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Survival of immobilized probiotics in chocolate during storage and with an in vitro gastrointestinal model
Food Bioscience 16 (2016) 37–43
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Survival of immobilized probiotics in chocolate during storage and with an in vitro gastrointestinal model ⁎
Varongsiri Kemsawasda, Pittaya Chaikhamb, , Paweena Rattanasenab a b
Institute of Nutrition, Mahidol University, Nakorn Pathom Campus, Nakorn Pathom 73170, Thailand Faculty of Science and Technology, Phranakhon Si Ayutthaya Rajabhat University, Phranakhon Si Ayutthaya 13000, Thailand
A R T I C L E I N F O
A BS T RAC T
Keywords: Lactobacillus casei 01 Lactobacillus acidophilus LA5 White chocolate Milk chocolate Dark chocolate
Probiotics are the bacteria that can provide health benefits to the consumers and they are suitable to be added to a variety of foods. In this research, viability of immobilized potential probiotics, including Lactobacillus casei 01 and Lactobacillus acidophilus LA5, in three different types of chocolate (white, milk and dark) were studied during storage and with an in vitro gastrointestinal model. The sensory attributes of probiotic-chocolates were also evaluated. Both cultures were found to be viable up to 60 days of storage at 4 °C ( > 6 log CFU/g) which were sufficient to potentially provide health benefits to the consumer. As a carrier, chocolates also may protect probiotics in both the stomach and small intestine environments. The overall sensory results suggested that the probiotic powders had no significant effect on sensory attributes. After 60 days storage, significant decreases of the overall liking scores in all the chocolate samples were observed. Therefore, this study may be useful for the future development of probiotic-supplemented chocolates as foods with health benefits for the consumers.
1. Introduction Presently, the worldwide market of functional food products with health benefits, especially those supplemented with probiotics, has been promptly expanding. In accordance with a joint report of Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO), the definition of probiotics is “live microorganisms which, when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2001). Sufficient amounts of viable and active probiotics should successfully reach the intestines for improving the microbial balance in the intestine environments, and this is greatly beneficial to the consumers (Chaikham, Apichartsrangkoon, Jirarattanarangsri & Van de Wiele, 2012; Fooks & Gibson, 2002; Fuller, 1992; Sekhon & Jairath, 2010). The health benefits of probiotics have previously been reported viz. preventing the infectious diarrhea, lowering the levels of blood cholesterol, controlling the intestinal infections, reducing the symptoms of lactose intolerance, enhancing some types of immune responses and functioning as antitumor/anticancer agents (Fuller, 1992; Patel et al., 2013; Sanders et al., 2013). Probiotics can be found in fermented dairy products, particularly yogurts and cheeses, and also some non-dairy products, which are currently receiving attention by people in all age groups. Some probiotics, especially Lactobacillus casei 01 and Lactobacillus acidophilus LA5, were suggested to have positive effects on human colon
⁎
microbiota because they could enhance short-chain fatty acids formation and increase beneficial colon bacteria, including lactobacilli and bifidobacteria (Chaikham et al., 2012). At the same time, certain harmful bacteria, such as fecal coliforms and clostridia, were noticeably suppressed (Chaikham & Apichartsrangkoon, 2013; Chaikham et al., 2012). Similar outcomes were observed by Bianchi et al. (2014) who investigated the fermented vegetal beverages (synbiotic aqueous extracts of quinoa and soy) enriched with L. casei 01. Therefore, it is interesting to evaluate the supplementation of both probiotics in other novel food products with health benefits. As food matrix, chocolate has been proposed to be supplemented with probiotics and it may be developed into different future products (Gadhiya, Patel, & Prajapati, 2015). Notably, chocolate was found to contain various bioactive compounds, such as polyphenols and flavonoids (i.e. catechin, epicatechin and procyanidin), both of which possessing high levels of antioxidant activities (Todorovic et al., 2015). Possemiers, Marzorati, Verstraete, and Van de Wiele (2010), Mandal, Hati, Puniya, Singh, and Singh (2013) and Laličić-Petronijević et al. (2015) reported that chocolate was an ideal carrier for protecting the probiotic cells during shelf storage and for enhancing their ability to tolerate the adverse environments of gastrointestinal tract. Moreover, several factors, including water activity (aw), oxygen tension and temperature were found to play the essential roles in forming probiotic-chocolates (Kingwatee et al., 2015). There are a variety of techniques, for example, encapsulation by spray-drying, which have been used for extending the
Corresponding author. E-mail address: [email protected] (P. Chaikham).
