Beneficial Microbes

Wageningen Academic P u b l i s h e r s

http://www.wageningenacademic.com/doi/pdf/10.3920/BM2012.0030 - Michael Chikindas - Wednesday, September 02, 2015 6:44:58 AM - BM editorial board IP Address:165.230.169.48

Beneficial Microbes, June 2013; 4(2): 127-142

Non-dairy probiotic beverages: the next step into human health D. Gawkowski and M.L. Chikindas Rutgers, The State University of New Jersey, School of Environmental and Biological Sciences, Department of Food Science, 65 Dudley Road, New Brunswick, NJ 08901, USA; [email protected] Received: 12 June 2012 / Accepted: 18 October 2012 © 2013 Wageningen Academic Publishers

Abstract Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. The two main genera of microorganisms indicated as sources of probiotic bacteria are Lactobacillus and Bifidobacterium. Historically used to produce fermented dairy products, certain strains of both genera are increasingly utilised to formulate other functional foods. As the consumers’ understanding of the role of probiotics in health grows, so does the popularity of food containing them. The result of this phenomenon is an increase in the number of probiotic foods available for public consumption, including a rapidly-emerging variety of probioticcontaining non-dairy beverages, which provide a convenient way to improve and maintain health. However, the composition of non-dairy probiotic beverages can pose specific challenges to the survival of the health conferring microorganisms. To overcome these challenges, strain selection and protection techniques play an integral part in formulating a stable product. This review discusses non-dairy probiotic beverages, characteristics of an optimal beverage, and commonly used probiotic strains, including spore-forming bacteria. It also examines the most recent developments in probiotic encapsulation technology with focus on nano-fibre formation as a means of protecting viable cells. Utilising bacteria’s natural armour or creating barrier mechanisms via encapsulation technology will fuel development of stable non-dairy probiotic beverages. Keywords: probiotic, non-dairy, beverage

1. Introduction Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (Brown and Valiere, 2004; FAO/ WHO, 2002). Historically used to produce fermented dairy products such as yoghurt and kefir, probiotics were found to improve human health and were then moved to the forefront of academic, pharmaceutical and food industry research. Probiotic strains are reported to have health benefits for a range of health disorders, spanning from intestinal to non-intestinal disorders. Intestinal health benefits include: prevention of various types of diarrhoea, reduction of inflammatory bowel disease symptoms, prevention of certain types of cancers in gastrointestinal (GI) tract, alleviation of lactose intolerance, and reduction of Helicobacter pylori infections (De Vrese et al., 2001; Kim et al., 2008; Reiff et al., 2009; Thirabunyanon et

al., 2009; Wenus et al., 2008; Zou et al., 2009). Certain probiotic strains were shown to have a positive effect on non-intestinal disorders by reducing serum cholesterol, stimulating immune system, reducing respiratory tract and urinary tract infections, treating and preventing allergy, and decreasing incidents of bacterial vaginosis (Cadieux et al., 2009; Galdeano and Perdigon, 2006; Hatakka et al., 2001; Huang and Zheng, 2010; Kalliomäki et al., 2003; Reid et al., 2003). In addition, a perinatal probiotic intervention study conducted by Luoto et al. (Luoto et al., 2010) showed a positive/beneficial effect on controlling childhood weight gain, indicating a possible way to prevent and manage childhood obesity. The main two genera of microorganisms indicated as sources of probiotic strains are Lactobacillus and Bifidobacterium. Some species of genera Lactococcus, Enterococcus, Saccharomyces, Streptococcus, Escherichia,

