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

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

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.

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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

The role of lutein in brain health and function

Correspondence to:
Samanta Maci
samanta.maci@kemin.com

Keywords: Lutein, Macular pigment optical density, Brain, Cognition

Abstract

Lutein selectively accumulates in the macula lutea and is a key component of the macular pigment. Recent research has indicated that lutein is also the predominant carotenoid in both the adult and the infant brain, and studies conducted in primates and humans have shown that lutein concentration in the retina is related to its concentrations in specific regions of the brain. A carotenoid-rich diet and high plasma levels of lutein are positively associated with cognitive status or function in healthy subjects, those with mild cognitive impairment, and subjects with Alzheimer’s disease.
Current research indicates that macular pigment optical density, a measure of dietary lutein (and zeaxanthin) deposited in the macula lutea, is positively associated with cognitive function. Additionally, interventional studies provide support that supplementation with lutein and/or zeaxanthin may enhance cognitive function and help maintain cognitive health. The beneficial effect of lutein is most likely linked to its antioxidant and anti-inflammatory properties, and its ability to integrate into cellular membranes, thereby influencing the structural properties and/or stability of those membranes, and possibly enhance gap junction communications. The aim of this review is to present the scientific evidence available to date.

Introduction

Lutein is well known for its role in eye health [1–4]. Three characteristics of lutein are reported to contribute to these health benefits, namely its antioxidant properties, anti-inflammatory benefits, and the nature of its interaction with lipid membranes [5–9]. New research shows that lutein crosses the blood–brain barrier and is the predominant carotenoid in the brain [5, 10], further suggesting that lutein has a critical role in overall brain health and cognitive function.
The eye and brain have a common developmental origin in the neural tube. Moreover, both organs are characterized by a high content of polyunsaturated fatty acids and high metabolic activity, making them particularly susceptible to oxidative stress and free radical damage [11]. Furthermore, carotenoids have been shown to enhance gap junction communication, which is proposed to be important for light processing in the retina and proper functioning of neural circuits in the visual system [5, 12], pointing to a joint role of lutein in both eye and brain health and function [13].
Cognition refers to the mental processes used to acquire knowledge and understanding. It is a multi-dimensional concept divided into domains of memory, attention, language, information processing, and executive function [14]. Each of these cognitive domains can be influenced by factors such as sleep, mood, stress and diet. As the brain ages, declines are observed in specific domains of cognition such as processing speed, episodic and working memory, and executive function. These changes are believed to be caused by physiological damage due to oxidative stress and inflammation, among other factors [15]. Molecules such as lutein with strong antioxidant and anti-inflammatory properties have great potential to play a beneficial role in cognitive health.

Lutein is the predominant carotenoid in the brain

In 2004, Craft et al. published a study that identified and measured a broad range of antioxidants in the adult brain [16]. Sixteen carotenoids, three tocopherols and retinol were identified in brain tissue. They found that xanthophylls (lutein, zeaxanthin and cryptoxanthin) account for 66–77% of the carotenoids in the brain regions studied, while data from previously published research reported that xanthophylls only account for about 40% of the total carotenoids in human blood [17, 18], prompting Craft et al. to suggest that there may be a ‘preferential accumulation of xanthophylls’ in the brain. Johnson et al. confirmed and extended these results in a 2013 study that measured carotenoid levels in both the serum and brain tissue of 42 centenarian decedents [5]. Carotenes (α-carotene, β-carotene and lycopene) were the predominant carotenoids in the serum, accounting for over half (57%) of total serum carotenoids. However, the ratio was reversed in the brain, with xanthophylls (lutein, zeaxanthin and cryptoxanthin) making up 72% of total carotenoids in the brain with lutein alone accounting for over one third (34%) of all brain carotenoids, a significantly greater proportion compared to other carotenoids (p<0.02), once again suggesting a selective uptake of lutein into the human brain. Data for carotene and xanthophyll concentrations in the serum and brain are given in Fig. 1 (data for α-carotene are not presented because it was detected in serum but not in brain tissue). The authors additionally assessed the relationship between brain carotenoid levels and 6-month premortem measures of cognitive function and found significant and positive correlations between several cognitive measures and lutein and zeaxanthin concentrations in the cortex. The mean concentration of all carotenoids progressively decreased with increased Global Deterioration Rating Scale (GDRS) scores from 1 (normal cognitive function) to 3 (mild cognitive impairment, MCI). Among all carotenoids, only the difference in brain lutein content between MCI and cognitively unimpaired subjects remained statistically significant (p<0.05) after adjusting for age, sex, education, diabetes and hypertension.

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A similar result confirming the preferential uptake of lutein in the brain was observed in a study on infant brains published in 2014 by Vishwanathan et al. [10]. Lutein comprised almost two-thirds (58%) of the carotenoids in infant brains, while zeaxanthin made up 16% of the brain carotenoids, resulting in lutein and zeaxanthin together representing 74% of the total carotenoids (Fig. 2) (data for lycopene, detected in some of the tissue in only three infants, are not reported). This high concentration of lutein relative to other dietary carotenoids is noteworthy if we consider a study of dietary intake of carotenoids in infants aged 2–11 months (NHANES 1988-94) [19] which showed β-carotene as the major dietary carotenoid and lutein (assessed together with its dietary isomer zeaxanthin) representing only 17% of the total carotenoid intake (Fig. 2).

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Scientific evidence

The importance of an adequate diet for maintaining general health and for sustaining optimal brain structure and function throughout life is well known [20]. Healthy subjects with high fruit and vegetable intake have higher blood antioxidant levels and better cognitive scores than healthy subjects with low fruit and vegetable intake [21]. Green and cruciferous vegetables are associated with a slower cognitive decline in ageing women [22]. These beneficial effects have been at least in part attributed to the nutritional compounds contained in these foods, including carotenoids [23]. For example, carotenoid-rich dietary patterns were positively associated with cognitive performance measured 13 years later [24]. Dietary intakes of lutein and zeaxanthin were found to be significantly lower in Alzheimer’s disease (AD) subjects compared to healthy controls [25]. However, other papers assessing the relationship between diet and brain health have provided confounding results [26, 27].
A search of third party literature was conducted on PubMed through August 2015 to identify articles that evaluated the effect of lutein (and its isomer zeaxanthin, commonly discussed together with lutein) on cognitive health and function or their relationship in healthy adult subjects, those with MCI or those with AD. The search terms were “lutein” OR “zeaxanthin” AND “cognition” OR “brain” OR “dementia”.
Only observational studies addressing the relationship between plasma lutein (and zeaxanthin) levels and/or their concentration in the macula expressed as macular pigment optical density (MPOD) and cognitive status/function, and interventional trials involving adult participants and published in English were selected. A total of 24 studies were identified that met the search criteria. So that the selection would be as comprehensive as possible, three known published papers not retrieved by the search [21, 28, 29] were additionally included for a total of 27 papers.

Observational studies

Twenty-three papers discussed the results of observational studies conducted to assess the relationship between plasma levels of lutein and zeaxanthin and/or MPOD levels and cognitive status/function (see flowchart in Fig. 3).