http://dx.doi.org/10.1016/j.fbio.2016.09.001 Received 25 February 2016; Received in revised form 10 September 2016; Accepted 14 September 2016 Available online 15 September 2016 2212-4292/ © 2016 Elsevier Ltd. All rights reserved.
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milk chocolate containing 10% (w/w) cocoa powder (Jiangsu Linzhi Shanyang Group Co., Ltd., Jiangsu, China), 49% (w/w) cocoa butter, 30% (w/w) sucrose, 10% (w/w) skim milk powder or probioticsupplemented skim milk powders, 0.9% (w/w) soy lecithin and 0.1% (w/w) ethyl vanillin; and (iii) dark chocolate containing 50% (w/w) cocoa powder, 9% (w/w) cocoa butter, 30% (w/w) sucrose, 10% (w/w) skim milk powder or probiotic-supplemented skim milk powders, 0.9% (w/w) soy lecithin and 0.1% (w/w) ethyl vanillin. All ingredients, except probiotic-supplemented skim milk powders, were mixed, heated, thoroughly stirred, immediately cooled down to ~35 °C and finally inoculated with 10% (w/w) probiotic-supplemented skim milk powders. After that, the chocolates and probiotic-chocolate mixtures were transferred to 2×2×0.5 cm plastic containers and kept in a refrigerator for 20 min. The final products were packed in aluminum foil with 0.2 mm thickness (Diamond, Zhangjiang, China) and then stored at 4 or 25 °C for 60 days.
viability and activity of these microbes in products during storage (Riveros, Ferrer, & Bórquez, 2009). However, the knowledge of supplementing the spray-dried probiotic cells into chocolate is still quite limited. Therefore, this research aimed to evaluate the survival rates of immobilized probiotics, L. casei 01 and L. acidophilus LA5 that were incorporated in different chocolate matrices (white, milk and dark) during storage and with an in vitro gastrointestinal model. The sensorial attributions of these probiotic-supplemented chocolates were also evaluated using 50 untrained volunteers. 2. Materials and methods 2.1. Activation and immobilization of probiotic cultures Briefly, freeze-dried L. casei 01 (Lot NO. 3230647) and L. acidophilus LA5 (Lot NO. 3251532) which obtained from Chr. Hansen (Hørsholm, Denmark) were rehydrated, activated and anaerobically incubated in de Man Rogosa and Sharp (MRS) broth (Hi-media, Mumbai, India) at 37 °C for 16 and 18 h, respectively, to reach their stationary phases (Chaikham, Apichartsrangkoon, George, & Jirarattanarangsri, 2013). The activated cells were then harvested by centrifugation at 4000g and 4 °C for 20 min (Centrifuge model Rotina 46 R, Tuttlingen, Germany), followed by washing twice using sterile 0.85% (w/v) saline water (Hi-media). After that, the cell pellets were diluted using deionized water (RCI Labscan, Bangkok, Thailand) to adjust the bacterial population at ~1012 CFU/ml. The skim milk (CP Meiji, Saraburi, Thailand) was processed using a ultra-high temperature (UHT) method and mixed with 20% (w/v) maltodextrin (10.5 DE: Ingredient Center, Bangkok, Thailand), then heated at 80 °C for 10 min before being cooled down to 35 °C. Both of probiotic cultures were separately inoculated into the skim milk mixture to obtain the cell concentration of ~1010 CFU/ml and subsequently dried into powders using a spray-dryer (JCM Engineering Concept, Bangkok, Thailand). Drying conditions for this experiment was set at 30 °C of feeding temperature, 1 l/h of feeding rate, 15 psi of atomizing pressure, and 170 °C of hot-air-inlet temperature to generate 90 °C outlet temperature. The powders were vacuum-sealed in laminated bags (nylon plus polyethylene: Siam Pack, Chiang Mai, Thailand) and stored in a refrigerator overnight before preparation into the probiotic-supplemented chocolates. The plate counts of survival L. casei 01 and L. acidophilus LA5 cells after spray-drying were 6.6 ± 1.2×1010 and 8.1 ± 1.6×1010 CFU/g, respectively.
