Bacterial therapy to help eradicate Helicobacter pylori and to reduce the gastrointestinal side effects of antibiotics: a possible treatment scheme?

Schermata 2018-01-19 alle 12.18.00Helicobacter pylori (Fig. 1), previously called Campylobacter pylori, is a gram-negative, microaerophilic bacterium found usually in the stomach. It was identified in 1982 by two Australian who found it in a patient with chronic gastritis and gastric ulcers, conditions not previously thought to have a bacterial aetiology. H. pylori is also linked to the development of duodenal ulcers and stomach cancer. It is present in the stomach of 50% of the world’s population and asymptomatic in over 80% of those infected. The standard first-line therapy to eradicate H. pylori, is the so-called triple therapy consisting of proton pump inhibitors (PPI), mainly omeprazole, along with the antibiotics clarithromycin and amoxicillin. Variations on the triple therapy have been developed over the years, using a different PPI, or replacing amoxicillin with metronidazole for those with an allergy to penicillin. Due to antibiotic-resistant bacteria, an additional round of antibiotic therapy, the quadruple therapy, consisting of a PPI, a bismuth colloid, metronidazole and tetracyclines, has been developed. Triple therapy is ineffective in 15%–30% of cases and quadruple therapy in 10%–25% of cases [1]. Therapy also often causes side effects such as antibiotic-induced diarrhoea [2]. Recently meta-analysis involving a pediatric population including 29 trials (3122 participants) and involving 17 probiotic regimens was published. Compared with placebo, probiotic-supplemented PPI and antibiotic therapy significantly increased H. pylori eradication rates and reduced the incidence of side effects [3].
Similarly, 13 randomized controlled trials involving 2306 of adult patients were recently included in another meta-analysis. These authors also reported that probiotic supplementation during H. pylori treatment may be effective for improving eradication rates, minimizing the incidence of therapy-related adverse events and alleviating most disease-related clinical symptoms [4].
One of the most investigated probiotics is Bifidobacterium animalis subspecies lactis BB12 (DSM 15954, hereafter referred to as BB12). Reported to reduce episodes of antibiotic-induced diarrhoea during anti-Helicobacter treatment by 60% [5], BB12 increased the eradication rate of H. pylori by 13% in the case of triple therapy, and by 14% in case of quadruple therapy [6,7], and decreased H. pylori urease activity after 6 weeks of therapy [8].
Helicobacter pylori uses molecular hydrogen as a respiratory substrate when grown in the laboratory. It is also known that hydrogen is available in the gastric mucosa and that its use greatly increases stomach colonization by H. pylori. Therefore, hydrogen present in animals as a consequence of normal colonic flora acitivity can facilitate the maintenance of a pathogenic bacterium [9].
Schermata 2018-01-19 alle 12.18.09The strongest hydrogen-producing organisms, are thought to be Escherichia coli, Clostridium, and Enterobacter species [10]; BB12 increased the numbers of stool bifidobacteria and suppressed coliform bacteria (Escherichia, Clostridium, Enterobacter) [11]. Therefore, since colonic bifidobacteria decrease colonic hydrogen production, BB12 might alter hydrogen production and can lessen the severity of H. pylori infection. Another strain has recently been shown to preserve the growth of bifidobacteria thus reducing of the number of opportunistic microorganisms: Enterococcus faecium L3 (LMG P-27496, hereafter referred to as L3) [12], which inhibits H. pylori growth in vitro (Fig. 2). On this basis, BB12 and L3 might be able to be used to better eradicate the gastric pathogen while reducing the number of side effects. The pre-antibiotic use of probiotics likely reduces the severity and/or the length of antibiotic-induced diarrhoea (by increasing the bacterial load), while their use following antibiotic administration likely increases the eradication rate (as they act on a pathogen already reduced in terms of vitality and strength). Thus, a possible treatment approach could be to use probiotics as add-on therapy in order to achieve better eradication of H. pylori while minimizing the gastrointestinal side effects of antibiotic use (Fig. 3).

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Bibliografia
1. Malfertheiner P, Bazzoli F, Delchier JC, Celiñski K et al (2011) Pylera Study Group. Helicobacter pylori eradication with a capsule containing bismuth subcitrate potassium, metronidazole, and tetracycline given with omeprazole versus clarithromycin-based triple therapy: a randomised, open-label, non-inferiority, phase 3 trial. Lancet 377(9769):905–913
2. Kwon SB, Lee KL, Kim JS, Lee JK et al (2010) Antibiotics-associated diarrhea and other gastrointestinal abnormal responses regarding Helicobacter pylori eradication. Korean J Gastroenterol 56(4):229–235
3. Feng JR, Wang F, Qiu X, McFarland LV et al (2017) Efficacy and safety of probiotic-supplemented triple therapy for eradication of Helicobacter pylori in children: a systematic review and network meta-analysis. Eur J Clin Pharmacol 73(10):1199–1208
4. Lü M, Yu S, Deng J, Yan Q, Yang C, Xia G, Zhou X (2016) Efficacy of Probiotic supplementation therapy for Helicobacter pylori eradication: a meta-analysis of randomized controlled trials. PLoS One 11(10):e0163743
5. de Vrese M, Kristen H, Rautenberg P, Laue C, Schrezenmeir J (2011) Probiotic lactobacilli and bifidobacteria in a fermented milk product with added fruit preparation reduce antibiotic associated diarrhea and Helicobacter pyloriactivity.JDairyRes78(4):396–403
6. Sheu BS, Wu JJ, Lo CY, Wu HW, Chen JH, Lin YS, Lin MD (2002) Impact of supplement with Lactobacillus- and Bifidobacterium-containing yogurt on triple therapy for Helicobacter pylori eradication. Aliment Pharmacol Ther 16(9):1669–1675
7. Sheu BS, Cheng HC, Kao AW, Wang ST, Yang YJ, Yang HB, Wu JJ (2006) Pretreatment with Lactobacillus- and Bifidobacterium-containing yogurt can improve the efficacy of quadruple therapy in eradicating residual Helicobacter pylori infection after failed triple therapy. Am J Clin Nutr 83(4):864–869
8. Wang KY, Li SN, Liu CS, Perng DS, Su YC, Wu DC, Jan CM, Lai CH, Wang TN, Wang WM (2004) Effects of ingesting Lactobacillus- and Bifidobacterium-containing yogurt in subjects with colonized Helicobacter pylori. Am J Clin Nutr 80(3):737–741
9. Olson JW, Maier RJ (2002) Molecular hydrogen as an energy source for Helicobacter pylori. Science 298(5599):1788–1790
10. Goyal Y, Kumar M, Gayen K (2013) Metabolic engineering for enhanced hydrogen production: a review. Can J Microbiol 59(2):59-78
11. Chen RM, Wu JJ, Lee SC, Huang AH, Wu HM (1999) Increase of intestinal Bifidobacterium and suppression of coliform bacteria with short-term AB ingestion. J Dairy Sci 82(11):2308–2314
12. Lo Skiavo LA, Gonchar NV, Fedorova MS, Suvorov AN (2013) Dynamics of contamination and persistence of Clostridium difficile in intestinal microbiota in newborn infants during antibiotic therapy and use of probiotic strain Enterococcus faecium L3. Antibiot Khimioter 58(11–12):13–18

Ribodiet®

Nucleotides are low-molecular-weight intracellular compounds. They are the building blocks for nucleic acids and play a key role in many biochemical pathways. They are phosphoric nucleoside esters, made up of three components: a weakly basic nitrogenous compound, a pentose sugar, and one or more phosphate groups. They are the basic units of the nucleic acids DNA and RNA. The most important nucleotides are adenosine, guanosine, inosine, cytidine and uridine monophosphates.
Nucleotides are considered ‘semi-essential’ nutrients. This means that endogenous production satisfies requirements in the maintenance condition, but exogenous administration is needed in childhood, in stress conditions or when tissues are damaged [1].
Every new cell requires around 1 billion nucleotides in order to duplicate. Some tissues have a limited capacity for de novo nucleotide synthesis, and thus require exogenously supplied bases that can be utilized by a salvage pathway. For example, the intestinal mucosa, haematopoietic cells of the bone marrow, leucocytes, erythrocytes and lymphocytes are incapable of de novo synthesis, and thus utilize the salvage pathway, suggesting that an exogenous supply of nucleotides via the diet might be important for these cells [2].
For over 15 years, Prosol has provided a benchmark in nucleotide blends, sold under the Ribocare® brand, for infant formula application. Following this success story, the company decided to deploy its extensive knowledge in the field of yeast cell extracts and, after years of trials, has launched Ribodiet®, a combination of natural nutritional ingredients that can have positive effects on different body tissues.