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Thirteen of the 16 observational studies retrieved in the literature search assessing plasma lutein and zeaxanthin levels and cognitive status/function indicate that higher plasma levels of lutein and, to some extent zeaxanthin, are positively associated with cognitive status/function [5, 7, 23, 25, 30–38], while three papers reported no or a negative association [21, 39, 40].
Plasma levels of lutein and/or zeaxanthin were found to be significantly lower in subjects with MCI, AD or vascular dementia when compared to age-matched healthy control subjects [7, 25, 30–33, 35, 36]. Studies assessing levels of oxidative stress show that AD subjects have higher plasma levels of 8-hydroxyl-2’-deoxyguanosine (8-OHdG), a well-established biomarker of oxidative stress in DNA [30], or higher levels of phospholipid hydroperoxide (PLOOH) in red blood cells (RBC) [7] when compared to healthy subjects. Furthermore, lower plasma lutein levels were significantly correlated with higher plasma levels of 8-OHdG in AD subjects (p<0.05). A similar inverse correlation was observed with levels of PLOOH in RBC of both AD subjects and healthy controls (p<0.05 and p<0.01, respectively). Finally, in 2015 Feart et al. reported that in a population of 1,092 elderly subjects without dementia, higher plasma lutein levels were significantly associated with a decreased risk of dementia and AD over a 10-year follow-up period [23]. In the EVA (‘Etude du Vieillissement Artériel’) study [34] which included 589 participants, Akbaraly et al. showed that participants with the lowest cognitive performance, defined as a score below the 25th percentile in the neuropsychological tests, had a higher probability of having plasma zeaxanthin levels also below the 25th percentile. Data for the oldest old from the Georgia Centenarian Study [5] including 78 octogenarians (age between 80 and 89 years) and 220 centenarians (98 years or older) showed that serum lutein and zeaxanthin concentrations were more consistently associated with better performance in the different cognitive tests conducted than α-tocopherol, retinol or the majority of the other carotenoids assessed. In the octogenarian group, only serum lutein was significantly related to better cognition and executive function and lower dementia severity.
Additional support has been provided by the research conducted in the last decade and consolidated in the last 2 years that indicates that MPOD, a measure of lutein and zeaxanthin deposited in the macula lutea, is positively associated with cognitive function. All 10 observational studies measuring MPOD identified in the literature search confirmed this positive correlation [25, 28, 38, 40–46].
Studies conducted in healthy subjects across different age ranges have shown that MPOD is positively correlated with temporal processing speed and reaction time [28, 41, 43–45]. This indicates that subjects with higher MPOD have faster processing speeds and reaction times. It also supports the connection between eye and brain functionality.
In a study conducted in Ireland and enrolling 4,453 subjects 50 years of age or older [42], MPOD was found to be significantly and positively associated with two tests assessing global cognition – the Mini Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) – as well as other tests assessing specific cognitive domains. Subjects with lower MPOD had lower MMSE and MoCA scores and poorer performance in tests of prospective memory, executive function, mental processing speed and sustained attention. MMSE scores ranged on average from 28.6 to 28.2 by MPOD quintiles, while MoCA scores ranged from 25.2 to 24.6. The relationship between MPOD and different measures of cognitive performance in healthy subjects observed in this large study were additionally supported by two smaller studies published in 2014 [40] and 2015 [38] which were conducted in older and younger populations, respectively.
Research conducted in participants with MCI [46] has shown that MPOD is correlated with the composite MMSE score, the total score of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and index scores of visuospatial and constructional abilities, language ability and attention. Finally, a study comparing AD patients and healthy controls [25] indicates that MPOD is significantly lower in AD subjects.
A summary of all the observational studies addressing the relationship between MPOD and cognitive function is reported in Table 1.
The relationship between MPOD and cognitive function is further supported by studies conducted in primates [47] and humans [48]. These studies have confirmed that lutein concentration in the retinal region is consistently related to its concentrations in the occipital cortex (the primary visual processing area of the brain). Additional macular–brain associations were found in primates for lutein and zeaxanthin and the cerebellum, lutein and pons, as well as for zeaxanthin and the frontal cortex. These findings suggest that MPOD has the potential to be used as a non-invasive biomarker for lutein and zeaxanthin concentration in the brain and potentially explain the observed relationship between MPOD and cognition.

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Interventional studies

Interventional studies also provide support that supplementation with lutein and/or zeaxanthin may enhance cognitive function and help maintain cognitive health. A total of six randomized, double-blind, controlled interventional studies have been conducted to date to investigate the role of lutein and zeaxanthin in cognitive functions [29, 44, 45, 49–51]; five of these studies used FloraGLO® Lutein as the source of lutein at a dose ranging from 8 to 12 mg. A study by Johnson et al. [49] provided 4 months of supplementation with lutein, DHA or a combination of both to a group of healthy 60–80-year-old women.
The research found a statistically significant improvement in verbal fluency scores for both the lutein and the DHA groups as well as significant improvements in three other measures of cognition for the lutein plus DHA combination group. Serum lutein and DHA levels also increased significantly. This study represents the first of its kind conducted to determine the role of lutein on cognitive measures in older adults. It showed that lutein supplementation significantly improved verbal memory, a measure of executive function and frontal lobe capabilities.
In addition, Bovier et al. [44] conducted a 4-month supplementation regimen in healthy 18–32-year-old men and women. Subjects were supplemented with high-dose zeaxanthin, lutein plus high-dose zeaxanthin and omega-3 fatty acids, or placebo. Neural processing speed was assessed at the end of the supplementation period. The researchers found that critical flicker fusion thresholds and missed coincidence anticipation time, two measures of neural processing speed, were improved upon supplementation. A second publication by the same group in 2015 using the same formulations assessed the effects of supplementation on visual processing speed [45]. Significant improvements were seen in temporal processing speeds via increased temporal contrast sensitivity function scores after 4 months of supplementation. The researchers observed a 20% increase in visual processing speed in the young healthy subjects. The baseline data in these two studies were also considered in the observational studies pool discussed above.
Three interventional studies have observed mixed effects on cognition after supplementation with lutein or zeaxanthin [29, 50, 51]. These studies are similar in that they were all conducted in non-healthy subject populations diagnosed with either age-related macular degeneration (AMD) or AD, so it may not be appropriate to translate the results to a general healthy population. Only one of the studies used a true placebo-controlled design and all studies were conducted in an older subject demographic.
In 2015, Chew et al. [50] conducted a secondary analysis on the AREDS2 trial data and failed to find a statistically significant difference on a composite cognitive score between a lutein/zeaxanthin group and a non-lutein/zeaxanthin group following 5 years of supplementation. The study only included subjects with medically diagnosed AMD, a neurodegenerative disease with unknown consequences on cognition in a population with an average age of 72 years. In addition, as an ancillary evaluation, the authors state that this portion of the study was not sufficiently powered to assess the cognitive benefits of supplementation. In order to control for multiple comparisons, the researchers set the α level for the study at p<0.001. With those limitations, the researchers still observed trends in recall (p=0.06) and Wechsler logical memory (p=0.02) test scores in the lutein and zeaxanthin treated group.
A 1-year supplementation study with zeaxanthin, lutein and zeaxanthin, or lutein by itself by Hoffman et al. [29] was also conducted in an older population diagnosed with mild AMD. The researchers found a significant improvement from baseline in delayed memory in the zeaxanthin only group but failed to find any other statistically significant changes in cognitive performance among the other two groups. Also, a study by Nolan et al. [51] with AD subjects (MMSE scores between 14 and 24) and healthy controls utilized 6-month supplementation with meso-zeaxanthin, lutein and zeaxanthin or placebo. The researchers observed significant improvements in serum concentrations, macular pigment and contrast sensitivity but failed to find a significant change in the cognitive measures assessed. The lack of cognitive findings could be due to the start of treatment after the onset of AD. The authors concluded that the results were not surprising given the fact that for lutein to have a beneficial effect, the optimal conditions are to start treatment with lutein prior to the onset of a neurodegenerative disease state. In addition, the small samples sizes (average of n=13 per group) resulted in some of the cognitive tests not being evaluated due to statistical assumption violations.
A summary of the interventional studies is shown in Table 2. In order to keep the table concise, only lutein or zeaxanthin outcomes in the listed papers have been summarized.

