Elsevier

Food Bioscience

Volume 40, April 2021, 100845
Food Bioscience

Viability of Lactobacillus rhamnosus GG in provitamin A cassava hydrolysate during fermentation, storage, in vitro and in vivo gastrointestinal conditions

https://doi.org/10.1016/j.fbio.2020.100845Get rights and content

Abstract

The effect of alginate encapsulation on the viability of Lactobacillus rhamnosus GG in provitamin A cassava hydrolysate used as a carrier during processing, storage, in vitro and in vivo gastro-intestinal conditions was assessed. Several studies in the literature have reported the viability of probiotics in non-dairy food matrices such as fruits and vegetables. However, there is need for further studies exploring the maintenance of probiotics in other non-dairy options and to evaluate the resistance of probiotics during passage through the gastro-intestinal tract. Lactobacillus rhamnosus GG (LGG) cells were microencapsulated in alginate beads (using emulsion and extrusion techniques), characterized and inoculated into provitamin A cassava starch hydrolysate. The survival/viability rates of LGG in provitamin A hydrolysate during storage (4 °C, 90 days), also in gastric and intestinal conditions (in vitro and in vivo) were measured. Free LGG cells in provitamin A cassava hydrolysates decreased rapidly in viability within the first 30 days of storage whereas encapsulated cells showed a gradual but insignificant decrease in viability. LGG cells maintained viability above 5 Log CFU/mL in hydrolysates with free LGG, and above 7 Log CFU/g in hydrolysates with encapsulated LGG by the end of 60 days of storage. Encapsulated LGG had 77.4 and 8.5% survival after 120 min in simulated gastric and intestinal juices, respectively. Growth direction index expanded up to 1.66 for LGG but decreased below −0.05 for total aerobes and Enterobacteriaceae in faeces of rats administered hydrolysate with encapsulated or free LGG by day 30. Encapsulated LGG showed better resistance during transit through 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)

  • R.R. Mokarram et al.

    The influence of multi-stage alginate coating on the survivability of potential probiotic bacteria in simulated gastric and intestinal juice

    Food Research International

    (2009)
  • T.D.T. Nguyen et al.

    Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects

    International Journal of Food Microbiology

    (2007)
  • J.P. Paques et al.

    Nanospheres of alginate prepared through w/o emulsification and internal gelation with nanoparticles of CaCO3

    Food Hydrocolloids

    (2014)
  • M.A. Patel et al.

    The effect of ionotropic gelation residence time on alginate cross-linking and properties

    Carbohydrate Polymers

    (2017)
  • J.M.C. Puguan et al.

    Characterization of structure, physico-chemical properties and diffusion behaviour of ca-alginate gel beads prepared by different gelation methods

    Journal of Colloid and Interface Science

    (2014)
  • P. Rayment et al.

    Investigation of alginate beads for gastro-intestinal functionality, Part 1: In vitro characterisation

    Food Hydrocolloids

    (2009)
  • E.F. Talsma et al.

    Biofortified yellow cassava and vitamin A status of Kenyan children: A randomized controlled trial

    American Journal of Clinical Nutrition

    (2016)
  • Z. Zhao et al.

    Immobilization of Lactobacillus rhamnosus in mesoporous silica-based material: An efficiency continuous cell-recycle fermentation system for lactic acid production

    Elsevier Journal of Bioscience and Bioengineering

    (2016)
  • A. Adak et al.

    Dynamics of predominant microbiota in the human gastrointestinal tract and change in luminal enzymes and immunoglobulin profile during high-altitude adaptation

    Folia Microbiologica - Springer

    (2013)
  • M.O. Adegunwa et al.

    Effects of fermentation length and varieties on the pasting properties of sour cassava starch

  • T. Ayichew et al.

    Bacterial probiotics their importance and limitations: A review

    Journal of Nutritional Health and Science

    (2017)
  • M.A.K. Azad et al.

    Probiotic species in the modulation of gut microbiota: An overview

    BioMed Research International

    (2018)
  • R.B. Canani et al.

    Potential beneficial effects of butyrate in intestinal and extraintestinal diseases

    World Journal of Gastroenterology

    (2011)
  • L. Chen et al.

    Lactobacillus rhamnosus GG treatment improves intestinal permeability and modulates microbiota dysbiosis in an experimental model of sepsis

    International Journal of Molecular Medicine

    (2019)
  • M.T. Cook et al.

    Microencapsulation of probiotics for gastrointestinal delivery

    Journal of Controlled Release

    (2012)
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