Composition and technical specifications

Ribodiet® is a natural product extracted from yeast cells with a gentle, standardized and highly controlled process which is free of chemical solvents. Ribodiet® is a source of nucleotides, nucleosides, oligo nucleotides, ribonucleic acid fragments, amino acids, minerals and group B vitamins (Table 1). It is derived from Kluyveromyces fragilis or Saccharomyces cerevisiae yeast cells.

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Yeast RNA, extracted from the cell only by physical means, is concentrated, and free nucleotides are obtained via enzymatic hydrolysis. The product is then spray-dried in order to maintain its chemical and physical characteristics and make it stable and storable at room temperature. The concentration process, complete standardization, and the hydrolysis of nucleic acids allows Prosol to obtain an ingredient with a high content of free nucleotides (>40%), qualitatively and quantitatively standardized.
Ribodiet® is a gluten-free product and suitable for vegans. It is certified halal and kosher. The Ribodiet® trade mark is registered in the European Union. Table 2 summarizes the technical features of Ribodiet®.

 

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Mechanism of action

Nucleotides are mainly used in infant foods because research in human nutrition has demonstrated that the inclusion of nucleotides in parenteral and infant milk formulas improves intestinal health and the development of the immune system [3].
Nucleotides do not all have the same effect, so a mixture of nucleotides is used to provide the best result. Many studies in healthy term infants have demonstrated that nucleotide supplementation may reduce the risk of diarrhoea by approximately one quarter because immune maturation is enhanced [2].
Nucleotides have a direct effect on the maintenance of intestinal mucosal integrity. It has been demonstrated that nucleotide supplementation in young rats increases the weight of the intestinal mucosa, villus height (by 25%), and the activity of brush border enzymes (maltase, sucrase and lactase), suggesting acceleration of gut growth and differentiation [3].
Supplementation with a nucleoside–nucleotide mixture increases recovery after food deprivation, infection or protein deficiency. Small intestine atrophy and decreased activity of brush border enzymes in rats quickly resolved with nucleotide supplementation [4].
As regards microbiota health status, in vivo studies show that dietary supplementation with nucleotides improves the intestinal flora and stimulates the growth of bifidobacteria [5]. Dietary nucleotides favour the development of faecal flora with a predominance of bifidobacteria and lactobacilli and lower percentage of Gram-negative enterobacteria.
Finally, in relation to immune system modulation, nucleotides influence both humoral and cell-mediated immunity: they accelerate T-cell-dependent antibody production and seem to exert actions on T-helper cells at antigen presentation, perhaps during cognitive cell–cell interactions.
A nucleoside-nucleotide mixture stimulates the proliferation, differentiation and maturation of neutrophils [3]. Nucleotides cause a transient increase in natural killer cell cytotoxicity, interleukin-2 production and interferon-gamma secretion, and lower macrophage activation [6].
Supplementation with dietary nucleotides increases resistance to bacterial infection in animals and humans.

Efficacy

Two pre-clinical trials have been performed on Ribodiet® at the Food Chemistry and Nutraceuticals laboratories (Department of Drug Sciences – Pavia – Italy) to evaluate efficacy in modulating some parameters involved in the immune response. The cell line used in this assay was THP-1, a human monocytic cell line, which was incubated for 24 hours in a complete medium plus a non-cytotoxic concentration (1.25 mg/ml) of Ribodiet®. Before the end of the treatment, a lipo-polysaccharide (LPS), one of the best characterized macrophage-activating factors, was added to the samples to promote the inflammatory response.

The following markers of the inflammatory process were detected:

– TNF-α, a pro-inflammatory cytokine recognized as a central mediator of inflammation;
– IL-10, a cytokine that down-regulates the production of the pro-inflammatory cytokines;
– NO (nitric oxide), a compound generated endogenously as part of the inflammatory response against pathogens, such as bacteria and viruses;
– ROS (reactive oxygen species) secreted upon macrophage activation as part of the host cell defence mechanism.

The results are summarized in Fig. 1.

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Ribodiet® treatment induced a significant reduction of over 90% in TNF-α expression compared to the control group. The yeast product also modulated oxidative process markers. After inflammation was induced, the nucleotide complex reduced NO levels by 22.5% and ROS levels by 55%. However, the anti-inflammatory IL-10 cytokine was significantly increased by 23.5% compared to control.
The reported results indicate that Ribodiet®, in the investigated in vitro system, exerts an anti-inflammatory, anti-oxidant and immuno-modulatory action on the cell line stimulated with LPS.
A second pre-clinical study was carried out to evaluate Ribodiet® efficacy in combination with a source of zinc, an ingredient with European Food Safety Authority (EFSA) – approved claims regarding the immune system (Commission Regulation (EU) no.432/2012).
A THP-1 human monocyte line was treated with LPS to induce an inflammatory response at the end of 24 hours of incubation in the culture media (Fig. 2). The zinc source (0.039 mg/ml, Zn at 20%) alone reduced TNF-α levels by 16.8%. However, when combined with Ribodiet® (1.25 mg/ml), the zinc reduced TNF-α expression by 91.6% compared to control.
The results demonstrate that Ribodiet® significantly boosts the anti-inflammatory activity of zinc, thus reducing TNF-α expression induced by LPS.

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Safety

Nucleotides are ingredients generally recognized as safe. The use of nucleotides in infant formulas has not been reported to cause an increase in gastro-intestinal intolerance. Furthermore, elderly residents in a long-term care facility were fed enterally for 12 weeks with either a standard formula without any added nucleotides or an immune-modulating formula that contained 1.3 g/l of nucleotides as yeast RNA. No differences related to feeding parameters or safety measures were observed [7].

Applications and usage

In light of the above evidence, Ribodiet® is an interesting ingredient for addition to products targeted at immune system functionality.

Other potential applications are to improve:

– Gut barrier health
– Sports nutrition (recovery after performance)
– Cognition/concentration (lack of synthesis of nucleotides in brain tissue)
– Iron absorption.

The suggested daily dosage is 50-350 mg. The ingredient can be used in both solid and liquid formulations.

References

1. Sánchez-Pozo A, Gil A (2002) Nucleotides as semiessential nutritional components. Br J Nutr 87(Suppl 1):S135–137
2. Koletzko B, Baker S, Cleghorn G et al (2005) Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group. J Pediatr Gastroenterol Nutr 41:584–599
3. Jyonouchi H (1994) Nucleotide actions on the humoral immune response. J Nutr 124(1 Suppl):138S–143S
4. Belo A, Marchbank T, Fitzgerald AJ et al (2006) Gastroprotective effects of oral nucleotide administration. Gut 55:165–171
5. Uauy R (1990) Dietary nucleotides and requirements in early life. In: E. Lebenthal (ed) Textbook of gastroenterology and nutrition in infancy. Raven Press, New York, pp 265–280
6. Matsumoto Y, Adjei AA, Yamauchi K et al (1995) Nucleoside-nucleotide mixture increases peripheral neutrophils in cyclophosphamide-induced neutropenic mice. Nutrition 11:296–299
7. Hess JR, Greenberg NA (2012) The role of nucleotides in the immune and gastrointestinal systems: potential clinical applications. Nutr Clin Pract 27:281–294

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Proflora™

Apart from its primary function of nutrient absorption, the intestinal mucosa also has an important immune function. This is secondary to antigenic stress induced by food intake (with its microbial load) and by the presence of a varied flora of resident microorganisms (microbiota) which are transient or adhere to the intestinal walls. It is estimated that about 98% of the population has dysbiosis (i.e., a depletion of beneficial microbial species), which leads to various types of bowel dysfunction that may have a negative impact on health.

Proflora™ is an innovative synbiotic consisting of six micro-encapsulated probiotic strains associated with prebiotic fibre, for the natural rebalancing of the ecosystem of different parts of the intestine.


Composition and technical specifications

Proflora™ contains six exclusive, selected probiotic strains (over 2 billion /sachet) with prebiotic fibre FOS (short-chain fructo-oligosaccharides) derived from sugar beet. The probiotic strains (registered in an international culture collection) are in a gastro-protected micro-encapsulated form for maximum probiotic biological activity. It has been reported in vivo that the colonization kinetics of 1×109/CFU of probiotics in a gastro-protected micro-encapsulated form is comparable to that of 5×109/CFU of non-micro-encapsulated probiotics [1, 2].

Proflora™ is allergen free (according to Commission Directive 2007/68/EC), odourless and tasteless. Tables 1 and 2 show the ingredients and the nutritional composition of Proflora.