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Conclusions

The body of literature presented demonstrates that lutein crosses the blood–brain barrier and is the predominant carotenoid in the adult and infant human brain. This supports lutein’s role as an important nutrient for brain health and function throughout the lifespan. Observational data are consistent in suggesting that plasma lutein (and zeaxanthin) levels and MPOD are positively associated with cognitive function. Studies conducted in primates and humans indicate that macular pigment carotenoids in the retina are significantly correlated with lutein and zeaxanthin concentrations in the brain, further indicating that MPOD may serve as a non-invasive biomarker for lutein and zeaxanthin concentration in the brain and potentially for cognitive health. Furthermore, interventional studies investigating the effects of lutein and possibly zeaxanthin on cognitive function provide initial support that supplementation with these xanthophylls may enhance cognitive function and help maintain cognitive health. Although most of the data discussed were on older adults, there is evidence from observational and intervention studies in support of the benefit of lutein and zeaxanthin on processing speed and reaction time in a young adult population. Additional studies aiming to further elucidate these benefits are now being conducted. Little is currently known about the role of lutein in early life. Lutein is present in cord blood and is the predominant carotenoid in human colostrum and breast milk. Cheatham and co-authors [52] explored the relationship between lutein, choline and DHA levels in human milk and recognition memory in 5-month-old infants and showed that high levels of lutein and choline are associated with better recognition memory. The neuroprotective effect of lutein and zeaxanthin is most likely linked to its antioxidant and anti-inflammatory properties, its ability to integrate into cellular membranes thereby influencing the structural properties and/or stability of those membranes, and possibly to enhance gap junction communications. In an exploratory metabolomics analysis conducted on postmortem infant brain tissues, Lieblein-Boff and co-authors [53] indicated that lutein is concentrated in neural tissues important for learning and memory and is correlated with fatty acids, phospholipids, antioxidants and amino acid neurotransmitters in the brain. Additional work demonstrated significant correlations across the lifespan for brain concentrations of lutein and StARD3, a specific binding protein for lutein previously identified in retinal tissues, suggesting a possible mechanism for the selective accumulation of lutein in the brain [54]. As both lutein and StARD3 are found in membranes, future studies investigating lutein’s role in modulation of the functional properties of synaptic membranes could help to elucidate the specific mechanisms of action underlying the beneficial role of lutein in brain health and function.

Conflict of Interest
Samanta Maci, Brenda Fonseca and Yong Zhu are employees of Kemin Food L.C.

Human and Animal Rights
This article does not contain any studies with human or animal subjects performed by any of the authors.

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*Kemin Human Nutrition and Health, a division of Kemin Foods L.C., Campo Grande 35- 8ºD, 1700-087 Lisbon, Portugal.
tel: +35 1214157500; fax: +35 1211412172

Nutrafoods (2016) 15:179-188
DOI 10.17470/NF-016-1014-3

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Probiotics to prevent tooth decay

Introduction

Dental decay is a chronic degenerative infection with a multifactorial aetiology. Oral health education begins in the prenatal period with advice from the gynaecologist and the dentist. Use of fluorine, which can be administered in a variety of ways, is one of the main methods to prevent tooth decay. However, although it has proven protective effects, its administration is not always optimal, likely due to poor scientific knowledge and fear of dental fluorosis. Can the nutraceutical sciences can offer something different? Or something more? Overgrowth and disequilibrium of pathogenic microorganism species in the oral cavity can manifest as a variety of different oral diseases, including dental caries. Streptococcus salivarius strain M18 is a bacterial strain with clinically significant probiotic applications for curtailing this pathogenic bacterial growth.

The oral community and probiotics

The vast microbiome of bacteria, fungi and other organisms residing in the oral cavity contributes significantly to oral health. At any given point in time, approximately 700 different taxa of bacterial species simultaneously inhabit the human mouth [1]. Some of these taxa are ‘good’ bacteria that benefit the oral cavity (and the body as a whole), but some are ‘bad’ bacteria that exert detrimental effects. The ‘bad’ bacteria can be problematic by causing dental caries, periodontal disease, strep throat and a wide range of other diseases. The ‘good’ bacteria confer a host of beneficial effects on humans, including immunomodulation and the prevention of pathogen colonization [1]. Certain ‘good’ microorganisms can dampen the effects of ‘bad’ microbes, while providing many other positive effects for the oral cavity and the body as a whole. Because of the beneficial qualities of certain ‘good’ bacterial species, microbiologists have sought to utilize supplements containing these species for human consumption. A discrete dose of viable ‘good’ bacteria which confer benefits on the individual receiving the supplement is referred to as a probiotic.

An important benefit of probiotics is their ability to combat inflammatory diseases and infections. Different probiotics can exert beneficial effects in specific targeted areas of the digestive tract, beginning at the oral cavity and ending at the colon. Most primary research focuses on the gastrointestinal benefits of probiotics, but probiotics can exert a wide array of benefits on other parts of the body, such as the oral cavity. Compared to their gastrointestinal probiotic counterparts, oral probiotics are relatively new probiotic formulations capable of opposing ‘bad’ bacteria and disease in the oral region, which is very important from a health perspective. S. salivarius is a particularly important ‘good’ bacterial species that is the subject of extensive research and is utilized as a commercial oral probiotic. This species, surely the most abundant in the oral cavity, is a spherical, gram-positive, oxidase-negative and catalase-negative bacterium. S. salivarius is one of the earliest colonizers of the epithelial lining of the human mouth and nasopharynx. The bacterium colonizes the dorsum of the tongue and the pharyngeal mucosa of infants, who acquire the bacterium from their mother within 2 days after birth. M18 is the best studied of the S. salivarius strains currently employed as probiotics to prevent dental decay.