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2012.0030127


D. Gawkowski and M.L. Chikindas

Bacillus, Leuconostoc and Propionibacterium were also recognised for their health benefits (Prado et al., 2008b; Sturm et al., 2005). Since many species of Bifidobacterium are found in the human GI tract, they were the first to be screened for probiotic functionality, safety and food application (Ventura et al., 2004). Bifidobacterium was shown to colonise the intestinal tract to synthesise and excrete vitamins, inhibit harmful bacteria and aid in absorption of food constituents (Gibson and Roberfroid, 1995). Lactobacilli are isolated from plant materials, animal, human, food fermentation and spoilage sources (Hammes and Hertel, 2006). Species belonging to lactic acid bacteria (LAB) are Gram-positive, generally do not form spores and ferment glucose through homo- or heterofermentation. Homofermentative LAB strains utilise glucose to mainly produce lactic acid, whereas heterofermentative cells produce lactic acid, carbon dioxide, ethanol and/or acetic acid during the fermentation pathway. These metabolites play an important role during fermented food production by lowering pH and negatively affecting pathogenic bacteria. The above described ‘natural preservation’ increases safety and stability for dairy and non-dairy fermented foods (Cleveland et al., 2001; Leroy and De Vuyst, 2004). Many of Lactobacillus species, found in the healthy human gut, inhibit growth of pathogenic bacteria, stimulate immune functions and play an important role in digestion of food ingredients and absorption of minerals (Gibson and Roberfroid, 1995). It is commonly believed that the majority of the human immune system is found in the digestive tract, where healthy GI tract microbiota play a vital role in human health and disease prevention (Hooper et al., 2012). The main aspects influencing the development of human microbiota after birth include: maternal microbiota, genetics, delivery mode, antibiotic and medication use, breastfeeding and maternal weight (Collado et al., 2010; Penders et al., 2006; Roger et al., 2010; Songjinda et al., 2005). During the human lifespan, factors such as ageing, diet, family size, and hygiene level are associated with affecting the balance between beneficial and harmful bacteria in the GI tract, which could lead to development of allergies, chronic diseases and infections (Bernstein and Shanahan, 2008; De Filippo et al., 2010; Mariat et al., 2009). Growing consumer interest in health and wellness including disease prevention and treatment is projected to catalyse more interest in probiotic products. However, not all consumers can take advantage of these beneficial microorganisms. Although widely available in developed countries, traditional dairy-based fermented foods such as yoghurt and kefir are not suitable for lactose intolerant consumers. In addition, fermented foods also require refrigeration, which further limits the initial convenience. To address these challenges, food manufactures began to formulate non-dairy probiotic foods with selected Lactobacillus and Bifidobacterium species. This is a 128

challenging task due to microbial susceptibility to high temperature, high acidity, high oxygen content, low nutrient availability, light exposure, high water activity, the presence of antimicrobial compounds and other factors contributing to microenvironment (Shah, 2001; Vasiljevic and Shah, 2008). Ingredient application and product development research continues to uncover formulation solutions to increase the survival of these health-conferring microorganisms, but keeping probiotics viable in nondairy, non-refrigerated beverages remains a tremendous obstacle. Different approaches that exclude or minimise the detrimental effects of environmental factors on probiotic cells include the use of more resistant probiotic strains and cell encapsulation. This review summarises current knowledge on the most stable known probiotic bacteria strains, and discusses cell encapsulation in synbiotic emulsions and electrospun fibres as potential solutions to developing non-dairy, non-fermented beverages containing efficacious probiotic bacteria.

2. Current non-dairy probiotic beverages Many probiotic microorganisms were originally isolated from fermented dairy foods and now are increasingly incorporated into dairy and non-dairy products. Fermentation increases nutritional content, improves sensory characteristics and preserves many non-alcoholic, cereal-based beverages (Gadaga et al., 1999). A variety of traditional non-dairy fermented beverages are produced around the world including: boza, bushera, mahewu, pozol, togwa and hardaliye. Traditional food fermentation utilised to produce boza, bushera, mahewu, pozol and togwa, categorised as non-dairy probiotic beverages, provides a means for obtaining nutritious and safe beverages consumed in developing countries where starvation and under-nutrition are common challenges. At the same time, the Western world, which is battling modern diseases associated with obesity and impaired immunity, is taking notice of probiotic benefits in preventing and treating a number of health conditions. Increasing health-consciousness and growing awareness of the benefits of probiotic yogurts and fermented milk, combined with vast use of probiotic supplements, are main contributors of the growth of probiotic market in USA and other developed countries (Anonymous, 2009). The majority of probiotic foods carry digestive and immune health claims (Sleigh and Barton, 2011). Between 2003 and 2008, the US probiotics market increased by 8.7% and reached $5 billion. This positive trend is expected to continue to 2013 with a 5% growth rate and is expected to reach an estimated value of $6.4 billion (Tallon, 2009). The non-dairy probiotic beverages commercially available in the USA are refrigerated juice and juice drinks containing non-starter, viable cultures mainly belonging to Lactobacillus Beneficial Microbes 4(2)