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Mechanism of action

Lactobacillus rhamnosus LR06 and Lactobacillus plantarum LP02 are both able to produce active substances (bacterio cins) that limit the spread of coliform bacteria in various intestinal segments [3], which is the reason why Proflora™ is particularly recommended for patients with frequent episodes of infections of the genitourinary tract due to Escherichia coli. Each of the six microbial species, belonging to the genera Bifidobacterium and Lactobacillus, are gastro-protected and act in synergy for higher efficacy and colonization of the different intestinal segments. The effect is maintained over time because of the strong ability of Lactobacillus salivarius LS03 to adhere to the intestinal mucosa.

FOS in Proflora™ are not hydrolyzed by digestive enzymes or absorbed by the mucosa of the small intestine, and so reach the colon intact where they selectively stimulate the development of probiotic strains present in Proflora™ and beneficial lactobacilli and bifidobacteria in the resident microflora. The FOS positively affect carbohydrate and fat metabolism, improve the function of the intestinal mucosa by increasing the ‘barrier’ effect, and facilitate the absorption of certain minerals, especially calcium and magnesium.

Safety

Proflora™ is patented by Probiotical S.p.A. and is free of all allergens according to current legislation (Annex II Reg. EU 1169/2011): wheat, rye, barley, oats, spelt, kamut and hybridised strains, crustaceans and products based on shellfish, eggs and egg products, fish and seafood, peanuts, soy and soy products, milk and dairy including lactose, nuts, celery, mustard, sesame seeds, lupins, molluscs and products based on molluscs, and sulfur dioxide and sulphites at concentrations above 10 mg/kg or 10 mg/litre.

Proflora™ is recommended for children, the elderly, pregnant and breast-feeding women, and those with food intolerances, food allergies or coeliac disease.

Applications and dosage

Proflora™ can provide healthy support in case of antibiotic and/or laxative administration, diarrhoea, gastrointestinal disorders, digestive difficulties, irritable colon, uro-genital infections, respiratory allergies, adverse reactions to foods, abdominal bloating, meteorism, mental and physical stress and dry eye syndrome [4].

The suggested dosage is one sachet daily (preferably half an hour before meals) for 30 consecutive days. In cases of acute symptoms, one or two sachets should be taken each day for 8–10 days depending on symptoms.

References

1.DelPiano M, CarmagnolaS, Andorno S, Pagliarulo M, Tari R, Mogna L, Strozzi GP, Sforza F, Capurso L (2010) Evaluation of the intestinal colonization by microencapsulated probiotic bacteria in comparison with the same uncoated strains. J Clin Gastroenterol 44:S42–46

2. Del Piano M, Carmagnola S, Ballarè M, Balzarini M, Montino F, Pagliarulo M, Anderloni A, Orsello M, Tari R, Sforza F, Mogna L, Mogna G (2012) Comparison of the kinetics of intestinal colonization by associating 5 probiotic bacteria assumed either in a microencapsulated or in a traditional, uncoated form. J Clin Gastroenterol 46:S85–92

3. Bottazzi V (2009) Il microbiota intestinale. Novara, Mofin Alce Group

4. Chisari G, Chisari EM, Greco C, Madeddu R, Motta M, Chisari CG (2016) Coadministration of Lactobacillus and Bifidobacterium strains in combination with short-chain fructo-oligosaccharides reduces the ocular surface damage caused by dry eye syndrome. Minerva Oftalmol 58(2) 31-38

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Harmonization of the EU nanomaterial definitions:

Introduction

In European Union legislation, the regulatory status of nanomaterials in sectoral regulations is established by their definition, indicating what is considered a nanomaterial, and thus subject to the application of specific nano provisions in each product category. Therefore, there is more than one regulatory definition of nanomaterial. The first definitions were those of cosmetics [1] followed by the food labelling regulation [2]. The European Commission then released a Recommendation for nanomaterial definition [3]. It must be kept in mind that a Recommendation is not binding unless included in a Regulation. The Recommendation is based on particle number size distribution as the primary parameter and specific surface area as the secondary parameter. In addition, it was stated that >50% particles by number must be in the size range 1–100 nm for a substance to be considered a nanomaterial, which made the parameters measurable and thus the EC Recommendation applicable. The recommendation was taken up by more recent legislation, such as the Biocide Product Regulation [4] and the Medical Device Regulation (approved on 5 May 2017). Since 2011, the use of the EC Recommendation has resulted in issues and the need for clarification, which in turn led to the ongoing revision that should be released soon by the Environment Directorate-General. The expected revision will not include major modifications but will only add some text clarifications. Also, guidelines for implementation of the Recommendation will be issued, setting out the best testing strategy to date to identify a substance as a nanomaterial. After the revision is published, it is expected the Commission will harmonize the definition of nanomaterial across sectoral regulations.

Regulatory definition(s) of nanomaterial

The definitions of nanomaterials used in cosmetics and food are more a statement of principle than regulatory enforceable definitions. In cosmetics, nanomaterials are defined as intentionally produced substances which have particle size between 1 and 100 nm and are biopersistent and insoluble, but there is no particle number benchmark. Therefore, in principle, even one nanoparticle (if detectable) in the substance is sufficient to make it a nanomaterial, with the consequent burden of safety dossier notification. However, other nanomaterials such as nanosomes are not considered nanomaterials under the cosmetic regulation since they are not biopersistent. The result is that even if the technical function of the nanomaterial is necessary to the product, and while the safety of the ingredient has to be demonstrated, it is not necessary to provide a notification with the full nano safety dossier 6 months before placing the product on the market. The definition of nanomaterials used for food is even less clear, since it includes intentionally produced (and not ‘intentionally in the nano-range’) materials, and does not establish a particle number benchmark. In addition, it specifies that nanostructured or aggregated materials with nano-properties have to be considered nanomaterials. Therefore, in principle even a natural ingredient intentionally produced to be used in food, with one particle in the nanosize range, would be considered a nanomaterial and thus be labelled as such. Consequently, the entering into force of the Novel Food Regulation [5] which refers to the same nanomaterial definition, would make almost all substances with detectable nanoparticles novel foods. It is also not clear if nanosomes or other soft nanomaterials will be considered nanomaterials or not. This uncertainty results in the non-applicability of nano specific requirements in food. For example, there are foods using additives that are known to contain detectable fractions of nanoparticles, such as E551 (silica dioxide). However, the labels of such marketed products do not mentioned them as being nano, even though they should. This example shows the current non-application of the nano definition in food.

There are other examples suggesting that even if not official, the tendency is to consider the EC Recommendation as the de facto working definition in food. The best example is the recent EFSA opinion on TiO2 (E171) re-evaluation, which found E171 to be safe for use. The EFSA Scientific Opinion on E171 [6] reported that, based on information collected from stakeholders and other sources, E171 is not a nanomaterial because the number of particles is below 50% (i.e., ranging between <1% and 36%). Therefore, literature data on nano-TiOwas considered not relevant (and therefore not used) in the safety assessment of E171.Nano-related data were only used in the comparative assessment of the behaviour after ingestion of nano and bulk forms (essentially, absorption, distribution, metabolism and excretion (ADME)). To complete the safety assessment, EFSA launched a call for data on reproductive toxicity and on particle size distribution because of the need to indicate nano fraction in E171specifications‘ due to its potential importance for toxicokinetics and toxicity’[7].

Conclusions

The EFSA Opinion on E171 shows there are different ways of defining nanomaterials in food. According to the food definition of nanomaterial, E171 must be classified as a nanomaterial. Therefore, for consumer information [2], (nano) should be placed beside E171 on the label’s ingredient list. However, this has not been done for products on the market. On the other hand, when determining which safety data to use, the EFSA Panel decided to follow the EC Recommendation instead (which is not mandatory for food, as it is only a Recommendation), thus deciding that E171 is not a nanomaterial. Which view is the correct one?

It is thus clear that the Commission’s intention to make the revised EC Recommendation enforceable in all regulations is necessary to eliminate the unjustified difference in the safety assessment of nanomaterials for companies in different industry sectors. The fact that the same substance can be considered a nanomaterial in one regulation and not in another with different safety requirements is not acceptable.