Strain M18 

The S. salivarius strain M18 exhibits a particular bacteriocin and enzymatic profile, secreting the bacteriocins A2, 9, MPS and M together with the enzymes urease and dextranase [2]. Salivaricin A2, MPS and 9 are all plasmid-encoded and capable of inhibiting Streptococcus pyogenes growth. While MPS, a 60 kDa peptide non-lantibiotic salivaricin, has an inhibitory effect specific to S. pyogenes, A2 and 9 are both broader in their actions and capable of inhibiting respiratory tract pathogens (i.e., Haemophilus influenza, Moraxella catarrhalis and Corynebacterium spp.). Salivaricin M is responsible for inhibiting mutans streptococci and likely Actinomyces (Fig. 1), and its expression is chromosomally regulated [2]. The release of urease and dextranase allows the strain to counteract oral acidity and to destroy dextran, a substrate which attaches to tooth enamel together with decay-promoting bacteria, allowing them to proliferate.

When M18 is introduced into the oral cavity, it must colonize specific oral regions and be tolerated by the human host. Once the bacteria have become established, they can then confer distinct oral health benefits on the human host. Colonization of the oral cavity by M18 is dose dependent and after 1×109 CFU/subject have been administered daily for 30 days, about 80–90% of treated subjects are colonized (Fig. 2). Despite colonization, there is little to no widespread perturbation of the oral microbiome. Instead of large-scale shifts in bacterial composition, the proportions of bacterial species only shift slightly. This experimental data supports the clinical safety of the strain, as extensive disturbances in the microbiota of healthy subjects are not desirable, significant alterations could potentially yield negative impacts on the probiotic consumers. Finally, in terms of oral persistence, M18 is detectable (1000 CFU/mL) in saliva of treated subjects still 27 days later the last administration [3].

The oral health benefits of strain M18

Once it has become established in the oral cavity, S. salivarius M18 can exert beneficial probiotic health effects on the human host, as demonstrated in in vitro, in vivo and clinical experiments. One particularly significant benefit is the reduction in dental caries, gingivitis and periodontitis. Dental caries is one of the most common childhood diseases and is characterized by the breakdown of tooth enamel and dentine due to ‘bad’ bacteria. These ‘bad’ bacteria release organic acids that reduce the pH of the oral cavity. The lowered environmental pH causes the dissolution of hydroxyapatite matrices of enamel and dentine. Typically, a combination of mutans streptococci (particularly Streptococcus mutans and Streptococcus sobrinus) and individual factors (i.e., saliva composition, fluoride exposure, dietary and hygiene habits, etc.) can stimulate this decrease in the pH of the oral cavity. Treatment with M18 effectively reduces a patient’s risk of developing dental caries through a molecular mechanism that increases oral pH and reduces plaque formation. In one study, the risk of a patient developing dental caries was assessed using Cariogram, a software program that identifies the relative risk of developing caries based on nine pathological and protective factors, coupled with the expertise of the dentist. According to Cariogram, when children at high risk of developing caries were treated for 90 days with M18 they were less likely to develop dental caries. In the untreated control group, on average, there was a 20% chance of avoiding cavities at day zero, which percentage only slightly increased to 37% after 90 days. In the M18-treated group, subjects also on average had a 20% chance of avoiding new cavities, but this percentage significantly increased to 70% after 90-day treatment with M18. The amount of plaque and mutans streptococci both decreased by approximately 50% and 75%, respectively, in the treated group, while the untreated control group did not exhibit any differences in amount of plaque or mutans streptococci [4].

Treatment of dental caries using the M18 probiotic appears to yield greater benefit and effectiveness in certain groups of patients. Those with high plaque scores benefit more from M18 treatment as they exhibit higher levels of plaque reduction. Additionally, patients colonized by M18 demonstrate greater plaque reduction compared to those not colonized but merely exposed to the bacterial probiotic. Similarly, M18-colonized patients exhibit a greater reduction in S. mutans. Studies indicate that higher levels of colonization result in greater reductions in this caries-causing bacterium in saliva, and thus an overall reduction in the development of dental caries. This significantly decreased risk of developing dental caries due to a 90-day S. salivarius M18 regimen can be attributed to several proteins produced by the strain. As previously mentioned, M18 releases salivaricin M, which limits the growth of the caries-causing bacterial species S. mutans and S. sobrinus. Moreover, the strain secretes dextranase and urease. While dextranase catalyzes the breakdown of dextran, urease facilitates the hydrolysis of urea. As dental plaques are rich in dextran, dextranase can help solubilize the plaques that contribute to the breakdown of tooth enamel and dentine. Similarly, urease can increase the pH of saliva by breaking down urea into carbon dioxide and ammonia, and thus prevent hydroxyapatite dissolution. Therefore, dextranase and urease are two M18 enzymes that are effective in decreasing rates of dental caries by reducing plaque accumulation and plaque acidification, respectively [5].

Gingivitis is characterized by inflammation of the gingiva as a result of excess plaque, while periodontitis is a more severe form of gum disease that involves the gingiva pulling away from the teeth. Periodontal disease can be caused by several bacterial species, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum [3]. These bacteria induce inflammation of the gums by releasing multiple cytokines, including IL-6 and IL-8. The accumulation of plaque on the surfaces of human dentition, especially near the gingiva, also contributes to the development of gingivitis and periodontitis. When the M18 strain is administered to patients, measures of gingivitis including supra-gingival plaque, gingival inflammation, sulcular bleeding and probing pocket depth using a Williams periodontal probe are all significantly reduced compared to baseline levels prior to consuming the probiotic. How does strain M18 work on periodontal disease? The M18 strain is capable of reducing P. gingivalis, A. actinomycetemcomitans and F. nucleatum-induced IL-6 and IL-8 levels, which are important indicators of the level of inflammation in periodontal disease. Moreover, as S. salivarius M18 reduces plaque in human hosts, this can also reduce the gingival inflammation involved in gingivitis. In summary, the probiotic M18 reduces levels of gingival inflammation and plaque, which in turn leads to a reduction in the severity measures of gingivitis and periodontitis [6].

Conclusions

On the basis of the available scientific literature, strain M18 can be considered, alone or as add-on therapy to fluorine administration, as a safe and effective nutraceutical tool to counteract tooth decay, gingivitis and periodontitis.

Conflict of interest

Francesco Di Pierro is owner of Velleja Research.

References

1. Cosseau C, Devine DA, Dullaghan E, Gardy JL, Chikatamarla A, Gellatly S, Yu LL, Pistolic J, Falsafi R, Tagg J, Hancock REW (2008) Commensal Streptococcus salivarius K12 downregulates the innate immune responses of human epithelial cells and promotes host-microbe homeostasis. Infect Immun 76:4163–4175

2. Wescombe PA, Hale JDF, Heng NCK, Tagg JR (2012) Developing oral probiotics from Streptococcus salivarius. Future Microbiol 7:1355–1371

3. Burton JP, Wescombe PA, Macklaim JM, Chai MHC, MacDonald K, Hale JDF, Tagg J, Reid G, Gloor GB, Cadieux PA (2013) Persistence of the oral probiotic Streptococcus salivarius M18 is dose dependent and megaplasmid transfer can augment their bacteriocin production and adhesion characteristics. PLoS ONE 8:e65991.