and Bifidobacterium genera. One of the beverages is a 100% juice smoothie containing Bifidobacterium lactis HN019, fructo-oligosaccharides and a blend of juices. The package displays a structure/function claim and focuses on promoting a healthy digestive and immune system. In multiple studies, B. lactis HN019 showed health benefits attributed to its ingestion, and recently published research conducted by Liu et al. (Liu et al., 2010) explored adherence and immuno-modulatory properties of this Bifidobacterium strain. The strain used was found to have excellent stability in dairy, supplements and non-dairy products (Gopal et al., 2005). The formulation contains fructo-oligosaccharide, which contributes to the sensory experience and nutritional composition of the juice, and possibly changes its physicochemical properties through water activity reduction. This, therefore, possibly increases the stability of probiotic bacteria (Crittenden and Playne, 1996). Benefits of formulating products with oligosaccharides extend beyond product application. Fructo-oligosaccharides (FOS) were shown to have prebiotic properties. A prebiotic is a defined as ‘a selectively fermented ingredient that allows specific changes, both in the composition of and/or activity in the GI microbiota, that confers benefits upon host wellbeing and health’ (Gibson et al., 2004). Another non-dairy probiotic juice drink that is commercially available in the USA contains Lactobacillus plantarum 299v. Similarly to most commercial probiotic products, the product claim focuses on promoting core digestive health and supporting the immune system. In addition to 25-30% juice, all variants contain organic oat flour and organic barley malt. Both oat and barley were found to support propagation of L. plantarum, suggesting possible enhancement of probiotic survival in beverage matrix as well as prebiotic activity after consumption (Angelov et al., 2006; Charalampopoulos et al., 2003). Probiotic juices and juice drinks are often lactose-free, soy-free and vegan, and these attributes are becoming increasingly important to health-centric consumers concerned with their intake of cholesterolcontaining foods, suffering from lactose intolerance, or following a specific diet. However, similarly to the dairy probiotic products, the current commercially-available non-dairy probiotic beverages require refrigeration and have a short shelflife measured in weeks. When stored at refrigeration temperature (4 °C) as compared to room temperature (25 °C), viability of Lactobacillus and Bifidobacterium cells in fermented milks was extended from few days to over 30 days for Lactobacillus casei YIT9018 (Yakult strain) and L. plantarum MDI133, and from 3/4 days to 15 days for the Lactobacillus acidophilus CH5 and B. lactis Bb12 strains (Salminen and Von Wright, 1998). An additional benefit of storing probiotic beverages at lower temperatures is a reduction in the lactic acid production, which contributes to the product’s sour taste and low pH. Fruit juices are typically between pH 2.5 and 3.8 and further acidification exacerbates Beneficial Microbes 4(2)

Non-dairy probiotic beverages

the detrimental effects of pH on the survival of bacteria. Nevertheless, cold storage temperature requirements and the short shelf-life increase handling costs incurred by manufacturers and retailers, and limit convenience desired by busy consumers on the go. To assure food safety and acceptable shelf-life, thermal processing is applied to fruit juice in order to destroy enzymatic activity and remove pathogenic and food spoilage bacteria. This, however, does not prevent fermentation by probiotics from taking place. Temperatures above 45/50 °C are detrimental to probiotic survival as well, and probiotic cells should be added after the heat treatment step during beverage or food manufacturing process. Food manufacturers are challenged to deliver adequate numbers of viable probiotic bacteria throughout product shelf-life. Commercial yoghurt products stored at a cold temperature (4 °C) and tested for viable numbers of lactobacilli showed a decline in cell count at the end of their shelf-life indicating high susceptibility (Schillinger, 1999). Effective delivery of health-conferring bacterial cells to the consumer requires selection of a resilient probiotic strain and identification of suitable formulation, processing, packaging, and handling technologies.

