a cultivation broth is inoculated with the seed culture and maintained under controlled conditions. After the fermentation stage, cells are harvested by centrifugation and suspended in a solution containing cryoprotectant components. Finally, the samples are subjected to a freeze drying process.
General process conditions are described in deliverable 4.1 for the laboratory scale production of bifidobacteria. The deliverable concludes that these are the optimum conditions for the cultivation of the bacteria, and thus they will be used throughout this document.
The partners within the QUALVIVO project have been able to identify the optimum conditions for small scale production of two sets of probiotic bacteria. Current tests conducted at their facilities confirm a significant yield of these bacteria from small scale batches. Suitable recommendations for scale up have been identified from current industrial practice and from the experiences provided by consortium members.
WP6 - Technology validation
The QUALVIVO consortium plan to execute the human study being part of WP6. The lead partner for the study is LUH. The amount of work in the project has led to delays with the impact that the human study has not yet been executed.
The human study is planned to take place during the next few months. Depending on how fast participants who match the inclusion criteria can be recruited, it will have impact on how long time the actual study will take. Possible delays in ethical approval will also impact the time schedule of the study.
Bacteria with resistance to antibiotics survives antibiotic treatment and propagates. This can lead to unspecified diarrhoea, overgrowth of large intestine bacteria higher up in the instestines and in severe cases antibiotic associated colitis in the colon.
The purpose of the human study is to study the amount of symptoms of diarrhoea and stomach-pain when antibiotic intake is complemented with QUALVIVO synbiotic as well as to study if the QUALVIVO synbiotic can reduce this bacterial unbalance.
The QUALVIVO synbiotic is a food supplement consisting of normal intestinal lactic acid bacteria (lactobacillus and bifidobacteria) and specially selected small carbohydrates (oligosaccharides, starch) for the chosen lactic acid bacteria.
We have investigated that the most common pathogenic bacteria being the reason for diarrhea (Salmonella, Campolybacter jejuni, et.al) not can grow on the chosen small carbohydrates included in the QUALVIVO synbiotic pilot product. The QUALVIVO synbiotc lactic acid bacteria have been given to immunosuppressed mice (cytostatic treated). They survived and showed no sign of disease.
The human study is a simple non-placebo controlled study where otherwise healthy people, their antibiotics intake not related to symptoms in the intestines, are included in the study.
Project website:
http://www.qualvivo.eu(se abrirá en una nueva ventana)
Contact details:
QUALVIVO Exploitation Manager
Mats Wallström
LAVIVO AB
Box 5031
SE-42605 Västra Frölunda
Sweden
e-mail: mats.wallstrom@lavivo.com
The effects of antibiotic associated diarrhoea have a major detrimental socio-economic impact on the European Union (EU) due to additional treatment costs, increased length of stay in hospital and the use of vancomycin, which is traditionally regarded as an antibiotic associated diarrhoea treatment of last resort.
The rising occurrence and severity of antibiotic associated diarrhoea poses an increased risk for the European Community. According to the United Kingdom (UK)'s Department of Health there were 52 000 antibiotic associated aiarrhoea cases in 2006 in the UK.
Each patient suffering from antibiotic associated diarrhoea (Clostridium Difficile) requires on average an additional 21 days in hospital, taking up hospital beds and requiring nursing care. The cost of using these resources and treating antibiotic associated diarrhoea has been estimated at EUR 5920 per patient.
QUALVIVO will develop a next generation of synbiotic products that can be consumed not only when a person is already infected with antibiotic associated diarrhoea but also as prophylaxes (preventive measures).
Activities during the two year project duration:
Analysed the market situation - the lack of such a type of product currently on the market that will have no side effects is believed by both the consortium partners and the European Commission (EC) evaluators to have the potential to create excellent business opportunities by taking a niche in the market that is still unexplored.
Screened lactic acid bacteria and bifidobacteria - a very large part of the first year's work has been the screening of about 100 existing and new lactic acid bacteria (LAB) and bifidobacteria. This has enabled the project to establish a short list to be used.
Selected the bacteria with the desired properties together with complementary ingredients - the selected bacteria with desired antimicrobial activities (AMA) were verified in-vitro during the first year. Complementary ingredients have been selected for inclusion in the final composition.