References

1. Regulation EC 1223/2009. OJ L 342, 22.12.2009, p. 59–209

2. Regulation EU 1169/2011. OJ L 304, 22.11.2011, p. 18–63

3. Commission Recommendation of 18 October 2011 on the definition of nanomaterial. OJ L 275, 20.10.2011, p. 38–40

4. Regulation EU 528/2012. OJ L 167, 27.6.2012, p. 1–123

5.RegulationEU20283/2015.OJL327,11.12.2015,p.1–22

6. EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food) (2016) Scientific Opinion on the re-evaluation of titanium dioxide (E 171) as a food additive. EFSA Journal 2016;14(9):4545, 83 pp. doi:10.2903/j.efsa.2016.4545

7. EFSA (2017) Call for scientific and technical data on the permitted food additive titanium dioxide (E 171). https://ec.europa.eu/food/sites/food/files/safety/docs/fs-improv-additive-20170130-call_sci-tech-data-e171.pdf

Bifidobacterium longum W11: an antibiotic-resistant probiotic

Preface

Possible unwanted consequences of antibiotic use include: (a) the selection of antibiotic-resistant pathogenic bacteria; (b) increased susceptibility of the host to new infections; (c) gram-negative bacterial overgrowth; (d) diarrhoea; and (e) Clostridium difficile colonization [1]. Theoretically, except for antibiotic resistance, all
these effects could be alleviated with probiotics. However, even a small delay between antibiotic administration and supplementation with probiotics severely reduces the positive impact of the probiotics as they are unable to integrate into the gut microbiota. The high sensitivity of probiotics to antibiotics prevents stable colonization of the gut, thus ensuring only non-significant and transient effects. However, the use of antibiotic-resistant bacteria could be beneficial. Of course, for safety reasons, this resistance must not be transferable and must not be located in plasmid DNA as probiotics could otherwise be responsible for dangerous horizontal gene transfer (Fig. 1) to pathogens [2]. Antibiotic-resistant probiotics sound very attractive, even tempting pharmaceutical companies to falsely claim some probiotic strains have antibiotic-resistant properties. Indeed, a brochure recently suggested that physicians could use Bifidobacterium longum BB536 in conjunction with antibiotics. This clearly suggests that BB536 is antibiotic resistant, even though it is known to be susceptible to antibiotics [3]. The questions are then: do we have any antibiotic-resistant probiotic strains for use with specific antibiotics? Are we sure that these antibiotic-resistant probiotics cannot transfer their resistance to pathogens? The answers to botSchermata 2017-07-20 alle 15.56.57h questions are yes. Some species of lactic acid bacteria commonly used in the food industry or naturally found in raw food are resistant to vancomycin and include Lactobacillus casei, L. rhamnosus, L. curvatus, L. plantarum, L. coryniformis, L. brevis, L. fermentum, Pediococcus pentosaceus, P. acidilactici, Leuconostoc lactis and L. mesenteroides. This vancomycin resistance found in lactobacilli, leuconostocs and pediococci is intrinsic, chromosomally encoded and not transferable [4]. Unfortunately, vancomycin is an antibiotic frequently used in hospitals, and is rarely (ifatall) prescribed by family physicians. As it is often used in combination with other antibiotics such as linezolid and meropenem, having vancomycin-resistant probiotics is not that relevant.

Bifidobacterium longum W11

Bifidobacterium longum is a commensal bacterium present in the human gut. It is one of the 32 species belonging to the genus Bifidobacterium. It is an early colonizer of the gastrointestinal tract of infants and one of the major constituents of newborn intestinal microbiota, where it is predominant especially in the first 6 months of life. Bifidobacterium longum W11 (LMG P-21586) is of particular interest for use as a probiotic [5]. It tolerates low pH and is resistant to bile salts, two characteristics which allow it to reach and survive in the intestine. In addition, as is well known, it is important that probiotic bacteria are able to adhere to human intestinal cells and then proliferate: adhesion enables probiotic strains to colonize the intestinal tract, stabilize the intestinal mucosal barrier, competitively exclude pathogenic bacteria, and provide improved metabolic and immune-modulatory activity. The W11 strain is able to colonize the gut and has impressive persistence (persistence indicates the length of time the strain is recoverable from faeces after wash-out). Indeed, several studies have shown that some strains of Bifidobacterium spp. can produce exopolysaccharides, sugar polymers which facilitate strong anchorage, and then create persistence, to intestinal epithelial cells (Fig. 2). Microscopy indicated that the W11 strain Schermata 2017-07-20 alle 15.57.10Schermata 2017-07-20 alle 15.57.18strongly adheres to human enterocytes (Fig. 3) and that the production of this exocellular polymers contributes to its adhesion and persistence properties [6]. Likely due to its production and release of exopolysaccharides, the W11 strain has been shown to be a strong colonizer also in severe conditions: in elderly patients on total enteral nutrition it increased the bifidobacterial count by more than 10-fold while simultaneously reducing the number of Clostridia [7]. In addition to its probiotic characteristics, the W11 strain also has important biological properties. From an immunity perspective, it seems to promote a Th1 response while lowering the Th2 response [8]. Clinically, the W11 strain has been shown to improve constipation in those following a low-calorie diet for the treatment of obesity [9], and to increase stool frequency (by 25% on average) in patients with constipation-variant IBS, reducing abdominal pain and bloating in those with moderate-severe symptoms [10].

Resistance of W11 to rifaximin

It has recently been shown that the W11 strain is totally resistant to rifampicin, rifapentine, rifabutin and rifaximin at concentrations ranging from 32 to 256 mg/ml and is also partially resistant to the same drugs at a concentration of 512 mg/ml (Fig. 4) [11]. A mutation in the rpoB gene (DNA-mediated RNA polymerase subunit β) is responsible for this resistance and has already been described in Staphylococcus aureus and Escherichia coli [12, 13]. Analysis of W11 shows a chromosomal DNA mutation which causes a change in the triple of a specific amino acid (P564L) of the protein leading to resistance to rifamycin. In W11 the exact position of the rpoB gene on the chromosome was identified and a targeted search was conducted for transposable elements 200 kbp upstream and downstream of the rpoB gene, using TransposonePSI software. No transposable elements were identified, confirming that the rpoB gene is not flanked by mobile genetic elements [11].

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Possible clinical uses of strain W11

In the last decade, the rifamycin-derivative rifaximin has been registered in many European countries and in the United States. It has attracted interest due to its pharmacological, toxicological and clinical characteristics. It has an excellent safety profile due to negligible intestinal absorption after oral administration [14]. Its wide antimicrobial spectrum covers gram-positive and gram-negative bacteria, including aerobes and anaerobes [15]. Rifaximin has been used successfully in the treatment of several intestinal disorders, including traveller’s diarrhoea, diverticular disease, small intestinal bacterial overgrowth (SIBO), C. difficile infection, Crohn’s disease, IBS, functional dyspepsia and hepatic encephalopathy. From a pharmacological perspective, rifaximin is not a bioavailable antibiotic and its mechanisms of action involve not only direct bactericidal activity but also alteration of the virulence factors of enteric bacteria, reduction of pathogen adhesion and internalization to the intestinal epithelium, and reduction of inflammatory cytokine release. Therefore, rifaximin could be used as a novel treatment for all those intestinal diseases mainly characterized by dysbiosis and inflammation. Consequently, a rifaximin-resistant probiotic strain, W11, could be used as adjuvant therapy when administered along with the antibiotic. Indeed, this association has been evaluated in IBS where patients administered rifaximin plus W11 reported greater improvement in symptoms than patients administered only rifaximin (plus placebo, of course) [16].

Conclusions

Bifidobacterium longum W11 is the first probiotics train identified as having antibiotic-resistant properties. This characteristic is chromosomally based and not transferable. W11 can be safely used in combined therapy with rifaximin in conditions responsive to rifaximin and in dysbiosis. This would open new treatment approaches in the era of probiotics.

Conflict of interest

Francesco Di Pierro is owner of Velleja Research.