3. Commercial beverage requirements Currently available traditional and formulated probiotic beverages contain live probiotic microorganisms that interact with components of the food matrix and are affected by environmental factors. Live microorganisms metabolise components such as sugars and produce acids, which in larger amounts may have a detrimental effect on taste and ultimately reduce product shelf-life. A model formulation can be described based on the needs of consumers, manufacturers and retailers. To develop a successful product, one must identify the population segment most likely to benefit from consuming non-dairy probiotic beverages. In the USA, the median age increased from 35.5 in 2000 to 37.2 in 2010 (Howden and Meyer, 2011) and was projected as 39.1 in 2035, with elderly population reaching 50 million by 2019 (National Institute of Aging; United States Bureau of the Census, 1997). The age factor, combined with unhealthy lifestyles, is projected to increase the number of persons suffering from chronic diseases such as diabetes, cancer, and high cholesterol. Combined with lifestyle changes, consumption of probiotic products may offer an alternative method to help restore and maintain healthy gut microbiota that plays a role in the occurrence of the above mentioned chronic diseases (Arunachalam et al., 2000; Huang and Zheng, 2010; Thirabunyanon et al., 2009). Probiotic beverages should contain viable bacteria counts based on levels identified to be efficacious in human studies. The effective dose is likely to vary depending on the strain used. For instance, a proprietary formula of L. acidophilus 129



CL1285 and L. casei LBC80R was documented to reduce the risk of antibiotic-associated diarrhoea and Clostridium difficile-associated diarrhoea at 5.0×1010 to 1.0×1011 cfu/day with a dose-ranging effect (Gao et al., 2010). L. plantarum 299v administered at a dosage of 2.0×1010 cfu/day was found to have a cholesterol-lowering effect (Naruszewicz et al., 2002). Manufacturers marketing probiotic beverages should provide cell counts for each strain to inform the consumer about product potency. Despite the absence of a legal definition for ‘probiotics’ in the USA, the Food and Drug Administration (FDA) holds regulatory authority over probiotic product labelling and quality whether in food, dietary supplement, or drug form. The only type of claim allowed to be used for marketing probiotic foods in the USA is structure/function claim. This claim relates probiotic food or probiotic strains to the normal functioning of the human body, such as promoting healthy digestive system. Approval or notification is not required for structure/function claims on foods, however FDA notification is required for the same claims on dietary supplements (Sanders, 2007). Currently available nondairy probiotic beverages are considered conventional foods and their quality and labelling thus falls under the jurisdiction of the FDA. Manufacturers marketing beverages containing probiotics should follow guidelines created in 2002 by a working group joined by members of the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). The guidelines include strain identification with the deposit of all strains in an international culture collection, strain safety assessment and functional characterisation, health benefits validation in human studies with Phase 2 for efficacy and Phase 3 for effectiveness, and proper labelling including truthful and not misleading health claims (FAO/WHO, 2002). In addition to the health benefits attributed to the probiotic strain, formulating a gluten-free, organic, dairyfree, natural and/or vegan beverage is likely to broaden the consumer base. While clearly-communicated product benefits attract first time buyers seeking alternatives to conventional medicine, repeated purchase can be often attributed to taste and effectiveness. Another important aspect of a probiotic product is its nutritional profile. Released at the end of 2010, the 2010 ‘Dietary Guidelines for Americans’ recommends making healthier food choices and complementing them with physical activity to reduce the prevalence of overweight and obese children and adults. The guideline recommends increasing consumption of vegetables, fruit, whole grains, milk and milk products to obtain more potassium, dietary fibre, calcium and vitamin D, and to consume less sodium, saturated and trans fats, added sugars, and refined grains (USDA and USDHHS, 2010). Many ingredients belonging to the recommended healthy food groups were shown to have prebiotic effects on various strains of bacteria and to 130