4. Di Pierro F, Zanvit A, Nobili P, Risso P, Fornaini C (2015) Cariogram outcome after 90 days of oral treatment with Streptococcus salivarius M18 in children at high risk for dental caries: results of a randomized, controlled study. Clin Cosmet Investig Dent 7:107–113

5. Burton JP, Drummond BK, Chilcott CN, Tagg JR, Thomson WM, Hale JDF, Wescombe PA (2013) Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children: a randomized double-blind, placebo-controlled trial. J Med Microbiol 62:875–884

6. Scariya L, Nagarathna DV, Varghese M (2015) Probiotics in periodontal therapy. Int J Pharm Biol Sci 6:242–250

Bacterial therapy in pregnancy to drive the intra-partum colonization of newborns

Introduction

Recently, a very interesting paper has been published on Nature Medicine. The Authors have clearly shown the relevancy of a close contact between mother and newborn to effect appropriate passage of microbes to her infant [1]. They have demonstrated that it is possible to get partial restoration of the microbiota of cesarean-born infants by exposing them to maternal vaginal fluids. Vaginal microbe transfer to cesarean-born infants makes them “microbially” similar to vaginally delivered infants. Epidemiological studies have clearly shown an association between cesarean-section delivery and increased risk of obesity, asthma, allergies and immune deficiencies [2-5]. The authors’ attempts to establish an appropriate microbiota in cesarean-born infants by exposing them to the mothers’ vaginal fluids demonstrates the importance of mother-infant microbe transfer and highlights, as a next step, the importance of specifically-directed “manipulation” of the pre-term maternal microbiota in order to further optimize and enrich this transfer process.

Failure of probiotic therapy

Unfortunately, despite their undoubted commercial success, the use of probiotics has not always lived up to the expectations of those who saw in their application a panacea against multiple ailments. This partial failure of probiotics has several explanations. For example, it was for a long time considered sufficient to consume probiotic microbes incorporated in capsules, tablets or sachets in order to alleviate a wide variety of disorders, especially of the intestinal tract. However, a beneficial outcome can only be achieved by carefully taking into consideration certain specific parameters. The probiotic strain should for example be derived following a careful selection process which includes established steps such as: 1) ability to survive both in gastric and enteric environments 2) a high in vivo proliferative potential, 3) capability of adhering to intestinal mucosa and 4) absence of (transferable) antibiotic resistance determinants. However, satisfying these selection criteria alone is not sufficient to establish an effective probiotic. The stability and the dosage of the probiotic strain in the chosen finished delivery format are also important factors. Anyway, the parameter which most certainly will contribute to probiotic therapeutic failure is the absence of colonization. Following their administration, probiotic cells are in fact typically faced with the problem of achieving colonization in tissues that are already highly colonized by the host’s indigenous microbes. This process is really difficult. The established resident bacteria leave little or no physical space on the surface of our tissues for newcomers to colonize and only following prolonged periods of administration of high doses of probiotic strains that have been selected for their high adhesion and proliferation indices will there any real prospect of success.

A totally different moment: the birth

In everyone’s life, however, there is one moment, albeit brief, in which this situation is reversed. This is a short time in which colonization does not seem to be so difficult but, on the contrary, takes place with ease. This is the moment of our birth. Infants are known to be microbiologically sterile until a few moments before their birth. During childbirth, and in the period immediately thereafter, the baby is predominantly colonized by the mother’s own microbes, these initially being of vaginal and rectal origins, but then also including microbes from the mother’s mouth. It is believed that most of the microbes associated with newborns in the first few days after birth directly reflect the composition of the maternal flora. This situation can be considered to have both pros and cons. A cause for concern is that, unfortunately, as well as the commensals, potentially pathogenic microbes can also colonize the infant with relative ease [6, 7]. For example, Streptococcus agalactiae, also known as the group B streptococcus, is the leading cause of severe neonatal bacterial infections in developed Countries. Infants can be colonized during passage through the birth canal by group B streptococci that are present in the mother’s gastrointestinal and/or genital tract. While vaginal infection in pregnancy is usually asymptomatic, in the newborn group B streptococci can produce extremely serious clinical pictures: early-onset infections are characterized by sepsis, pneumonia, and, less frequently, meningitis; in late-onset infections the main clinical manifestations include osteomyelitis, septic arthritis, cellulitis and other localized infections. Studies of western populations of pregnant women have estimated the prevalence of vaginal group B streptococcus colonization to be 15-25%. Approximately one third of infants of these women are colonized at birth and during the first 7 days of life, about 3% of colonized infants develop early-onset infections that can either be fatal or induce severe consequences. Infections occurring beyond the first 7 days of life however seem not to be related to the mother’s intra-partum colonization, but rather to group B streptococci acquired in the post-partum phase. A recent study of newborns under 3 months of age found that the incidence of streptococcal disease was 0.5 per 1,000 live births [8]. To prevent group B streptococcal disease the method adopted in many countries is based on vaginal-rectal screening performed between weeks 35 and 37 of gestation, with intra-partum antibiotic treatment given only for women testing positive. Several randomized clinical trials have shown that this prophylactic approach reduces the risk of early infection from 4.7 to 0.4%.

The case of Enterococcus faecium L3

Schermata 2016-04-19 alle 10.36.35For the past decade, Enterococcus faecium, a Gram positive, non-hemolytic, commensal of the human gut has been the subject of study by a group of Russian researchers. A particular strain, identified later as L3, was shown to release two low molecular weight, thermostable bacteriocins named enterocin A and enterocin B. This strain has been shown to effectively compete with Streptococcus agalactiae. Agar co-culture studies clearly showed that L3 kills S. agalactiae [9]. Subsequent studies have demonstrated that the L3 antibiotic-like activity can also affect the growth of other potential pathogens of the gut and vagina, including Escherichia, Shigella, Salmonella, Proteus, Klebsiella, Mycoplasma and Candida. It is possible that these pathogens compete with Enterococcus faecium for the same niche. To help illustrate what it could mean to fight for the same ecological niche, Figure 1 displays colonies of enterococcus surrounded by “scorched earth” zones of interference with the growth of other bacteria in a mixed fecal population growing on the surface of MRS agar. Other studies have also shown that administration of L3 in premature infants undergoing antibiotic therapy, is associated with both a significant weight increase and a reduction in the frequency of infections (20.7% in the L3 treated infants versus 53.9% of controls). In these same studies a significant reduction in the persistence of Clostridium difficile was also observed. Moreover, in both premature and mature, L3 therapy reduced the risk of dyspeptic disorders and increased the populations of intestinal bifidobacteria and lactobacilli [10]. These results collectively show that L3 administration during pregnancy could potentially reduce maternal dysbiosis and the presence of pathogens, thereby reducing the need for intravenous antibiotic intra-partum therapy. Further, L3 vertical transmission from mother to newborn during passage through the birth canal could contribute positively to newborn weight gain, in addition to increasing gut levels of Bifidobacterium and Lactobacillus species and effecting a reduction in neonatal infections, Clostridium difficile diarrhea and various dyspeptic manifestations.