References
1. Becattini S, Taur Y, Pamer EG (2016) Antibiotic-induced changes in the intestinal microbiota and disease. Trends Mol Med 22(6):458–478
2. Modi SR, Collins JJ, Relman DA (2014) Antibiotics and the gut microbiota. J Clin Invest 24(10):4212–4218
3. Office of Food Additive Safety (2008) Generally Recognized as Safe (GRAS) Notification for the Use of Bifidobacterium longum BB536 in Selected Foods. Prepared by Spherix Inc. for Morinaga Milk Industry Co., Ltd. Tokyo, Japan. https://www.fda.gov/downloads/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm269214.pdf (accessed 20 Jun 2017)
4. Tynkkynen S, Singh KV, Varmanen P (1998) Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes. Int J Food Microbiol 41(3):195–204
5. Medina M, Izquierdo E, Ennahar S et al (2007) Differential immunomodulatory properties of Bifidobacterium longum strains: relevance to probiotic selection and clinical applications. Clin Exp Immunol 150:531–538
6. Inturri R, Stivala A, Sinatra F et al (2014) Scanning electron microscopy observation of adhesion properties of Bifidobacterium longum W11and chromatographic analysis of its exopolysaccaride. Food Nutr Sci 5:1787–1792
7. Del Piano M, Ballarè M, Montino F et al (2004) Clinical experience with probiotics in the elderly on total enteral nutrition. J Clin Gastroenterol 38(2):S111–S114
8. Medina M, Izquierdo E, Ennahar S, Sanz Y (2007) Differential immunomodulatory properties of Bifidobacterium longum strains: relevance to probiotic selection and clinical applications. Clin Exp Immunol 150(3):531–538
9. Amenta M, Cascio MT, Di Fiore P, Venturini I (2006) Diet and chronic constipation. Benefits of oral supplementation with symbiotic zir fos (Bifidobacterium longum W11 + FOS Actilight). Acta Biomed 77(3):157–162
10. Colecchia A, Vestito A, La Rocca A et al (2006) Effect of a symbiotic preparation on the clinical manifestations of irritable bowel syndrome, constipation-variant. Results of an open, uncontrolled multicenter study. Minerva Gastroenterol Dietol 52(4):349–358
11. Graziano T, Amoruso G, Nicola S et al (2016) The possible innovative use of Bifidobacterium longum W11 in association with rifaximin. A new horizon for combined approach? J Clin Gastroenterol 50:S153–S156
12. Wichelhaus TA, Schafer V, Brade V et al (1999) Molecular characterization of rpoB mutations conferring cross-resistance to rifamycins on methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 43:2813–2816
13. Kothary V, Scherl EJ, Bosworth B et al (2013) Rifaximin resistance in Escherichia coli associated with inflammatory bowel disease correlates with prior rifaximin use, mutations in rpoB, and activity of Phe-Arg-b-naphthylamide-inhibitable efflux pumps. Antimicrob Agents Chemother 57:811–817
14. Gillis J, Brogden RN (1995) Rifaximin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic potential in conditions mediated by gastrointestinal bacteria. Drugs 49:467–484
15. Scarpignato C, Pelosini I (2005) Rifaximin, a poorly absorbed antibiotic: pharmacology and clinical potential. Chemotherapy 51:36–66
16. Fanigliulo L, Comparato G, Aragona G et al (2006) Role of gut microflora and probiotic effects in the irritable bowel syndrome. Acta Biomed 77(2):85–89

Oximacro®, a natural cranberry extract with a very high content of proanthocyanidin A

Urinary tract infections (UTIs) are widespread and affect a large portion of the human population. About 150 million people worldwide develop UTIs each year, with high societal costs, and an estimated 40% of women develop at least one UTI during their lifetimes. UTIs refer to the presence of a certain threshold number of bacteria in the urine (usually >105/ml) and consist of cystitis (or lower UTIs, with bacteria in the bladder), urethral syndrome and pyelonephritis (or upper UTIs, with infection of the kidneys). Bacterial cystitis (also called acute cystitis) can occur in women and men, and some people also develop recurrent UTIs with an average of two to three episodes per year. Herpes simplex virus (HSV) infection is lifelong and its spectrum of clinical manifestations is wide, ranging from asymptomatic infection or mild mucocutaneous lesions on the lips, cornea, genitals or skin, up to more severe, and even life-threatening, infections, including encephalitis, neonatal infections, and progressive or visceral disease in immunocompromised hosts. Products extracted from the fruit of American cranberry (Vaccinium macrocarpon Ait., Ericaceae), in different formulations, are rich in compounds that are thought to exert numerous health benefits, such as the prevention of microbial infections and beneficial activity against inflammation [1]. Indeed, the berries of cranberry (Vaccinium macrocarpon Aiton) have been used for hundreds of years as a remedy for diseases of the urinary tract and have attracted attention due to their potential health benefits [2, 3]. The beneficial mechanism of cranberry was historically thought to be due to the fruit’s acids causing a bacteriostatic effect in the urine. However, recently, a group of proanthocyanidins (PACs) with A-type linkages (PAC-A) was isolated from cranberries and shown to exhibit bacterial anti-adhesion activity against both antibiotic-susceptible and antibiotic-resistant strains of uropathogenic P-fimbriated Escherichia coli bacteria, including multidrug-resistant E. coli [4–8]. Central to the efficacy of cranberry extract/juice is the determination of the optimum dose of PAC-A, which is an essential requirement in establishing botanical supplements as viable supports to conventional therapies [9]. Oximacro® is a cranberry extract produced by Biosfered (Turin, Italy) that possesses the highest content of PACs and the highest percentages of PAC-A dimers and trimers available on the market. Oximacro® has been shown to exert both antiviral and UTI prevention capability in in vitro and in preclinical studies.

Composition and technical specifications

Schermata 2017-01-13 alle 09.16.08Oximacro® is produced by Biosfered with a proprietary method of extraction. It is characterized by a high content of PACs (>36% according to the BL-DMAC method; >80% according to the Bates-Smith method; >99% according to the European Pharmacopoeia) and the highest percentages of PAC-A dimers and trimers available on the market (>85%, analysed by HPLC coupled to ESI-tandem mass spectrometry). Oximacro® fractionation (Fig. 1) shows that the extract consists of five main fractions: fraction 1 is mainly composed of delphinidin and cyanidin glycosides, and rutin; fraction 2 contains quercetin and isorhamnetin; fractions 3 and 4 are dominated by several isomers of PAC-A dimers and trimers; and fraction 5 shows no detectable compounds (Fig. 1). In vitro tests show that only the fractions containing PACs possess biological activity [10]. Oximacro® is available as both a powder extract and a fluid extract (Oximacro®-FL). The latter is standardized and titrated to provide 36 mg PAC-A per gram of product. Stress tests with increasing temperature from 22°C to 55°C show that Oximacro® is stable and that the content of PACs is not affected by temperature. The technical specifications of Oximacro® and Oximacro®-FL are reported in Table 1.

Schermata 2017-01-13 alle 09.17.20Efficacy In vitro studies and mechanism of action

We recently reported on the ability of Oximacro® to inhibit herpes simplex type 1 (HSV-1) and type 2 (HSV-2) replication in vitro [9]. Analysis of the mode of action revealed that Oximacro® prevents adsorption of HSV-1 and HSV-2 to target cells. Further mechanistic studies confirmed that Oximacro® and its PACs-A target the viral envelope glycoproteins gD and gB (Fig. 2), thus resulting in the loss of infectivity of HSV particles. Moreover, Oximacro® completely retains its anti-HSV activity even at acidic pHs (3.0 and 4.0) and in the presence of 10% human serum proteins, conditions that mimic the physiological properties of the vagina (a potential therapeutic location for Oximacro®). Figure 2 shows the interaction between the A-type PACs present in Oximacro® and HSV envelope glycoproteins.

Clinical studies

Oximacro® was shown to be effective in the prevention of UTIs and support for patients with UTIs. A balanced group of female (ranging from 19 to over 51 years of age) and male volunteers (over 51 years of age) was divided into two groups. The experimental group received one capsule containing Oximacro® (36 mg PACs-A) twice per day (morning and evening) for 7 days, while the placebo group was given the same number of capsules with no PACs. After 7 days of administration, a significant difference was found between the placebo and Oximacro® groups for both females (p<0.001; N=60) and males (p=0.016; N=10) concerning reduction of UTI symptoms (e.g., dysuria, frequency, cloudy urine and occasionally haematuria). Furthermore, CFU/ml counts from the urocultures were almost cleared and showed a highly significant difference between the experimental and the placebo groups (p<0.001) [10].

Safety

Oximacro® is produced according to strict procedures and the safety of the product is guaranteed through the use of top technology detection systems (microbiological, chemical and molecular). The product has been recently approved by the US FDA as a New Dietary Ingredient and is Generally Recognized as Safe (GRAS).

Application and use

Careful determination of the total PAC content using the BL-DMAC method and the authentication of PACs-A with mass spectrometry of Oximacro® are necessary to prepare effective doses for antiviral efficacy and UTI prevention. Our published findings indicate Oximacro® is an attractive candidate for the development of novel microbicides of natural origin for the prevention of HSV infections and UTIs and for active support for patients with UTIs. The recommended dosage of Oximacro® is 120 mg/dose, which corresponds to 36 mg PAC-A. This dosage has been demonstrated to be effective when administered twice a day for at least 7 days.