serve as substrates during the fermentation process, as well as protecting viable cells from exposure to acid, digestive enzymes and bile during passage through the digestive system (Rivera-Espinoza and Gallardo-Navarro, 2010). Since modern lifestyles demand convenience, consumers continue to look for ‘portable’ products consumed on-thego or requiring minimum preparation time. Shelf-stable formulated probiotic beverages provide a convenient way to supplement daily diets and to improve digestive health and immunity. In addition to protection, containment and utility, packaging provides a means of communication that influences consumer behaviour and can impact repeated purchase. To sustain a successful formulated probiotic beverage business, it is recommended that manufacturers partner with companies that have expertise in supplying safe and consistent ingredients, providing scientific and technical support including assistance with application, testing, and label/claim development. Probiotic suppliers usually offer probiotic microorganisms as freeze-dried or spraydried powders, or as a frozen ‘direct vat set’ concentrate (Kaya and Aksu, 2005). To maintain cell viability upon receipt of the probiotic ingredient, manufacturers should adhere to the storage and handling instructions outlined by the supplier. The advantages of producing probiotic beverages with added bacteria strains as an ingredient include a simplified production process due to the lack of substrate use, and a longer shelf-life due to the elimination of the fermentation process. This results in the prevention of generation of by-products and the ability to control the amount of added cells. Manufacturers marketing probiotic beverages should employ production, filling, and storage processes that do not have detrimental effects on viable probiotic cell counts. Stability testing should be conducted on the final formulation to assess whether probiotic cells can tolerate the food matrix and applied handling conditions. To ameliorate reduction of the viable cell count, manufacturers should consider making changes to their current processing, storage, handling techniques, and/or food composition including bacteria overage. For quality control purposes, a proper enumeration technique suited for the particular strain must be identified and validated to ensure that the claimed strain and cell levels are delivered. FAO/WHO guidelines include labelling regulations that should be used by all manufacturers marketing probiotic foods. These requirements include designating genus, species and strain on the label, along with the viable number of each probiotic strain at the end of the product shelf-life, truthful and not misleading health claims, the serving size that delivers the effective dose related to the stated heath claim, proper storage conditions, and manufacturer contact information (FAO/WHO, 2002). Other crucial factors in the go-to-market strategy are marketing and advertising during which consumers Beneficial Microbes 4(2)


learn about the product and benefits associated with its consumption. The success of a company such as Yakult provides an example of a properly selected and executed communication campaign that extends beyond traditional marketing and advertising practices (Tallon, 2009). Product distributors and retailers play an integral part in the success of probiotic beverages by adhering to proper handling and storage recommendations, merchandising, restocking, ordering, and pricing. For a retailer, an ideal probiotic beverage has an adequate shelf-life, does not require unique storage or handling, has an affordable price and short order lead-times. Formulated non-dairy probiotic beverages can serve as successful vehicles to deliver health-promoting benefits, nutrition, great taste and convenience.

4. Product optimisation. Approach 1: probiotic bacteria selection and beverage formulation Despite an increasing amount of research dedicated to understanding factors influencing probiotic cell survival, incorporating live probiotic microorganisms into beverages continues to pose a colossal challenge for research and product development scientists. To develop stable probiotic beverages that contain a viable cell count at the end the product shelf-life, product composition must be carefully considered. While packaging and environmental conditions were shown to impact probiotic survival, beverage composition plays an integral role in sustaining cell viability. Even if probiotic ingredients belong to the same genus and species, differences in strain can impact susceptibility to environmental factors and compatibility with the selected food matrix. When selecting a strain for a particular beverage formula, bacteria robustness must be considered. A key desirable feature of the probiotic strains would be their ability to resist exposure to stomach acidity (pH ~2.0) and bile salts in the jejunum (e.g. Dunne et al., 1999). Shelf-stable beverages are typically acidified to a pH below 4.4 and thermally processed to control growth of pathogenic bacteria, yeast, and mould. The acidification practices applied to functional beverages including nondairy probiotic beverages limit strain selection. In general, lactobacilli are more robust than bifidobacteria, and more Lactobacillus species are thus applicable for food use including L. acidophilus, L. johnsonii, L. rhamnosus, L. casei, L. paracasei, L. fermentum, L. reuterii and L. plantarum (Crittenden, 2008; Ross et al., 2005). Despite a high cytoplasmic buffering capacity (pH 3.72-7.74), L. acidophilus shows less resilience than other species when used in non-dairy foods such as oat-based beverages or fruit drinks (Champagne and Gardner, 2008; Gokavi et al., 2005; Lankaputhra and Shah, 1995; Rius et al., 1994). The most commonly used Bifidobacterium is B. animalis ssp. lactis (Crittenden, 2005). The optimal temperature and pH growth conditions of 37-43 °C and pH 5.5-7.0 make the Bifidobacterium species susceptible to acidic food matrix, process and handling practices. Since most species show Beneficial Microbes 4(2)