Exploiting childbirth?

The fact that childbirth allows for close contact between a mother who is abundantly colonized by microorganisms and her baby, whose tissues are sterile, but receptive for microbial colonization, could be “exploited” to try to colonize the baby with strains that are selected for particular characteristics that in some way are useful for the baby. In this view, strain L3 is just one example. Strains encoding and producing beta-galactosidase and/or proteases capable of digesting immunogenic milk proteins could be another example [11].

The enzymatic pools created by such strains may be useful, after newborn colonization, to promote the digestion of milk (both maternal and artificial) thereby reducing the risk of development of lactose intolerance and allergy to milk proteins. Similarly, colonization by strains capable of shifting immune reactivity from a primarily Th2 response (allergic) to a Th1 response (non-allergic) could help reduce the incidence of asthma and other allergies. Moreover, strains capable of improving the specific immune response to vaccination could be considered potentially useful. One strain which appears capable of reducing asthma and allergy and of improving the immune response to vaccines is Bifidobacterium animalis lactis BB12 [12]. Same concept could be applied for the oral microbiota. In 1983 [13] it was shown that babies are typically colonized by Streptococcus salivarius strains derived from their mother. This evidence opens the prospect for colonization of mothers with beneficial probiotic strains of salivarius, such as K12 or M18, in the period immediately preceding delivery as a strategy for achieving natural colonization of the infant from the first days of life.

Could an exogenous probiotic be “vertically” transmitted?

While it has been shown that vertical transmission of well-established endogenous, microorganisms from mother to newborn regularly occurs during delivery, what is less well known is the extent to which similar “contamination” of babies can occur from strains that have been just recently introduced to the mother’s native flora. That is, with strains voluntarily administered during pregnancy for the express purpose of influencing the colonization of the newborn. Although it may seem “a miracle” the phenomenon seems to occur. For several years we have known that certain strains administered to mothers only until the day of birth, were later found in their child’s stool, even 24 months after the last maternal self-administration [14]. Obviously this outcome cannot necessarily be expected to apply to all other strains, but it does mean that colonization strategies such as this are indeed possible.

Acknowledgment

The author wishes to thank Professor John Tagg for his kind and careful review of this paper.

Conflict of interest

Francesco Di Pierro is owner of Velleja Research.

References

1. Dominguez-Bello MG, De Jesus-Laboy KM, Shen N, Cox LM, Amir A, Gonzalez A, Bokulich NA, Song SJ, Hoashi M, Rivera-Vinas JI, Mendez K, Knight R, Clemente JC (2016)
Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer. Nat Med 22(3):250-253
2. Thavagnanam S, Fleming J, Bromley A, Shields MD, Cardwell CR (2008) A meta-analysis of the association between Caesarean section and childhood asthma. Clin Exp Allergy 38(4):629-633
3. Pistiner M, Gold DR, Abdulkerim H, Hoffman E, Celedón JC (2008) Birth by cesarean section, allergic rhinitis, and allergic sensitization among children with a parental history of atopy. J Allergy Clin Immunol 122(2):274-279
4. Huh SY, Rifas-Shiman SL, Zera CA, Edwards JW, Oken E, Weiss ST, Gillman MW (2012) Delivery by caesarean section and risk of obesity in preschool age children: a prospective cohort study. Arch Dis Child 97(7):610-616
5. Sevelsted A, Stokholm J, Bønnelykke K, Bisgaard H (2015) Cesarean section and chronic immune disorders. Pediatrics 135(1): e92-98
6. JM Ross, JR Needham (1980) Genital flora during pregnancy and colonization of the newborn. J R Soc Med 73(2) 105-110
7. R Mändar, M Mikelsaar (1996) Transmission of mother’s microflora to the newborn at birth. Biol Neonate 69(1) 30-35
8. A Six, C Joubrel, A Tazi, C Poyart (2014) Maternal and perinatal infections to Streptococcus agalactiae. Presse Med 43(6 Pt 1) 706-714
9. EI Ermolenko, AIu Chernysh, IV Martsinkovskaia, AN Suvorov (2007) Influence of probiotic enterococci on the growth of Streptococcus agalactiae. Zh Mikrobiol Epidemiol Immunobiol 5 73-77
10. LA Lo Skiavo, NV Gonchar, MS Fedorova, AN Suvorov (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
11. Li J, Zhang W, Wang C, Yu Q, Dai R, Pei X (2012) Lactococcus lactis expressing food-grade β-galactosidase alleviates lactose intolerance symptoms in post-weaning Balb/c mice. Appl Microbiol Biotechnol 96(6):1499-1506
12. Chattha KS, Vlasova AN, Kandasamy S, Rajashekara G, Saif LJ (2013) Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model. J Immunol 191(5):2446-2456
13. Tagg JR, Pybus VP, Fiddes TM (1983) Application of inhibitor typing in a study of the transmission and retention in the human mouth of the bacterium Streptococcus salivarius. Archives Oral Biol 28:911-915
14. Schultz M, Göttl C, Young RJ, Iwen P, Vanderhoof JA (2004) Administration of oral probiotic bacteria to pregnant women causes temporary infantile colonization. J Pediatr Gastroenterol Nutr 38(3):293-297

Red pigment production by Monascus purpureus using sweet potato-based medium in submerged fermentation

Correspondence to:
Muthukumaran Chandrasekaran Government College of Technology
Coimbatore, Tamil Nadu INDIA
© Springer – CEC Editore 2015

Sharmila Govindasamy, Muthukumaran Chandrasekaran (•)
Department of Industrial Biotechnology Government College of Technology
Thadagam Road 641013 Coimbatore, Tamil Nadu, INDIA.
E-mail: biopearl1981@gmail.com
Prateek Srivastav, Vivek Kumar Yadav
Bioprocess Laboratory, Department of Biotechnology
School of Bioengineering
SRM University, Kattankulathur 603203, Chennai
Tamil Nadu, INDIA.

Abstract

Monascus pigments have potential application as natural colorants in food industries. Sweet potato-based medium was optimised by statistical methods for maximised production of water-soluble red pigment from Monascus purpureus. Significant medium components (sweet potato, K2HPO4 and MgSO4·7H2O) were identified by employing a Plackett-Burman screening experiment for pigment and biomass production. A five-level central composite design of the response surface method was applied to evaluate the optimal concentration and the interaction effects between the selected components. Maximum pigment absorbance of 4.488 (ODU/ml) was predicted at the optimum level of sweet potato, 3.341%, K2HPO4, 0.082% and MgSO4·7H2O, 0.033%. Model verification was performed at the predicted optimal level and the model was well fitted with the experimental results. The results of this study showed that sweet potato can be utilised as a low-cost substrate for red pigment production.