References

1. Blumberg JB, Camesano TA, Cassidy A, Kris-Etherton P, Howell A, Manach C, Ostertag LM, Sies H, Skulas-Ray A, Vita JA (2013) Cranberries and their bioactive constituents in human health. Adv Nutr 4:618–632

2. Jass J, Reid G (2009) Effect of cranberry drink on bacterial adhesion in vitro and vaginal microbiota in healthy females. Can J Urol 16:4901–4907

3. Jepson RG, Williams G, Craig JC (2013) Cranberries for preventing urinary tract infections. Sao Paulo Med J 131:363

4. Gupta K, Chou M, Howell A, Wobbe C, Grady R, Stapleton A (2007) Cranberry products inhibit adherence of P-fimbriated Escherichia coli to primary cultured bladder and vaginal epithelial cells. J Urol 177:2357–2360

5. Stapleton AE, Dziura J, Hooton TM, Cox ME, Yarova-Yarovaya Y, Chen S, Gupta K (2012) Recurrent urinary tract infection and urinary Escherichia coli in women ingesting cranberry juice daily: a randomized controlled trial. Mayo Clinic Proc 87:143–150

6. Howell AB, Reed JD, Krueger CG, Winterbottom R, Cunningham DG, Leahy M (2005) A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 66:2281–2291

7. Howell AB, Botto H, Combescure C, Blanc-Potard AB, Gausa L, Matsumoto T, Tenke P, Sotto A, Lavigne JP (2010) Dosage effect on uropathogenic Escherichia coli anti-adhesion activity in urine following consumption of cranberry powder standardized for proanthocyanidin content: a multicentric randomized double blind study. BMC Infect Dis 10:94

8. Gurley BJ (2011) Cranberries as antibiotics? Arch Int Med 171:1279–1280

9. Terlizzi ME, Occhipinti A, Luganini A, Maffei ME, Gribaudo G (2016) Inhibition of herpes simplex type 1 and type 2 infections by Oximacro®, a cranberry extract with a high content of A-type proanthocyanidins (PACs-A). Antiviral Res 132:154–164

10. Occhipinti A, Germano A, Maffei ME (2016) Prevention of urinary tract infections with Oximacro®, a cranberry extract with a high content of type-A proanthocyanidins (PACs-A): a pre-clinical double-blind controlled study. Urol J 13:2640–2649

Schermata 2017-01-13 alle 09.17.36

Use of Microorganisms for Carotenoids Delivery

The CaroDel project lasted 2 years, ended in January 2016 and received €2 million in financial support from the EU. The aims of the project were to develop an efficient oral delivery strategy for highly active carotenoids and to evaluate the potential beneficial (probiotic) effect on health of a Bacillus delivery vehicle, with the ultimate aim of improving biomarkers associated with cardiovascular disease. The CaroDel consortium of eight partners (including five SMEs) from six European countries brought together complementary expertise and was coordinated by ProDigest BVBA, Belgium, a spin-off of Ghent University.

Characterized by their orange, yellow and red pigments, carotenoids are mainly synthesized in plants but also have been isolated from other organisms, including some bacteria and fungi. Humans, like other animals, are unable to synthesize carotenoids but absorb them from their diet. Over 700 different carotenoids have been described, but only a few have been studied in relation to their impact on human physiology. Carotenoids act as antioxidants within the body, protecting against cellular damage, ageing and even some chronic diseases (including cardiovascular disease). Plant-derived carotenoids are widely associated with cardiovascular benefits, yet their low stability and poor bioavailability (absorption in the human body) hamper successful product development.

Several bacteria from the Bacillus species were shown to produce carotenoids which are highly stable throughout the gut and show higher antioxidant activity and bioavailability than common dietary carotenoids.

These discoveries provided strong and compelling reasons for supporting further development and commercialization of these bacteria-derived carotenoids.

By combining in vitro gut models and in vivo animal studies, the CaroDel project developed an efficient oral delivery system for these highly active carotenoids, in the form of Bacillus spores which can also exert probiotic effects.

CaroDel aimed to valorize the results of the earlier FP7 COLORSPORE project, in which initial isolation and characterization of Bacillus strains producing gastric-stable carotenoids was carried out. As particular Bacillus carotenoids were shown to have better stability in the gastrointestinal tract (GIT), antioxidant activity and bioavailability than common dietary carotenoids, the COLORSPORE project provided strong and compelling evidence to support further development and commercialization of these bacteria-derived carotenoids.

The CaroDel project therefore focused on developing an efficient oral delivery strategy for these highly active carotenoids, and on evaluating the potential direct (probiotic) beneficial effects on health of the Bacillus delivery vehicle, with the ultimate goal of improving biomarkers associated with cardiovascular disease (CVD).

The study compared the effective delivery of the carotenoids in the human body following administration as (i) vegetative Bacillus cells, (ii) Bacillus spores or (iii) extracted bacterial carotenoids. In parallel, the ability of the Bacillus strain to exert bona fide effects (i.e. effects on the host microbiota, metabolism and immunity) was investigated using in vitro gut models and in vivo rat studies. Based on the results, the best delivery strategy was selected and validated in a human study, in which carotenoid bioavailability was assessed as well as endpoints related to CVD biomarkers and potential probiotic activity. In combination with a full safety assessment, a proof-of-concept production strategy and an exploitation plan, the scientific evidence compiled in this project provides a framework for efficient further commercialization of a well-characterized Bacillus carotenoid product.

The main objectives of the project were:

1. To optimize the pilot scale production process of the strain and its carotenoids;

2. To determine the best delivery approach for the carotenoids in the human GIT by means of validated in vitro gut models and in vivo rat studies;

3. To determine the effect of the carotenoids and the Bacillus strain on host endpoints and the composition/activity of the gut microbial community;

4. To demonstrate that the selected Bacillus strain is safe for human consumption, which will allow future registration under quality and patient safety (QPS) regulations;

5. To model the scale-up of the production process in order to bring the product to market;

6. To evaluate the effect of the Bacillus strain in humans in relation to CVD biomarkers, modulation of the intestinal environment and host health endpoints; and

7. To generate the necessary knowledge to develop an intellectual property (IP) protection strategy and a business model to commercialize and sustain the product.

To address these objectives, the CaroDel project was divided in two phases: Phase 1 (selection of the best formulation) and Phase 2 (translational phase for taking the product to market), by combining five research work packages (WPs).

During Phase 1 of the CaroDel project, WP2, WP3 and WP4 were run in parallel (WP1 was a management work package).

WP2 (‘Production’) was designed to optimize the production of the different carotenoid formulations that were tested in the other research WPs.

WP3 (‘Evaluation of the carotenoids’ delivery and effect in the GIT’) was designed to elucidate – through in vitro research and animal studies – the intestinal fate and bioavailability of the carotenoids when administered as purified carotenoids extracted from the Bacillus strain, when contained in the vegetative cells, or when contained in the spores of the strain. The effects of the Bacillus strain on the intestinal environment were also assessed upon administration as spores or vegetative cells. The aim of this part of the project was to determine the optimal formulation to be used in the human trial, in terms of carotenoid bioavailability profile and, if possible, potential to modulate the intestinal environment.

In parallel, in WP4 (‘Evaluation of the safety of the carotenoid-producing Bacillus strain’), all the steps necessary to demonstrate the safety of the carotenoids and the carotenoid-producing strain in relation to novel food and QPS regulations, respectively (e.g. toxicology, antibiotic resistance, genome sequencing, and characterization of the carotenoids) were performed.

In Phase 2 of the project, the best formulation identified in WP3 and supported by the data on safety determined in WP4 was tested in a human study within WP5 (‘Human intervention trial’). Analyses were conducted by the different partners to determine the bioavailability of the ingested carotenoids, the effects on the human host in terms of CVD biomarkers, and the impact on gastrointestinal microbial composition and metabolic activity.

Finally, in WP6 (‘Regulatory and life cycle assessment’), the consortium defined the regulatory strategy, evaluated consumer perception of the novel concept, identified the necessary additional R&D steps and, finally, prepared a life cycle assessment plan. In WP7, an IP strategy was developed to protect the outcomes of the project and exploit the results.

In summary, successful achievement of the above objectives has brought the Bacillus carotenoid product close to commercialization and exploitation as a unique health ingredient.

Main results achieved 

In year 1, the CaroDel consortium devoted its efforts to the production of the different formulations of carotenoids and the carotenoid-producing Bacillus strain, and to the determination of the optimal formulation to be used in the human trial. As the carotenoid-producing Bacillus strain did not encounter viability problems, the extracted bacterial carotenoids were excluded from the trial. In order to decide whether the spores or the vegetative cells of the Bacillus strain were the best option, the bioavailability and probiotic activity of both formulations were compared using in vitro gut models and in vivo animal studies. As both spores and vegetative cells exhibited probiotic activity, selection of the optimal formulation was primarily based on the best carotenoid bioavailability profile. It could be concluded that an optimized spore formulation was the better formulation in terms of bioavailability. Therefore, it was decided to use this optimized formulation of the Bacillus spores in the human trial.

In silico genomic screening and in vitro toxicity assessments suggest that the specific Bacillus spores used in this product are safe for human consumption and have an even better safety profile than other Bacilli.