Animal models for safety and proof of concept - safety is a key component for the project and animal models have been prepared for safety and proof of concept trials.
Work package (WP)4 outlines development of the optimum isolation and packaging conditions for the final symbiotic product. It is envisaged that a freeze drying technique will be employed for isolation.
WP5 brought together all the outputs from WP1 - WP4 for a prototype evaluation. The culmination of the extensive and sometimes complex scientific and technology development in the preceding WPs leading to a relatively simple prototype manufacturing process.
The QUALVIVO research project is being part financed by the EC under the Seventh Framework Programme (FP7) for Research for the benefit of SMEs (Contract No. 232087).
A consortium of 6 organisations comprising companies and research partners has developed the technology and will disseminate the results.
The technical work programme over the period 1 July 2009 to 31 June 2011 has involved the following:
WP1: Scientific basis for 'tailor made' pro and prebiotics
WP2: Technology development - probiotic methodology
WP3: Technology development - prebiotic methodology
WP4: Technology development - isolation
WP5: Technology integration
WP6: Technology validation.
Other, management and research and technology development (RTD0 work programmes over the same period has been as follows:
WP7: Innovation related activities
WP8: Consortium management
WP9: Project management.
The activities in the QUALVIVO project have been conducted by a consortium consisting of six organisations from four different European countries. The project has been coordinated by Probiotics International Ltd.
Project context and objectives:
The main objective of the QUALVIVO project is the development of a next generation probiotic supplement for treatment and prevention of antibiotic associated diarrhoea. Antibiotic associated diarrhoea and clostridium difficile associated disease can be hazardous for the aging population and immuno-compromised patients. Due to severe dehydration, antibiotic associated diarrhoea can be especially hazardous, leading in some cases to fatalities.
The increase and spread of antimicrobial resistance is a rapidly growing global problem following the antibiotic medication and over-consumption of antibiotics. At the European Union (EU) - United States of America (USA) summit on 3 November 2009, the EU and USA decided to establish a joint task force to combat antimicrobial resistance - 'The EU and the USA will, along with the World Health Organization (WHO), take the lead in dealing with the disastrous situation we find ourselves in.' One in every 250 death certificates now cites clostridium difficile as a contributory or main factor.
The QUALVIVO product will be a highly sophisticated synbiotic product, it will through dosage control and a target delivery mechanism deliver medicinal effects.
Project results:
Work progress and achievements during the period
WP1 - Scientific basis for 'tailor made' pro and prebiotics: New scientific understanding
1. Thew following section describes the mouse and artificial gut models that are used in QUALVIVO project for in vivo studies in order to validate the use of preferred candidate probiotic strains and prebiotics, selected in the first WP. The entire study was approved by the ethical committee (months 16 - 10). C57BL/6 mice were segregated into four groups. Mice that received the antibiotic mixture for four days were given autoclaved water for one day. Then all mice including the control group were given a single dose of clindamycin intraperitoneally one day before challenging with106 cfu/ml of either CD 2167 and CD 027 by gastric gavage.
From all the groups, fecal samples were collected before the antibiotic administration, after antibiotic administration, after two day of C. difficile infection, after one day of synbiotic administration and at the time of sacrifice. The animals were sacrificed by carbon dioxide asphyxiation and the proximal and rectal part of the colons were excised and stored in histopathological studies.
The faecal samples were suspended and cultured anaerobically on selective cycloserine-cefoxitin-fructose agar plate for C. difficile counts. LAB counts were determined on MRS agar, total aerobes and anaerobes on blood agar and total bifidobacteria on Beeren's agar. PCR, strain specific PCR, qPCR were used to follow the changes in faecal microbial populations.
Laborious screening process lead to the identification of probiotic strains with:
(i) good cell surface hydrophobicity;
(ii) degradation of at least one prebiotic;
(iii) antimicrobial activity against CD, ESBL E. Coli, Salmonella, Campylobacter;
(iv) growth in presence of mucin, bile, tolerance to acid;
(v) mucin binding ability; and
(vi) certain strains showed production of antioxidants compounds and cytokines.
Based on this, the candidate pro/prebiotics combinations will be tested in artificial gut system (GITS), animal model and clinical trials.