Introduction

Industrial utilisation of agricultural crops attracts much attention due to their high nutritional values and low cost. Agroproducts and byproducts of agricultural processing have been used as substrates for the production of various bioproducts such as pigments, biopolymers, organic acids, solvents, and enzymes [1]. Starchy tubers such as cassava, sweet potato, yam and potato are utilised as alternate carbon sources since these tubers are rich in starch, which can be converted into fermentable sugars [2]. Sweet potato (Ipomoea batatas L.) is an important starch-producing crop worldwide. Approximately 95% of the world’s sweet potato production is in Asia and African countries [3]. Sweet potato is an ideal source of glucose for several industrial applications since it contains 20–30% starch [4, 5]. Pigments derived from microbial sources are termed as microbial pigments and are a potential alternative source to synthetic colouring agents, which cause health hazards for humans [6]. Monascus species are a well known microbial source for the production of natural pigments and are widely used as food colorants in East Asian countries [7]. The fungus Monascus produces at least six types of closely related polyketide pigments ranging in colour from yellow to red. Monascus pigments can be classified into three categories based on colour – (i) yellow: monascin and ankaflavin; (ii) red: monascorubramine and rubropunctamine; and (iii) orange: monascorubrin and rubropunctatin [8]. Monascus purpureus is the main source of red pigments as secondary metabolites.
Red pigments gain more attention than other pigments because of their application in food industries to attract consumers; they are also used as a substitute for nitrites in red meat processing industries [9]. Monascus red pigments are highly stable to temperature and pH and it is also reported that they can be used as a potential therapeutic agent for tumour treatment [10–12]. Monascus red pigments are mainly produced through fermentation, specifically solidstate fermentation. Studies on red pigment production by submerged fermentation is limited, but this method has many advantages over the solid-state process such as easy control of process parameters, high productivity, large volume processing, reduced fermentation time and cost [13]. Carbon and nitrogen sources constitute the major cost of the fermentation medium and several studies on alternate sources reported economical production of red pigment using various agroproducts and byproducts [14–19]. In our study, we used sweet potato powder as an alternate low-cost carbon source for red pigment production by submerged fermentation which has not been reported previously in the literature. Medium design and optimisation are the most important steps for any fermentation process; they directly influence the product yield and the process cost [20]. Application of statistical tools for screening and optimisation studies helps to analyse the results with less experiments and time. Plackett-Burman (PB) design is a useful method for screening significant variables from a large number of variables with fewer experiments [21–24]. Response surface methodology (RSM) is a collection of statistical tools used to optimise, develop and improve the process [25]. RSM is successfully applied for the optimisation of medium components and process parameters in various bioprocesses [26–29]. It also provides details on the interaction effect between the variables involved in the process. The present study assesses the potential utilisation of sweet potato as a low-cost substrate for red pigment production by M. purpureus and aims to determine the optimal concentration of medium components by RSM.

Materials and methods

Microorganism
A fungal strain of M. purpureus (MTCC 369) procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India, was used in this study. It was maintained on potato dextrose agar (PDA) medium preserved at 4°C and sub-cultured once every 3 weeks.

Preparation of sweet potato flour 
Sweet potato purchased from a local market in Guduvancherry, Chennai, was used as substrate. After removing the outer skin, it was cut into small pieces, sun-dried for 3 days and ground well to make very fine particles. It was sieved using a 0.09 mm size sieve and the fine sweet potato powder was used as substrate.

Fermentation conditions
To a fully sporulated PDA agar slope culture, 10 ml of Tween 20 (0.1%, v/v) in sterile distilled water was added and the spores were scraped off under aseptic conditions to produce a spore suspension to be used as the inoculum (1×106 spores/ml). The inoculum medium consisted of glucose 30 g/l, MSG 4 g/l, KH2PO4 2.4 g/l, K2HPO4 2.4 g/l, MgSO4·7H2O 1 g/l, KCl 0.5 g/l, ZnSO4·7H2O 10 mg/l, FeSO4·7H2O 10 mg/l and MnSO4·H2O 3 mg/l, and the medium’s pH was adjusted to 6. Sweet potato-based medium was prepared by replacing the glucose in synthetic media with sweet potato powder. PBD and RSM studies were carried out in 250 ml conical flasks each con taining 50 ml of production medium as per the experimental design (Tables 1 and 3). Five percent spore suspension was used to inoculate the flasks and kept in a temperature-controlled rotary shaker at 150 rpm at 30°C for 7 days. Samples were taken every 24 h and analysed for pigment and biomass.

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Pigment estimation
Extracellular pigment estimation was done according to the method described by Tseng et al. [30]. Pigment production was analysed by measuring the absorbance maxima (480 nm) of pigment extract in a UV-vis spectrophotometer and multiplied by the dilution factor. Pigment yield was expressed as OD units at its maximum wavelength per millilitre of clarified fermented broth.

Biomass estimation
The fermentation broth was filtered through Whatman No.1 filter paper and the mycelial pellets were washed twice with the distilled water. It was dried at 80°C in a hot air oven to constant weight and measured as biomass.

Plackett-Burman design
Plackett-Burman (PB) experimental design was successfully employed to screen the significant parameters in the pigment production process with fewer experiments [31]. For the PB experiment, seven medium variables (%, w/v), sweet potato powder (X1), MSG (X2), KH2PO4 (X3), K2HPO4 (X4), MgSO4·7H2O (X5), KCl (X6) and ZnSO4·7H2O (X7), were selected to evaluate the influence of each variable on pigment production. Minitab 14 software was used to prepare the experimental design matrix for the PB experiment. The experimental design consists of 12 runs in which each medium variable was studied at two levels: high (+) and low (–). Table 1 represents the range of variable levels and PB experimental design. The relation between the pigment OD (Y) and the medium components (Xi) in PB design is generally expressed by the following simple polynomial model (Eq. 1):

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where Y is the dependent variable, α0 and αi are regression coefficients for the intercept and linear effects respectively and Xi is coded independent variables.

Schermata 2016-03-25 alle 17.41.12Response surface optimisation
Central composite design (CCD) of RSM was employed to determine the optimal level of three significant medium variables (sweet potato powder, K2HPO4, MgSO4·7H2O) screened from the PB experiment. A five-level optimisation (+2, +1, 0, –1, –2) with 20 experimental runs was shown in a CCD matrix (Table 3). The relation between the response and the independent variables is represented by a second-order polynomial model, given in Eq. 2. where Y is the response, α0, αi, αii and αij are regression coefficients for the intercept, linear, quadratic and interaction effects respectively and Xi and Xj are coded independent variables.