2.4. Determination of antioxidant properties of chocolates 2.4.1. Extraction of samples The chocolates were extracted according to the method of Todorovic et al. (2015). First, chocolate samples were ground using a coffee grinder (Bosch, Farmington Hills, MI, USA). Two g of each ground sample were extracted three times using 10 ml n-hexane (Merck, Darmstadt, Germany) for eliminating the lipids from the samples. The defatted samples were then air-dried overnight to evaporate the n-hexane residue and then extracted twice using 5 ml of a mixture of 70% (v/v) acetone (Merck), 29.8% (v/v) distilled water (RCI Labscan) and 0.2% (v/v) acetic acid (Merck) for 30 min using a ultrasonic bath (FALC, Treviglio, Italy). After that, the mixtures were centrifuged at 4000g for 15 min. The supernatants of the extracts were used for determinations of bioactive components and antioxidant activities by the following methods. 2.4.2. Total polyphenols The levels of total polyphenol contents of the chocolate extracts (Section 2.4.1) were spectrophotometrically determined using FolinCiocalteu method (Chaikham & Apichartsrangkoon, 2012). Each supernatants of the extracts (0.5 ml) were thoroughly mixed with 10 ml of absolute ethanol (Merck), 2.5 ml of 10% Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA), and 2 ml of saturated sodium carbonate solution (Ajax, Sydney, Australia), and finally incubated at 25 °C for 2 h. The mixtures were measured for absorbance at 765 nm using a Perkin Elmer UV WINLAB spectrophotometer (Perkin Elmer, Waltham, MA, USA). Gallic acid (Sigma-Aldrich) was used as standard reagent. The regression line between absorbance (y) and gallic acid content (x) was y=0.006x+0.018. The results were expressed as mg gallic acid equivalent per 100 g of sample (mg GAE/ 100 g).
2.2. Scanning electron microscopic (SEM) images A scanning electron microscope (Hitachi S-3000 N, Hitachi HighTechnologies Co., Ltd., Tokyo, Japan) was used to examine the microstructure characteristics of both immobilized probiotics. The powdered probiotics were placed on to the SEM sample holders and coated with 10 nm of gold particles using a Hitachi E1010 ion sputter (Hitachi Science Systems Co., Ltd., Tokyo, Japan). The external structures of gold-coated samples were then examined at an accelerating voltage of 15 kV (Kingwatee et al., 2015).
2.4.3. Total flavonoids The levels of total flavonoids of the chocolate extracts (Section 2.4.1) were determined following the modified method of Šarić et al. (2012). In brief, 2 ml of each extracts were thoroughly mixed with 4 ml of deionized water and 2 ml of 5% (w/v) sodium nitrite (Ajax) and allowed to react for 6 min. Two ml of 10% (w/v) aluminum chloride (Sigma-Aldrich) were added to each mixtures followed by shaking for 6 min and adding of 12 ml of 1 M sodium hydroxide (Merck). The mixtures were spectrophotometrically measured for absorbance at 510 nm. Catechin (Sigma-Aldrich) was used as standard reagent. The standard curve was plotted and the regression line between absorbance (y) and catechin content (x) was y=0.003x+0.120. The results were expressed as mg catechin equivalent per 100 g sample (mg CE/100 g).