WP2 - Technology development: Probiotic methodology
Closstridium difficile (CD) causes CD associated diarrhoea (CDAD) and pseudomembranous colitis in hospitalised patients when administered with proton pump inhibitors or antibiotics such as clindamycin, cephalosporins, fluoroquinolones and ampicillin. These drugs destroy the indigenous intestinal microbiota, thus leading to gut overgrowth of CD. Elderly populations (age = 65) and patients on prolonged antibiotic treatment are most susceptible for infection. Thus, CD is posing a challenge for the management in health care units. Colitis is mainly caused by two large protein toxins: TcdA (308 kDa, Toxin A) and TcdB (260 kDa, Toxin B). In addition, emerging hypervirulent CD strains like B1/NAP1 (Warny et. al., 2005) secrete a toxin that causes actin aggregates, that enhances the effects of Toxin A and B and destroys the cytoskeleton. Emerging hypervirulent strains and increasing relapses of severe CDAD demand the search for new alternatives. One approach could be the use of probiotics with prebiotics, a combination referred to as 'synbiotics'.
Prevention study
Administration of a synbiotic RSIG mix containing multi-strain probiotics and prebiotics conferred protective effect in the antibiotic treated mice against CD infection. The exact mechanism(s) of action is unknown but a possible modulation by the probiotic bacteria with AMA against CD 027 as well as inhibited toxin production might be one explanation.
Treatment study
In conclusion, the present study shows that treatment with only synbiotic IG successfully treated and reduced the CD 027 load and showed no toxin production and neat histopathology of the caecum samples. Synbiotic RSIG, probiotic and prebiotic treated groups showed reduced histopathological score but toxin titres and CD 027 colony counts were high. This implies that the treatment model requires further optimisation in a mouse model.
WP3 - Technology development: Prebiotic methodology
A. The main aim of this study is to identify starch degrading bifidobacteria and to select a suitable starch preparation for designing a synbiotic mix with starch as one of the ingredients.
B. To develop an in vitro co-culture method for the selection of an optimal synbiotic mix using a combination of probiotics and prebiotics.
C. To investigate the effect of in vitro screened combination of prebiotic mixes for their growth promoting effects of beneficial microbes in an in vivo murine model to be able to make prioritisation of prebiotics
Bifidobacteria are one of the prevalent culturable, first to colonise, anaerobic bacterial groups of the human and animal gastrointestinal tract (GIT). They exert health- promoting effects, such as antimicrobial activity against the pathogenic microorganisms (e.g. bacteriocins) and/or block the adhesion of pathogens, or the immuno modulation response. Growth of the probiotic bacteria including bifidobacteria can be stimulated by non-dietary carbohydrate oligosaccharides (NDO) known as prebiotics. 8 % of the genes in the whole genome of bifidobacteria code for carbohydrate metabolising enzymes and transport proteins. Starch, a storage polysaccharide of plants and is the energy source for humans. Undigested starch reaching the colon, referred to as resistant starch (RS), is further degraded by various colonic bacteria) to volatile acids of which butyric acid plays an important role in intestinal mucosal health. Epidemiological studies have shown a correlation between a high intake of dietary starch, e.g. up to 400 g per day for countries with high starch intakes and a lower incidence of colorectal cancer. Bifidobacteria is reported to degrade starch, amylopectin, amylose and pullalan by secreting amylolytic and amylopullalonolytic enzymes. In the present study, we have investigated the ability of bifidobacteria to degrade starches originated from different plant sources and also chemically modified starches.
WP4 - Technology development: Isolation
There is a growing interest for food products containing probiotics due to their beneficial effects on humans upon consumption like alleviation of antibiotic associated diarrhoea. Consequently, this has lead to focus on probiotic bacteria for formulation of efficient functional food products. Probiotics by defination are 'live microorganisms which upon ingestion in adequate amount provides health benefits to the host'. Bifidobacteria is one group of probiotic bacteria, branched or pleomorphic rods and present as a single chains or clumps. Bifidobacteria are Gram-positive, non-spore forming, non-motile and non-filamentous anaerobic bacteria.
