Probiotic gummy candy with xylitol : development and potential inhibition of Streptococcus mutans

Gummy candies are nutritionally poor foods; however, they have a large consumer market. Enhancing these products with nourishing and/or functional matrices is a challenge. This study aimed to develop the features of a gummy candy probiotic, containing Bifidobacterium Research, Society and Development, v. 9, n. 10, e7369108942, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8942 2 lactis HN019, and evaluate its effectiveness in inhibiting Streptococcus mutans UA159. The candy was suitable for probiotic development, whose viable bacterial count was 8.54 Log CFU/g in the ready-to-eat product; high viability was maintained during the 28-day shelf life. Additionally, the rate of probiotic survival under in vitro gastrointestinal simulation was 98% and 93% in the ready-to-eat product and after shelf life, respectively. The sensory analysis revealed scores over 8.0 in all attributes evaluated. Lastly, the inhibition test of S. mutans UA159 showed 25% and 98% inhibition, with 10% and 20% xylitol respectively. Probiotic and xylitol gummy candy is a potential functional food and can target a niche food market, such as teenagers.


Introduction
Probiotics show beneficial effects on human health and are associated with the microbial balance of the digestive tract and the immune system (Abuajah et al., 2014).
Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (Hill et al., 2014). To obtain the probiotic status, microorganisms must fulfill a number of criteria related to safety, functional effects, and technological properties (Foligné et al., 2013). In this sense, Bifidobacterium and Lactobacillus are the most popular probiotics employed in foods (Tufarelli & Laudadio, 2016) that can be added to nondairy products, including cereals, juices, chocolate, chewing gum, and dairy products such as yogurt, cheese, and ice cream (Ranadheera et al., 2010).
Xylitol, a natural sugar substitute, defined as a nutritive sweetener, has a low caloric value, thus helping consumers to reduce their energy intake and lose weight (EU legislation, 2008). Xylitol is widely known as an anticariogenic agent, is not fermented by S. mutans, on the other hand, exerts an inhibitory effect on the microbial group (Watthanasaen et al., 2017).
Recently, studies have also demonstrated the effectiveness of both probiotics and xylitol as anticariogenic agents preventing caries and other oral diseases (Witzler et al., 2017;Kaur et al., 2018). Scientific evidence demonstrates that xylitol meets the safety and efficacy prerequisites for use in caries prevention programmes in all age groups. These characteristics make xylitol a good substitute for sugars in candies, the consumption of which is common, especially among children.
Candies that usually do not provide any nutritional value are considered versatile Development, v. 9, n. 10, e7369108942, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8942 4 matrixes and thus can act as vehicles for the supply of vitamins, fibers, and other compounds with health benefits (Archaina et al., 2019). Considering these factors, the scope of the current work was to develop a gummy candy with Bifidobacterium lactis HN019 and xylitol and study its physicochemical properties, acceptance, probiotic in vitro stability, scanning electron microscopy of probiotic gummy candy, and the effect of xylitol on the growth of S. mutans UA159.

Methodology
This is a quantitative study, as it used the collection of numerical data that had been analyzed applying statistical analysis relevant to the study (Pereira et al., 2018)

Probiotic
B. lactis HN019 (Dupont, Brazil) was added (0.01% w/v) to skimmed milk powder reconstituted at 10 % (w/v), sterilized at 115 °C/10 min and cooled. The culture was incubated at 37 °C/24 h. For the development of gummy candy, the probiotic was mixed to whey protein isolate (WPI) (Alpha Galvano Chemical) in the ratio 1:1(v/w) as protector agent. The homogenized and lyophilized (Liobras L101, Liotop) mix was used in the gummy candy formulation (Patent BR1020160307902).
The candy was produced by agglutination of ingredients followed by molding, and stored in plastic bags (low density polyethylene) at room temperature (±25 °C).

Texture profile of probiotic gummy candy with xylitol
The analysis of texture (hardness, adhesiveness, elasticity and chewing) was carried out on the Brooksfield CT3 Texture Analyzer. The analysis parameters assigned to the equipment for determination of the Texture Profile Analysis (TPA) were: TA4/1000 probe; target 2.0 mm; 0.05 N trigger load; pre-test speed: 2 mm/s; test speed: 1 mm/s. Texture parameters were evaluated on 1, 7, 14, 21 and 28 days of storage at ±25 °C.

Sensory Acceptance
Sensory analysis was carried out with a panel of 100 untrained individuals (25 children, 25 teenagers and 50 adults). Evaluation in children was conducted using a hedonic scale with facial expression in two categories (extremely like and extremely dislike).
Teenagers and adults used an unstructured 9-point line scale ranging from 9 (extremely like) to 0 (extremely dislike) for each sensory characteristic: appearance, aroma, flavor, texture and overall acceptability. The product was analyzed for the presence of thermotolerant coliforms, coagulase-positive staphylococci, and Salmonella sp., as required by legislation (Brasil, 2005) prior to sensorial test, and were in accordance with this regulation. Approximately 10 g portions of the gummy candy were served in disposable transparent polyethylene plates. The test was carried out under controlled conditions, with mineral water and cream crackers available to the panelists. The Ethics Committee of the Universidade Pitágoras Unopar approved the study (Register 1.148.291), and consent forms indicating voluntary and fully informed participation were signed by all panelists. Children and teenagers were recruited from a school located in Londrina-PR, and parental consent to participate was obtained along with the children's assent. Students and staff from Unopar were recruited as adult panelists.

