Production of encapsulated probiotic chocolate containing Bifidobacterium bifidum alginate-whey and evaluation of physicochemical, sensory and shelf life

Chocolate is made of cocoa mass and sugar suspended in a cocoa butter matrix (Konar et al., 2016). One of the most important ingredients in chocolate manufacture is cocoa powder, which is a complex blend of proteins, polysaccharides, and lipids (Orkaz et al., 2020)In recent years, the global market of functional foods, especially supplements with probiotics, is expanding rapidly. Probiotics are the live microorganisms that, when administered in adequate amounts, provide health benefits to consumers (Tsuda et al., 2010). The health benefits of probiotics include preventing infectious diarrhea, decreasing cholesterol levels in the blood, reducing symptoms of lactose intolerance, increasing immunity to specific diseases, and acting as an antitumor/anticancer agent. Probiotic efficacy can be enhanced when these microorganisms are integrated into the diet, as interactions with food components can protect microbial cells as they pass through the gastrointestinal tract (Vindeola et al., 2011), influenced by the growing demand for functional chocolates that may help customers improve their health (Orkaz et al., 2020). milk chocolate, in particular, is known to be a source of a variety of physiologically active compounds with significant antioxidant activity, such as flavonoids and polyphenols (Todorovic et al., 2015).

Probiotic strains B. bifidum were obtained from the Persian Type Culture Collection (PTCC), Tehran, Iran. Bifidobacterium bifidum was grown in 18 mL MRS broth, supplemented with 0.05% L-cysteine hydrochloride (Sigma, Sydney, Australia) MMRS (Modified MRS) to provide an anaerobic environment, at 37 °C for 48 h under anaerobic conditions using the Gas Pak system (Anaerocult A, Darmstadt, Merck, Germany). The cultures were transferred into 180 mL MMRS for B. bifidum and incubated under the same conditions. The cultures were then reactivated by transferring 3–4 times in MRS broth and the cells were harvested by centrifuging at 1500 g for 15 min at 25_C (Eppendorf Centrifuge, 5810R, Hamburg, Germany) and washed twice with sterile 0.1% peptone solution. The final cell concentration was adjusted to 1.0 * 109 Cfu ⁄mL (Zomorodi et al., 2010). Preparation of the microencapsulated solution: The whey protein isolated micro-coating solution was prepared according to Tellioghlu harsa & cabuk (2015) method with some modifications. Thus, first a solution of 8% whey protein isolate was prepared and for protein denaturation, it was heated at 80 ° C for 30 minutes and then cooled to ambient temperature. To prepare the alginate coating solution, first a 2% alginate solution was prepared in sterile distilled water and after sterilization (at 121 ° C for 15 minutes) it was cooled. Microencapsulation of microorganisms: The extrusion technique was used to microencapsulate bacteria. After washing, the cultures were suspended in 5 mL of sterile 0.1% peptone solution and mixed with 20 mL of 2% (w⁄ v) sodium alginate solution and 20 mL of 8 % (w⁄ v) whey protein concentrate solution sterilized at 121_C for 15 min. The cell suspension was injected through a 0.11-mm needle into sterile 0.05 M CaCl2 (Merck, Germany). The beads were allowed to stand for 30 min for jellification, and then rinsed with, and subsequently kept in, sterile 0.1% peptone solution at 4_C. After filtering from sterile filter paper, the seeds were transferred to sterile plates and placed in the freeze dryer for 2 days at -130 ° C for drying, and after drying under sterile conditions, they were powdered.

Evaluation of survival of Bifidobacterium bifidum: Bacterial count results for probiotic chocolate increased from 7.33 Cfu/gr on the first day to 6.15 Cfu/gr on the 30th day and to 4.69 Cfu/gr on the 60th day, indicating that the probiotic chocolate retained its probiotic properties until the 30th day(P<0.05). Bacterial count results for microencapsulated probiotic chocolate increased from 6.48logcfu/gr to 6.33logcfu/gr on the 15th day and to 3.5 Cfu/gr on the 60th day, indicating that the microencapsulated probiotic chocolate retained its probiotic properties until the 15th day. Chocolate is one of the products that with increasing of water activity, the amount of damage to the product and the spoilage of the product is accelerated, and therefore it is necessary to pay much attention to the amount of aw. The results showed that probiotic chocolate has a higher water activity than plain chocolate, but not enough to cause probiotic bacteria activity in it (P<0.05). Investigation of acidity changes: The results indicate that the acidity changes for the control sample after 60 days of storage are 1.14 (in terms of oleic acid percentage). This value starts from 0.89 on the first day for microencapsulated probiotic chocolate and reaches 0.95 (in terms of oleic acid percentage) after 60 days of storage, which is not similar to the acidity changes for the unencapsulated sample, which means the trend of acidity changes is not too different for each of the samples. Probiotics remain in the incubator phase in the chocolate-based environment, so no activity occurs that produces lactic acid products that affect acidity. Investigation of pH changes: It is important to study the pH changes of chocolate samples because pH changes are one of the factors affecting product quality, spoilage and its shelf life. The pH values during the storage period for the probiotic chocolate samples show an almost constant trend. Investigation of moisture changes: The results showed that the moisture content of the control sample is higher than probiotic chocolate and microencapsulated probiotic chocolate, which is due to the smaller particle size in the control sample and thus the ability to absorb high moisture. The high moisture content of microencapsulated chocolate compared to the unencapsulated type is probably due to the presence of hygroscopic substances such as whey protein in the bacterial coatings, which have a high ability to absorb moisture and increase the amount of moisture. Investigation of texture hardness changes: Based on the results of texture hardness measurements, the highest hardness was observed in microencapsulated probiotic chocolate (after 60 days) and the lowest hardness was observed in plain chocolate (after 1 day), which could be due to the effects of the materials used to microencapsulation. Investigation of viscosity and yield value changes of chocolate: In this study the results of viscosity measurements show that due to the smaller size in the control sample and thus its ability to absorb high moisture, the viscosity and yield value of the control sample is higher than probiotic chocolate. In addition, microencapsulated probiotic chocolate has a high moisture absorption capacity compared to the unencapsulated type due to the presence of hygroscopic substances such as whey protein in the coating, resulting in higher viscosity and yield value. Sensory evaluation: results show that the probiotic chocolate containing Bifidobacterium bifidum is not significantly different from the microencapsulated type, by the method of sodium alginate - whey protein gel formation, and also the control sample which indicates that the addition of probiotics to chocolate or microencapsulation of probiotic bacteria did not have a significant effect on the desirability and organoleptic properties of chocolate.

There were not seen significant negative effects on physicochemical, rheological and sensory properties of probiotic chocolate, probiotic chocolate and microencapsulated probiotic during the storage time and therefore there is no need to change the technological and device conditions and also purchase additional equipment for probiotic chocolate production.