Viability of Lactobacillus rhamnosus GG in provitamin A cassava hydrolysate during fermentation, storage, in vitro and in vivo gastrointestinal conditions
Introduction
The natural gut microflora is beneficial to the host in the stimulation of intestinal maturation, breakdown of undigested food, synthesis of vitamins (especially vitamin K and biotin), drug metabolism, improved host immunity and resistance of colonization by pathogens (Maity et al., 2012). Healthy gut microbiota is a community in which beneficial microbes predominate, contrary to dysbiosis, which is characterized by a predominance of harmful microbes (Karl et al., 2013; Roberfroid et al., 2010). Alterations of gut microflora could be due to dietary changes, disease conditions, medications or stress (Lobionda et al., 2019).
Probiotics such as Lactobacillus and Bifidobacterium have the potential to restore altered gut microflora to a healthy gut microbiota, thereby showing a protective effect on the gut. Lactobacillus induces expression of anti-inflammatory genes which improves gut function and motility and modulates immune response (Tintore et al., 2017). Lactobacillus rhamnosus GG (LGG) is a probiotic beneficial in the treatment of several forms of diarrhoea, inflammatory bowel disease, pouchitis and ulcerative colitis in humans (Nguyen et al., 2007). It is stable to acidic and bile conditions, and it attaches to the human intestine while temporarily and effectively colonizing it (Goldhaber, 2003). Probiotics are considered useful to the host if ingested as live microorganisms, and remain viable until they reach the colon. Probiotics tend to lose viability during gastrointestinal transit and storage (Cook et al., 2012). Several factors reported to interfere with the viability of probiotics during storage are the level of oxygen that permeates the product as determined by the porosity of the packaging material, the acidity of the medium due to accumulation of organic acids such as lactic acid, the temperature and duration of storage (Xu et al., 2013).
Therefore, a major consideration in producing functional foods containing probiotic cells is the maintenance of functionality and viability of the probiotics through gastrointestinal transit particularly to the colon (Heidebach et al., 2009). Hence, encapsulation is needed to physically entrap microbial cells within a polymer matrix, which encloses the material as if in a capsule. This protects probiotic living cells against several environmental conditions such as pH, temperature, organic solvents, poison and even water molecules (Borgogna et al., 2010). When cells are immobilized in a matrix, a micro-environment is created which protects the cells and ensure viability during processing and gastrointestinal transit up to the time of release in the intestine (Fijalkowski et al., 2016). Encapsulation of cells offers several advantages, which include decreased susceptibility of the microorganism to contamination, increased fermentation rate, improved microorganism handling characteristics and reusability of the cells (Zhao et al., 2016). Different methods of achieving encapsulation exist such as emulsion, extrusion and spray drying methods (Burgain et al., 2011).
Various encapsulating materials are found among which sodium alginate is the most commonly used biopolymer in food systems owing to its non-toxic nature and its numerous advantages (Chavarri et al., 2010; Li et al., 2009; Mokarram et al., 2009). Sodium alginate is simple to use, inexpensive and forms gentle matrices with calcium chloride to trap sensitive materials such as living microbial cells (Soto et al., 2012). Probiotic microencapsulation techniques have been mostly used in matrices from dairy products such as milk and its derivative products, e.g., yoghurt, fermented milk, cheese, and whey-based products which have been conventionally used in the delivery of probiotics (Song et al., 2012). Milk proteins create a suitable environment that provides excellent protection for probiotic strains (Ayichew et al., 2017).
Recently, the matrix has expanded to accommodate non-dairy sources such as fruit, vegetable or root and tuber juices as carriers for probiotics (Rokka & Rantamäki, 2010). Provitamin A cassava roots are low-cyanide varieties (sweet varieties), high in bioactive compounds such as beta-carotene, and have been reported to mitigate micronutrient deficiency of vitamin A (Talsma et al., 2016). The hydrolysate could also enhance the viability of LGG, owing to its high glucose content, which improves probiotic viability when exposed to low pH (Corcoran et al., 2005). Based on these advantages, provitamin A cassava hydrolysate was selected in this study as a probiotic carrier.
This study was designed to further search for alternative food matrix that can be used as a probiotic carrier, which supports the survival of encapsulated microbes in the gastrointestinal tract. A ready-to-drink beverage was developed using cassava starch hydrolysates from three varieties of provitamin A cassava, a root/tuber source, and explored with emphasis on the ability to support the viability of alginate-encapsulated L. rhamnosus GG during processing, storage, in simulated and in vivo gastrointestinal conditions.