Additionally, in vivo safety assays conducted in mice showed no signs of toxicity. Furthermore, the safety of oral intake was confirmed in two phase I safety studies conducted in healthy individuals, who were and were not overweight. No treatment-related adverse effects occurred with repeated intake over 2 weeks.

In year 2, a 6-week phase II efficacy study was performed in healthy, but overweight individuals. The study was designed as a randomized, placebo-controlled, double-blind, parallel study. Carotenoid analysis showed accumulation of bacterial carotenoids in the plasma of individuals treated with the Bacillus spores throughout the study period. This indicated that the bacterial carotenoids were absorbed in the human GIT and can therefore exert a systemic effect. Additionally, analysis of faecal samples showed that the bacterial strain was able to survive transit through the human GIT, potentially exerting probiotic effects. Indeed, beneficial effects were observed on some biological endpoints after intake of the spore formulation. As this was the first time that such effects were seen in humans, the results provide compelling evidence for the further development and commercialization of the CaroDel product.

A life cycle assessment showed that the CaroDel product would be environmentally competitive when compared to other products with a claimed positive effect on CVD biomarkers, making it a sustainable business. The most environmentally friendly delivery option would be to sell the product as a supplement (in capsules). Moreover, assessment of market trends, current heart health products, consumer attitudes and competitive products, showed that the CaroDel product could be successfully introduced on to the market.

CaroDel was designed to fill existing gaps between the discovery phase and the translation of those findings into a marketable product. The probiotic activity of the Bacillus strain and its effect on cardiovascular endpoints were investigated in parallel with an in vivo study of the absorption, safety and mechanism of action of the biological compound. The result was CaroDel, a new health ingredient that is different from all probiotics and carotenoids currently on the market.

ProDigest in a nutshell

ProDigest is a product leader in the development of unique laboratory models of the human and animal gastrointestinal tract. Without the need for animal or human studies, these models allow a unique insight into gut processes associated with the intestinal fate, metabolism and bioavailability of active ingredients and facilitate study of the complete gut microbiota under controlled conditions and its link with human and animal health. ProDigest is globally active as a service provider for food and pharmaceutical companies and since 2014 also installs its technology in selected R&D facilities around the world. Furthermore, ProDigest has set up a number of in-house product development projects related to microbial biotechnology and gut microbiota management and the development of novel biotherapeutics.

For information
tel +32 9 241 1190
info@prodigest.eu
www.prodigest.eu; www.carodel.eu

Nutrafoods 3 – 2016

Safety of hydroxyanthracene derivatives

In 2013, the European Food Safety Authority (EFSA) issued a positive scientific opinion on a health claim application for authorization related to hydroxyanthracene derivatives and improvement of bowel function. The application was based on a food supplement containing a blend of botanical ingredients and micro-organisms.

In their opinion, the EFSA Panel concluded that a cause and effect relationship was established between consumption of the substance and the claimed beneficial effect, but that in order to bear the health claim 10 mg hydroxyanthracene derivatives per day from the named botanical sources should be taken for the target population of adults.

Furthermore, some restrictions of use were noted by EFSA, as these were considered necessary. Namely, that stimulant laxatives should not be consumed continually for periods longer than 1–2 weeks without medical supervision and that long-term use of stimulant laxatives should be avoided, and these should only be used if their effect cannot be achieved by a change of diet or the administration of so-called bulking agents. However, as is the case for all health claims applications, EFSA opinions and subsequent authorizations cannot be construed as marketing authorizations, positive safety assessments or decisions on classification as a foodstuff of the concerned substance, as this is not foreseen in the framework of the Nutrition and Health Claims Regulation.

Although it was the first favourable EFSA outcome for a health claim for substances derived from botanicals, the Scientific Opinion was considered somewhat controversial and during the risk management phase related to the authorization of the health claim, concerns were repeatedly raised by certain EU Member States.

The medicinal character of these substances was evoked during the European Commission (EC) meetings, as was the importance of setting appropriate conditions of use that would fully take account of the restrictions of use concerning stimulant laxatives stated in the EFSA opinion. Following lengthy discussions in the EC working group on claims, early this year two options were discussed to resolve the issue, either to reject the claims or use the so-called Article 8 procedure to address potential safety concerns.

The EC, on their own initiative, therefore decided to request an EFSA Scientific Opinion on the safety of hydroxyanthracene derivatives under Article 8 of Regulation (EC) No. 1925/2006 on the addition of vitamins and minerals and of certain other substances to foods. This procedure provides for the potential prohibition, restriction or placing under Community scrutiny, of substances other than vitamins or minerals when added to or used in the manufacture of foods, where these represent a risk to consumers.

EFSA has indicated a deadline of 30 June 2017 to deliver their opinion, and have just initiated discussions to identify the necessary expertise to conduct the assessment and establish a dedicated Working Group. The outcome of this assessment will determine if hydroxyanthracene derivatives will be added to Annex III of Regulation 1925/2006, and therefore be subject to prohibition, restrictions or scrutiny for use in foods including food supplements in the EU.

Nutrafoods 4 – 2016

Did cranberry fail to show its ability to protect against recurrent urinary tract infections?

On 27 October 2016, one of the most prestigious medical journals in the world, JAMA, published a negative double-blind and placebo controlled clinical study conducted by researchers from Yale (USA) in which a highly standardized, proanthocyanidin-A (PAC-A)-containing cranberry extract was used [1]. According to the conclusion of the trial: ‘Among older women residing in nursing homes, administration of cranberry capsules vs placebo resulted in no significant difference in presence of bacteriuria plus pyuria over 1 year’. An editorial by LE Nicolle in the same issue of JAMA flatly condemns the use of cranberry PACs to prevent urinary tract infections (UTIs) and calls on healthcare providers to stop using cranberry and switch back to antibiotics [2].

Are the results totally correct? Are the suggestions proposed (to stop cranberry use) appropriate? After reading the report of this clinical trial in JAMA, one can immediately understand why, in all likelihood, the study produced negative results: instead of recruiting women who had suffered from recurrent infection, 95% of the women included were healthy without any mention of prior UTI. From a medical perspective, the difference between healthy women and those with recurrent UTI is huge. Most clinical papers on the use of cranberry have found it has a role in preventing recurrent UTI [3]. Obviously, in order to be efficacious, cranberry must be administered when urine culture is negative and to patients in whom a new positive urine is expected within the next 4–8 weeks. This phenomenon is called recurrence and is different from relapse where infection by residual bacteria not eliminated by antibiotic therapy flares up again. In most cases, recurrence seems to be caused by bacterial transmigration, which occurs, mainly in females, due to the anatomical proximity of the intestine to the bladder, allowing bacteria to cross the septum separating the two organs [4]. PAC-A, by interacting directly with P-type fimbriae present in uropathogenic strains of Escherichia coli (as in other flagellated strains) prevents the fimbriae from binding to glycoprotein receptors on the bladder epithelium.

The efficacy of cranberry in preventing recurrence raises some points. Uropathogenic E. coli and many other flagellated strains typically involved in recurrent cystitis, are positive for at least two types of adhesins localized at the level of cilia and flagella: type-1 pili and P-type structures.

The latter, as mentioned above, interact directly with glycoprotein located on the uroepithelium which facilitates germ proliferation in the bladder. PAC-A also interacts with P-type structures to mechanically prevent binding to the uroepithelial receptor. Type-1 pilus, whose presence alone does not determine uropathogenic status, is a mannose-sensitive protein structure which allows the bacterium to touch the intestinal mucosal membrane, and in some circumstances, pass through it. The following question then arises: PAC-A in cranberry protect against recurrent cystitis but are polyphenolic structures and consequently have poor oral bioavailability, so how do they reduce the proliferation of bacteria in the bladder? PAC-A, like most other polyphenolic structures obtained by extraction, have poor bioavailability and mostly remain unabsorbed in the intestine [5]. However, recurrent cystitis depends on the intestine acting as a culture medium tank for germs. Recurrence is usually found in young women associated with their menstrual cycle, in elderly women and/or in subjects with poor intestinal motility.

The most likely hypothesis is that the bacteria themselves transfer PAC-A which attach themselves to the P-type structures when still in the intestine. However, the bacteria do not use the protein to bind to the receptors and the intestinal epithelium structures in order to proliferate and transmigrate but use type-1 pili which, being PAC insensitive, are available when the bacteria are in the intestine. Once they have transmigrated into the bladder, the bacteria must attach themselves to a structure in order to proliferate as they cannot simply float in the urine which typically occupies the bladder trigone. The problem is that while receptors on the uroepithelium are free, P-type fimbriae are not as they have already been occupied by PAC while the bacteria were still in the intestine. Consequently, the bacteria are expelled during urination as they cannot attach to the uroepithelium. The different roles of antibiotics and cranberry in the treatment and prevention of recurrent cystitis are shown in Figs. 1 and 2.