The bifidobacteria Bif LU 30 and Bif LU 10 strains are two top strains well characterised within this project (deliverables 1.3 1. 4 and 1.5). The Bif LU 10 is an auto aggregating (AA) strain. They colonise the intestine of mouse, exhibits high antimicrobial activity (AMA) against CD, degrade the prebiotics galacto-oligosaccharide (GOS), isomalto-oligosaccharide (IMOS), lactulose and fructo-oligosaccharide (FOS) and resistant starch (corn) .They multiply well in a mouse gut model when fed as probiotic strains in a synbiotic mix. The Bif LU 30 strain exhibits high AMA against CD, is able to degrade prebiotics namely xylo-oligosaccharide (XOS), GOS, IMOS, Lactulose and FOS, and express high cell surface hydrophobicity during stress conditions. This strain multiplied well in a mouse model when fed as one of the strains in a probiotic and synbiotic mix as reported in deliverable 3.2.
The Bif LU 10 and Bif LU 30 production was successfully upscaled and a stable biomass was produced under pH controlled fermentation conditions as well as the freeze drying with 1 % (w/v) glycine as the cryoprotectant. The freeze dried powder obtained was stable when stored at + 4 oC over a period of 30 days.
WP5 - Technology integration
The work done throughout the QUALVIVO project has involved the identification of viable strains of probiotic bacteria able to help in the treatment of antibiotic associated diarrhoea. Two strains of bifidobacteria (Bif LU 30 and Bif LU 10) have shown excellent resistance to conditions similar to the ones in the gastrointestinal tract and thus have been considered viable for the purpose of this project. Two strains of Lactobacilli (LU28 and LU33) have also been identified to be suitable for the project. The following sections describe the pilot plant manufacturing processes and capability for both sets of bacteria.
The process starts with the selected strains which have previously been isolated and stored at -20 oC as glycerol stock cultures. A single colony is then transferred to a cultivation medium to prepare the 'seed culture' under controlled conditions. A fermenter containing a cultivation broth is inoculated with the seed culture and maintained under controlled conditions. After the fermentation stage, cells are harvested by centrifugation and suspended in a solution containing cryoprotectant components. Finally, the samples are subjected to a freeze drying process.
General process conditions are described in deliverable 4.1 for the laboratory scale production of bifidobacteria. The deliverable concludes that these are the optimum conditions for the cultivation of the bacteria, and thus they will be used throughout this document.
The partners within the QUALVIVO project have been able to identify the optimum conditions for small scale production of two sets of probiotic bacteria. Current tests conducted at their facilities confirm a significant yield of these bacteria from small scale batches. Suitable recommendations for scale up have been identified from current industrial practice and from the experiences provided by consortium members.
WP6 - Technology validation
The QUALVIVO consortium plan to execute the human study being part of WP6. The lead partner for the study is LUH. The amount of work in the project has led to delays with the impact that the human study has not yet been executed.
The human study is planned to take place during the next few months. Depending on how fast participants who match the inclusion criteria can be recruited, it will have impact on how long time the actual study will take. Possible delays in ethical approval will also impact the time schedule of the study.
Bacteria with resistance to antibiotics survives antibiotic treatment and propagates. This can lead to unspecified diarrhoea, overgrowth of large intestine bacteria higher up in the instestines and in severe cases antibiotic associated colitis in the colon.
The purpose of the human study is to study the amount of symptoms of diarrhoea and stomach-pain when antibiotic intake is complemented with QUALVIVO synbiotic as well as to study if the QUALVIVO synbiotic can reduce this bacterial unbalance.
The QUALVIVO synbiotic is a food supplement consisting of normal intestinal lactic acid bacteria (lactobacillus and bifidobacteria) and specially selected small carbohydrates (oligosaccharides, starch) for the chosen lactic acid bacteria.
We have investigated that the most common pathogenic bacteria being the reason for diarrhea (Salmonella, Campolybacter jejuni, et.al) not can grow on the chosen small carbohydrates included in the QUALVIVO synbiotic pilot product. The QUALVIVO synbiotc lactic acid bacteria have been given to immunosuppressed mice (cytostatic treated). They survived and showed no sign of disease.
The human study is a simple non-placebo controlled study where otherwise healthy people, their antibiotics intake not related to symptoms in the intestines, are included in the study.
Project website:
http://www.qualvivo.eu(se abrirá en una nueva ventana)
Contact details:
QUALVIVO Exploitation Manager
Mats Wallström
LAVIVO AB
Box 5031
SE-42605 Västra Frölunda
Sweden
e-mail: mats.wallstrom@lavivo.com