Section snippets
Sources of raw materials and microorganism
Provitamin A cassava varieties IITA-TMS-I011368, IITA-TMS-I070593, and IITA-TMS-I011371 were obtained from International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. The lyophilized L. rhamnosus GG (LGG) used was obtained from Valio Ltd. (Helsinki, Finland). α-amylase and glucoamylase enzymes were purchased from Sigma Aldrich Co. (St. Louis, MO, USA).
Extraction and hydrolysis of provitamin A cassava starch
Cassava starch was extracted using the method of Adegunwa et al. (2010) with slight modification. Provitamin A cassava starch was
Properties of calcium-alginate beads
The microcapsules produced by both extrusion and emulsion techniques were spherical in shape (Fig. 1, Fig. 2). Microcapsules made by extrusion had a marginally smaller mean diameter and a lighter weight, with a higher encapsulation efficiency than microcapsules made using the emulsion technique (Table 1).
Effect of storage of microcapsules at 4 °C on the viability of L. rhamnosus GG
Initial viable cell counts of LGG cells were higher in the extrusion microcapsules than the emulsion microcapsules on day 0. However, a steady decline was observed in viable cell counts of LGG,
Discussion
A ready-to-drink probiotic beverage with LGG in provitamin A cassava starch hydrolysate was developed. Organism encapsulation was imperative to ensure probiotic organism reached the colon at therapeutic levels and encapsulation using extrusion was better than the emulsion method. Surface morphology of microcapsules made using extrusion showed a more uniform surface layer compared to microcapsules made by emulsion, which were spherical in shape with cracks giving a rough appearance. The visible
Conclusion
Alginate encapsulation significantly improved Lactobacillus rhamnosus GG viability during fermentation and storage in provitamin A cassava hydrolysates, as well as during exposure to in vitro and in vivo gastrointestinal conditions. Faecal microbial population and expansion of GDI showed that encapsulated LGG in the hydrolysate was able to out-compete total aerobes and other pathogenic organisms in the intestine and eventually colonizing the intestine. This study showed that TMS-I011368
Funding sources
This research did not receive any significant grant from funding agencies in the public, private or not-for-profit sectors.
CRediT authorship contribution statement
Modupeola A. Oguntoye: Conceptualization, Methodology, Software, Visualization, Resources, Investigation, Validation, Formal analysis, Writing - original draft, preparation, Writing - review & editing. Olufunke O. Ezekiel: Conceptualization, Methodology, Software, Supervision. Olayinka A. Oridupa: Conceptualization, Methodology, Software, Supervision, Writing - review & editing.
Declaration of competing interest
The authors confirm that there are no conflicts of interest with respect to the work described in this manuscript.
Acknowledgements
The authors are thankful to the Cassava Breeding Unit, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria, for providing the provitamin A cassava samples used in this study.
References (51)
- et al.
Investigation of swelling/degradation behaviour of alginate beads crosslinked with Ca2+ and Ba2+ ions
Reactive and Functional Polymers
(2004) - et al.
Food microencapsulation of bioactive compounds: Rheological and thermal characterization of non-conventional gelling system
Food Chemistry
(2010) - et al.
Encapsulation of probiotic living cells: From laboratory scale to industrial applications
Journal of Food Engineering
(2011) - et al.
Effect of homogenisation on bead size and survival of encapsulated probiotic bacteria
Food Research International
(2007) - et al.
Effect of storage in a fruit drink on subsequent survival of probiotic Lactobacilli to gastro-intestinal stresses
Food Research International
(2008) - et al.
Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions
International Journal of Food Microbiology
(2010) - et al.
Survival of probiotic lactic acid bacteria immobilized in different forms of bacterial cellulose in simulated gastric juices and bile salt solution
Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology
(2016) Trace element risk assessment: Essentiality vs. Toxicity
Regulatory Toxicology and Pharmacology
(2003)- et al.
Microencapsulation of probiotic cells by means of rennet-gelation of milk proteins
Food Hydrocolloids
(2009) - et al.
Study of the cultivable microflora of the large intestine of the rat under varied environmental hyperbaric pressures
Journal of Microbiology, Immunology, and Infection
(2012)