Non-dairy probiotic beverages

no growth at temperatures below 20 °C, storage in chilled conditions is recommended to obtain adequate shelf-life for acidified non-dairy probiotic beverages (Doleyres and Lacroix, 2005). Some probiotic lactobacilli can grow at mesophilic temperatures of 15 °C and higher, with some even growing at up to 44 °C (Savoie et al., 2007). Species within bifidobacteria genera exhibit varying sensitivity to acid, bile and oxygen, with B. animalis strains showing the most overall resistance. When bifidobacteria isolated from human intestinal samples were characterised and tested, three B. animalis ssp. lactis strains and Bifidobacterium longum ssp. longum E-001565 showed good or moderate survival in acid (pH 3.0), bile (pH 7.2) and during areotolerance tests (Mättö et al., 2004). Matsumoto et al. (2004) found weak acid tolerance of bifidobacteria in a Gifu aerobic media broth with varying pH levels, with the exception of B. lactis and B. animalis. The results also indicate that the acid tolerance level of bifidobacteria depends on species difference rather than on strain difference, and that the tolerance level is dependent on the H+-ATPase activity. Sheehan et al. (Sheehan et al., 2007) examined Lactobacillus and Bifidobacterium robustness to acid and heat. To test acid resistance, a number of strains were added to juice and stored at 4 °C for 12 weeks. B. animalis ssp. lactis Bb-12, L. rhamnosus GG and L. paracasei NFBC43338 remained viable at levels above 107 cfu/ml in orange juice and above 106 cfu/ml in pineapple juice for 12 weeks. Despite pH adjustment from 2.5 to 3.5, cranberry juice proved to be lethal with viable cell counts dropping to 106 cfu/ml within 4 days. Researchers attributed the poor probiotic survival in cranberry juice to the high benzoic acid content of approximately 34 mg/l in certain varieties. Thermal pasteurisation of orange juice containing probiotic strains at 76 °C for 30 s resulted in cell loss ranging from 4.2 to 6.4 log cycles. L. rhamnosus GG was reduced by over 5 log. In addition, high-pressure pasteurisation treatment at 400 MPa for 5 min caused extensive inactivation of all strains related to electrostriction, a change in the mean density of the system due to the electric field, which suddenly reduces pH during pressurisation. The results of this research further confirmed that probiotic cells should be added after the thermal and high pressure pasteurisation steps have been completed. Khalf et al. (2010) studied the viabilities of B. animalis ssp. lactis Bb12 and L. rhamnosus GG when added to liquid maple sap, which can be used to develop new probiotic products. During four weeks of storage at 4 °C in sap enriched with inulin, the B. lactis Bb12 viable cell count had decreased by 0.5 log10 cfu/ml by day 21, while the L. rhamnosus GG viable cell count at day 28 remained similar to the inoculation levels. In addition to testing strain survival during storage, researchers examined the viability 131



of probiotics under GI conditions. The viabilities of B. animalis ssp. lactis Bb12 and L. rhamnosus GG in maple concentrate and maple sap, both with and without inulin in acid and basic conditions, was tested using a multicompartmental dynamic TIM-1 model of the GI system. After 80 min of gastric digestion, both probiotics in maple concentrate with inulin exhibited the lowest reduction in viable cell count. The increased cell survival in maple concentrate containing inulin can be attributed to prebiotic properties of inulin and lower water activity due to the higher sugar concentration. A number of strains are being incorporated into probiotic non-dairy beverages marketed in Europe and the USA including L. plantarum 299v, B. animalis ssp. lactis Bi‑07, B. animalis ssp. lactis HN019, L. rhamnosus GG, L. plantarum HEAL9 and L. paracasei 8700:2. Launched in 1994 in Sweden, the first non-dairy probiotic beverage, contains L. plantarum 299v fermented oatmeal which was originally developed for enteral (nasogastric) feeding (Molin et al., 1993). The fermented oatmeal applies principles used to produce Tanzanian togwa, where oat is used as the fermentation substrate instead of maize or sorghum. For liquefaction, malted barley is used as opposed to germinated maize or sorghum, and L. plantarum 299v is utilised for fermentation instead of spontaneous mixed microbiota. To produce the probiotic beverage, 5% of fermented oatmeal gruel is added to fruit drink base with pH