Schermata 2016-03-25 alle 17.40.11

Results and discussion

Screening of medium variables by PB design
The relative importance of seven medium components on red pigment production was evaluated using PB design. Twelve experiments were performed according to the PB experimental design shown in Table 1 and the responses (pigment and biomass) were tabulated. Red pigment concentration ranged from 1.35 to 3.25 ODU/ml and biomass concentration ranged from 3.04 to 5.34 g/l. The responses were analysed by Student’s t-test using Minitab software and estimated coefficients, tvalue and p-value are tabulated in Table 2 for pigment and biomass. Using the estimated coefficients (in coded units), linear model equations for red pigment (Eq. 3) and biomass (Eq. 4) were constructed.

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In general, the statistical significance of the variables was determined using p-values. Table 2 shows that none of the medium variables involved in the process were statistically significant, i.e., (p>0.05). Stowe and Mayer reported that a confidence level greater than 70% may be acceptable in screening experiments. In our study, we selected the medium variables on the basis of confidence level greater than 70% for pigment and biomass production [32]. According to the confidence level, sweet potato was found to have a more significant effect and its presence in the medium contributed as much as other medium components because it acts as both a carbon and energy source for pigment and biomass production (Table 2). Other than sweet potato, KH2PO4 and MgSO4·7H2O were also considered as important components for both red pigment and biomass production. KH2PO4 acts as the buffering agent in the medium and also as a source of phosphate for the cell growth. The presence of MgSO4·7H2O may act as a cofactor for the enzymatic machinery in the metabolic pathway and also trace elements have an important role in the secondary metabolism of the microbes [33]. A Pareto chart is usually used to graphically represent the order of the significant parameters involved in the process [34]. It illuminates the information desirable to make priorities on significant medium variables.
Optimising these variables will have a great impact on the development of economical bioprocesses. The relative importance of medium components was identified by a Pareto chart (Fig. 1) and p-values are given in Table 2. Figure1a and b shows the order of important medium components for red pigment production and biomass respectively. From the PB screening experiment, three medium components, sweet potato, KH2PO4 and MgSO4·7H2O, were selected for further optimisation process by RSM.

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Optimisation by RSM
RSM is applied to evaluate the optimal level of the three variables (sweet potato, KH2PO4 and MgSO4·7H2O) screened from the PB experiment. Experiments were conducted according to the CCD of RSM shown in Table 3. Twenty experiments were carried out according to the combinations of variables, and the responses (pigment (ODU/ml) and biomass (g/l) are tabulated in Table 3. The experimental data obtained for 20 runs were analysed by regression to estimate the coefficients. Estimated regression coefficients and p-values are given in Table 4. The linear effect of sweet potato (p<0.05), quadratic effects of sweet potato (p<0.05), KH2PO4 (p<0.05) and MgSO4·7H2O (p<0.05) and interaction effects of sweet potato with KH2PO4 (p<0.05) and KH2PO4 with MgSO4·7H2O (p<0.05) were found to be significant (Table 4). A second-order polynomial model (Eq. 5) for the M. purpureus red pigment production was obtained using the regression coefficients (in coded units).

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The goodness of fit of the regression equation is usually correlated by the determination coefficient, R2. An R2 value closer to unity represents a better relation between experimental and predicted values by the second-order polynomial equation. In our study, the observed R2 value of 0.879 implies that the model was fitted for 87.9% of pigment production with the predicted and experimental results. ANOVA statistics represented in Table 4 indicate that the model was fitted well with high significance. The high F-test value for the regression indicates that the model is fit and can adequately explain the variation observed in pigment concentration with the designed levels of factors. These results show that the model chosen can satisfactorily explain the square (p<0.05) and interaction (p<0.05) effects of selected medium components on pigment production using sweet potato powder in shake flask cultures (Table 4). The relationships between the variables are graphically rep-resented by surface or contour plots [35]. Figure 2a–c represents the interaction effects of any two variables on red pigment production by keeping the third variable at a constant level. The shape of the surface or contour plot is used to indicate whether the interactions existing between the variables are significant or not. Yu et al. reported that there is no significant interaction between the variables observed if the contour lines are circular in shape [36]. A strong interaction was observed between sweet potato vs. KH2PO4 (Fig. 2a) and KH2PO4 vs. MgSO4·7H2O (Fig.2b). No significant interaction effect was observed with sweet potato and MgSO4·7H2O since contour lines were circular in shape (Fig. 2c).

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Model validation
The response tool of Minitab software was used to estimate the optimal level of medium variables to attain maximum red pigment production. The predicted maximum response of 4.488 (ODU/ml) was obtained at the optimal level of medium variables of sweet potato, K2HPO4 and MgSO4·7H2O of 3.341%, 0.082% and 0.033%, respectively. To verify the accuracy of the model, validation experiment was carried out at the predicted optimal levels of the medium components. In the validation experiment we obtained maximum red pigment absorbance of 4.12 (ODU/ml) and the accuracy of the model was found to be 91.96%. Results of validation explained that the predicted model for the red pigment production was well fitted with the experimental results.
The results of this study showed that sweet potato may be used as an alternate low-cost substrate for red pigment production by M. purpureus. Fourier transform infrared spectroscopy The FT-IR spectrum of the pigment was analysed in an IR spectrometer (Model: Spectrum one FT-IR Spectrometer, Perkin-Elmer, USA) in the wavelength range 450–4000 cm−1. Spectral lines were recorded at a resolution of 1 cm−1. The FTIR spectrum of the liquid sample containing the pigment produced by M. purpureus is shown in Fig. 3. The band at 3401 cm–1 suggests O-H and the one at 2137 cm–1 indicates the presence of alkynes (C≡C) in stretching mode of vibration. The wave number of 1646 cm–1 confirms the presence of a carbonyl group (C=O) with a stretching mode of vibration, while the wave numbers 1120 cm–1 and 1079 cm–1 show the presence of C–O and C–C stretching vibrations respectively.
The band at 726 cm–1 represents the presence of an amine group (N-H) with rocking mode of vibration. The presence of an N–H group (amine) and C–O group shows that the pigment belongs to the rubropuctamine (C21H23NO4) or monascorubramine (C23H27NO4) Monascus pigments.

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Conclusion

In this study, sequential optimisation methods were successfully carried out to obtain maximum red pigment production using a sweet potato-based medium. PB design was used to screen seven medium variables and three variables, sweet potato powder, K2HPO4 and MgSO4·7H2O, were found to be more significant than the other variables. CCD of RSM was applied to evaluate the optimal selected variables from the PB experiment. ANOVA statistics and the R2 value of the model were found to be significant and explain a good relation between the predicted and experimental results. Verification experiments were performed to check the accuracy of the model and it was found to fit with the predicted results. The results of this study revealed that sweet potato may be employed as an alternate substrate and the utilisation of sweet potato could be a better approach for cost-effective red pigment production in submerged fermentation.

Acknowledgement
The Authors acknowledge with thanks the management of SRM University and the director (E&T) for providing the necessary facilities to carry out this study. They also thank the Sophisticated Analytical Instrument Facility (SAIF) at IIT, Madras, for FTIR analysis.

Conflict of interest
The Authors declare that there is no conflict of interest regarding the publication of this article.

Human and Animal rights
This article does not contain any studies with human or animal subjects performed by any of the authors.

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