Schermata 2016-12-29 alle 10.05.02

Schermata 2016-12-29 alle 10.05.12

In conclusion, the negative results described in the JAMA paper could be the results of a mistake in enrolment. By recruiting subjects without recurrent UTI, the Authors have failed to demonstrate the true role played by cranberry PAC-A in limiting adhesion to bladder cells by bacteria migrating from the gut. Subjects without recurrence likely do not harbour the uropathogenic bacteria targeted by PAC-A in the gut.

References

1. Juthani-Mehta M, Van Ness PH, Bianco L, Rink A, Rubeck S, Ginter S, Argraves S, Charpentier P, Acampora D, Trentalange M, Quagliarello V, Peduzzi P (2016) Effect of cranberry capsules on bacteriuria plus pyuria among older women in nursing homes: a randomized clinical trial. JAMA 316:1879-1887

2. Nicolle LE (2016) Cranberry for prevention of urinary tract infection? Time to move on. JAMA 316:1873–1874

3. Singh I, Gautam LK, Kaur IR (2016) Effect of oral cranberry extract (standardized proanthocyanidin-A) in patients with recurrent UTI by pathogenic E. coli: a randomized placebo-controlled clinical research study. Int Urol Nephrol 48:1379–1386

4. Rossi R, Porta S, Canovi B (2010) Overview on cranberry and urinary tract infections in females. J Clin Gastroenterol 44:52–57

5. Feliciano RP, Krueger CG, Reed JD (2015) Methods to determine effects of cranberry proanthocyanidins on extraintestinal infections: relevance for urinary tract health. Mol Nutr Food Res 59:1292–1306

ProGo™ salmon protein hydrolysate

The use of salmon offcuts from the salmon fillet industry to produce human grade nutritional products is an aspirational goal of sustainability for the Norwegian salmon aquaculture industry. Hofseth BioCare has developed and commercialized a novel, patented process to produce a very palatable salmon protein hydrolysate powder, ProGo™, that received the Ingredient of the Year for Weight Management NutraIngredents Award at Vitafoods 2016.

Obesity and situational anaemia are both major global health problems with over 3 million adults dying each year from obesity-related complications and over 1 billion humans suffering from situational anaemia, where haemoglobin levels are low. Overweight and obese individuals with abnormal or excessive fat accumulation are at increased risk of type 2 diabetes, cardiovascular disease and metabolic disorders, particularly cancer. Situational anaemia in infants leads to developmental problems and in adults to low energy and poor mental focus, reducing well-being and quality of life.

Most efforts to overcome obesity and anaemia have focused on developing anti-obesity and iron-containing medicines such as hunger suppressants and iron fumarate supplements. However, these preparations are often associated with serious side effects. Functional foods are considered safe for general use in the control and amelioration of chronic health conditions such as obesity and anaemia and are seeing continuous validation through better clinical studies.


Composition 
and technical specifications

ProGo™ is a salmon protein hydrolysate powder obtained from the head and backbone offcuts left after salmon filleting, which are then subjected to gentle processing using only enzymatic hydrolysis, followed by separation of the water-soluble protein phase and spray-drying the concentrate. ProGo™ contains 621 distinct oligopeptides and peptides, 100% of which have a molecular weight less than 3,000 Da, as determined by MALDI-TOF (matrix assisted laser desorption/ ionization-time of flight) analysis. Gel permeation chromatography analysis further showed that 50% of these peptides have a molecular weight of less than 1,000 Da. ProGo™ is routinely tested for the absence of persistant organic chemicals, pesticides, heavy metals and microbial contamination. The technical specifications and amino acid composition of ProGo™ are reported in tables 1 and 2, respectively.

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Preclinical studies

Our earliest preclinical studies were carried out on elderly pointer dogs to assess the digestibility and palatability of ProGo™ as a pet food supplement.

ProGo™ has excellent digestibility (over 98%) and showed a remarkable array of bioactivity as demonstrated by serum level changes in important biomarkers such as C-reactive protein, ferritin/transferrin and myeloperoxidase. These studies were followed up with our TIM-1 study to measure the in vitro absorption rates of ProGo™ in a model human digestive system. The results showed that ProGo™ has the fastest uptake among protein powders currently commercially available [1]. These properties are being further exploited to develop a series of medical food supplements for use in a range of patients, from infants to geriatric subjects, using data from human clinical studies conducted at the Norwegian School of Sport Sciences in Oslo.

A pre-clinical proof-of-concept study has recently been published [2] that shows a statistically significant increase in serum haemoglobin levels (from 10.04 g/dl to 11.5 g/dl) following 6 weeks of supplementation with 16 g/day of ProGo™ in 24 iron deficient recipients. A larger clinical study with a lower, optimized dose of ProGo™ that will also measure increases in energy, vitality and well-being is ongoing.

Clinical studies and mechanism of action

A clinical study was recently published on the efficacy of ProGo™ to reduce body mass index (BMI) and the proposed mechanisms of action of this unique bioactive [3]. The results show that the BMI of 24 overweight subjects was significantly (p=0.005) reduced by approximately 6% after 6 weeks of daily supplementation with 16 g/day of ProGo™ salmon protein hydrolysate powder. Further results showed that metabolism-related, circulatory biomarkers – bile acid (p=0.027), adiponectin (n.s.), lipoprotein lipase (LPL) mass in preheparin serum (Pr-LPL Mass, n.s.) and interleukin-6 (p=0.038) – showed positive improvements, indicating that ProGo™ might be reducing BMI via interaction with the metabolic pathway.

Figure 1 compares the effects obtained with ProGo™ and whey protein isolate (WPI) on BMI and biomarkers. Results showed that 83% of subjects in the ProGo™ treated group showed a significant decrease in BMI, while only 25% in the WPI-treated group showed only a modest decrease. Moreover, WPI at the dose tested did not modify the examined biomarkers. This implies that the decrease in BMI obtained with ProGo™ may be related to modulation of inflammation and metabolic pathways, possibly via the presence of bioactive peptides in the salmon protein hydrolysate. Our current results clearly show that dietary supplementation with 16 g of ProGo™ per day decreases BMI in overweight individuals after only 6 weeks of treatment and also positively affects circulatory biomarkers associated with inflammation and metabolism.

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Safety

The literature has reported that protein hydrolysate powders often contain some ACE-inhibiting peptides that can cause precipitous and dangerous drops in blood pressure. Consequently, the presence of ACE-inhibiting peptides in ProGo™ was investigated using the ACE kit-WST (Dojindo Laboratories, Munich, Germany). The results showed that ProGo™ contained very little blood-pressure lowering ACE-inhibiting peptides and was thus GRAS-approved for use at up to 90 g/day. No adverse events related to the administration of ProGo™ have been reported in any clinical study.

Formulations 

ProGo™
salmon protein hydrolysate powder – over 93% pure protein

Lean Protein
salmon protein hydrolysate powder – 16 g/sachet for BMI reduction

Endurance Protein Tablets
salmon protein hydrolysate tablets – 1,000 mg per tablet 4 g/day for hemoglobin increase

Endurance Protein Drink
apricot and lemongrass flavoured sachets – 16 g/sachet

Endurance Protein Drink
blackcurrant and passionfruit flavoured sachets – 16 g/sachet

Applications and dosage 

Safety and clinical studies support the use of ProGo™ either as an unflavoured powder or in ready-to-drink flavoured formulations for a sustained reduction in weight and BMI. It can be recommended as a complementary tool in the overall management of weight loss (as ProGo™ flavoured and unflavoured powder administered at 16 g/day) as well as to improve well-being by nutritionally increasing iron (taken as four Endurance tablets/day) over 4–8 weeks of treatment.

References

1. Framroze B, Savard P, Gagnon D, Richard V, Gauthier SF (2014) Comparison of nitrogen bioaccessibility from salmon and whey protein hydrolysates using a human gastrointestinal model (TIM-1). Funct Foods Health Dis 4:222–231

2. Framroze B, Vekariya S, Swaroop D (2015) A placebo-controlled study of the impact of dietary salmon protein hydrolysate supplementation in increasing ferritin and hemoglobin levels in iron-deficient anemic subjects. J Nutr Food Sci 5:379

3. Framroze B, Vekariya S, Swaroop D (2016) A placebo-controlled, randomized study on the impact of dietary salmon protein hydrolysate supplementation on body mass index in overweight human subjects. J Obes Weight Loss Ther 6:296

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Nutrafoods 3 – 2016