Scanning electron microscopy (SEM)
The WPI containing the probiotic B. lactis HN019 and the probiotic gummy candy were fixed onto aluminum stubs and made electrically conductive by gold coating before examination on the SEM apparatus (FEI Quanta 200, FEG, Netherlands). The microstructures were imaged at an accelerating voltage of 30 kV.

Xylitol effect in the S. mutans inhibition
The inhibition of S. mutans was evaluated in the Brain Hearth Infusion (BHI) broth with 0 (control), 1, 5, 10, 15 and 20% xylitol, sterilized at 121 °C/15 min and tested with three S. mutans UA159 inoculum (2, 3 and 4 Log CFU/mL) incubated at 37 °C/24 h. The percent inhibition of S. mutans was calculated by the equation:

% Inhibition = [(A Abs -B Abs)/A Abs] x 100
A = absorbance in the broth with 0% xylitol (control), after incubation; B = absorbance in the broths with different concentrations of xylitol, after incubation.

Statistical analysis
The candy characterization data were analyzed using the analysis of variance (ANOVA) and Tukey procedure (p < 0.05) while the sensory data were analyzed using Student's t-test (p < 0.05) with the software Statistic 13.0.

Results and Discussion
The protein content in the probiotic gummy candy was due to the addition of gelatin and, mainly, WPI (Table 1), which is the unique feature of this product, as candies are usually not protein sources. A significant decrease (p > 0.05) in the viability of B. lactis HN019 was not reported during the 28 days of storage whose counts were 8.5 and 8.0 Log CFU/g in fresh gummy and after 28 days of storage, respectively, indicating a suitable stability of the probiotic in this matrix. During the ingredient preparation, the probiotic was completely absorbed by the WPI, and the same condition was maintained after probiotic addition to the gummy candies, as shown in Figure 1A and 1B, which the WPI may have had a protective effect on the probiotic cells.  During the 28 days of storage, an increase in hardness (from 1.65 to 6.94 N) and chewiness (from 2.65 to 13.77 mJ) of the gummy candies was observed (p<0.05) ( Table 2).
These results might indicate water loss during storage, which is a reflection of the storage conditions and kind of packing used. a b Development, v. 9, n. 10, e7369108942, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8942  Sensory analyses in previous studies demonstrated the effect of probiotic addition on the acceptance of gummy candies or similar products (Witzler et al., 2017;Bartkiene et al., 2018). Herein, we observed that 88% of the children liked the product (Table 3). The texture presented a difference when the results of teenagers and adults were compared, indicating greater acceptance by the adults. The presence of probiotic in food can affect the sensory characteristics due the production of metabolic components such as organic acids. In all simulated phases (Table 4), B. lactis HN019 was observed to show a high survival rate. In the gastric phase, a difference in probiotic counts in the enteric phases I and II Development, v. 9, n. 10, e7369108942, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8942 9 was noticed (p<0.05). However, noticeable changes in the total count were not reported, since the changes occur within one logarithm cycle. In Phase 3, an increase in B. lactis population at all evaluated periods was observed, indicating that the probiotic was not affected by the drastic pH conditions in the previous phases. The results showed that the probiotic resistance in the gastric and enteric juice conditions was not significantly affected during the storage time, indicating that the consumption of probiotic gummy candy after production and 28 days of storage has the same effect. The probiotic presented a high rate of survival of 98% in the ready-to-eat product and 93% after the shelf life under in vitro gastrointestinal simulation (Guo et al., 2009). In the study of Medeiros et al. (2019), the authors added B. lactis to curd cheese, and the minimal counting was observed only in day 1 of storage. The inhibition of S. mutans at different concentrations of xylitol and inoculum of S. mutans (Figure 2) was evaluated. At xylitol concentrations of 10% and 20%, inhibition of S. mutans inoculum 2 Log CFU/mL was 24.64% and 97.96%, respectively. At a xylitol concentration of 15%, the inhibition percentage decreased as the inoculum increased. These results indicate that the inoculum of S. mutans directly affects the inhibition caused by xylitol and lowers the initial cellular concentration of the microorganism, implying greater inhibition. Kaur et al. (2018) evaluated the effect of chewing gums containing probiotics and xylitol on Research, Society and Development, v. 9, n. 10, e7369108942, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8942 the salivary S. mutans counts and concluded that both were equally effective in reducing S. mutans counts in children. The authors suggest that fluoride supplements, chewing gums containing probiotics and xylitol can be an alternative regimen for prevention of caries in children.

Final Considerations
The gummy candy proved to be a stable and suitable matrix for the development and maintenance of the probiotic B. lactis HN019. The high protein content of the candy and the potential cariogenic effects are interesting characteristics of this type of product, especially with regard to the target audience, children and adolescents.