2.3. Preparation of probiotic-chocolates Three different types of chocolate viz. white, milk and dark chocolates were mixed with probiotic powders (Section 2.1) following the method of Laličić-Petronijević et al. (2015) with some modifications. These chocolates were prepared as following: (i) white chocolate containing 59% (w/w) cocoa butter (Foodchem International Corporation, Shanghai, China), 30% (w/w) sucrose (Mitr Phol, Bangkok, Thailand), 10% (w/w) skim milk powder or probioticsupplemented skim milk powders, 0.9% (w/w) soy lecithin (Modernist Pantry, Portsmouth, NH, USA) and 0.1% (w/w) ethyl vanillin (Chemipan Corporation Co., Ltd., Bangkok, Thailand); (ii) 38
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5 g of probiotic-supplemented chocolates were mixed with 25 ml of gastric solution (pH 1.4, 37 °C) in sterile glass bottles and then anaerobically incubated at 37 °C. After 2 h of incubation, 25 ml of simulated small intestinal solution (a mixture of 15 ml of duodenal solution and 10 ml of bile solution, pH 8.1, 37 °C) were added to the prepared bottles and they were then incubated for 4 h. For enumeration of survival cells, 1 ml of each incubated fluids was taken to be diluted with 9 ml of 0.2 M sterile phosphate buffer (pH 7; Merck) and used for plate count assay for viable cells using MRS agar. The results were expressed as CFU/ml.
2.4.4. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity The levels of DPPH radical scavenging activity of the chocolate extracts (Section 2.4.1) were determined by the method of Chaikham and Apichartsrangkoon (2012) with some modifications. The extract (0.5 ml) was well-mixed with 4.5 ml of 0.5 μM DPPH (Fluka, Buchs, Switzerland) solution (diluted using methanol) (Merck), then incubated at room temperature for 1 h and finally measured for the absorbance at 517 nm. Trolox (Sigma-Aldrich) was used as standard reagent and the DPPH radical inhibition was calculated from a calibration curve plotted between absorbance (y) and Trolox content (x). The regression line was y=147.1x–11.0. The results were expressed as μM Trolox equivalent/g sample (μM TE/g).
2.8. Statistical data analysis All data were shown as the averages of six replicates (n=6) with standard deviations. Analysis of variance (ANOVA) was carried out using a SPSS program version 15.0 (SPSS Inc., Chicago, IL, USA). Duncan's multiple range tests were used to examine the significant differences among the means of treatments (P≤0.05).
2.4.5. Ferric reducing antioxidant power Ferric reducing antioxidant power (FRAP) values of the chocolate extracts were assessed following the modified method of Benzie and Stain (1996). Two ml of each extracts (Section 2.4.1) were mixed with 8 ml of deionized water and 3 ml of FRAP reagent [a mixture of 300 mM sodium acetate buffer (pH 3.6; Sigma-Aldrich)], 10 mM 2,4,6tripyridyls-triazine solution (Sigma-Aldrich) and 20 mM ferric chloride hexahydrate solution (Merck) at a ratio of 10:1:1 and then incubated at 37 °C for 1 h in the dark. The mixtures were measured for absorbance at 593 nm. The FRAP values were calculated from a calibration curve (y=1.35x−0.03; where y is absorbance and x is Trolox content) and expressed as μM TE/g.
3. Results and discussion 3.1. Bioactive components and antioxidant capacity of chocolates Several studies have reported that cocoa possessed high levels of polyphenols and flavonoids, which were shown to have beneficial effects towards human's health, in particular prevention of cardiovascular disease and cancer (Andújar, Recio, Giner, & Ríos, 2012; Gadhiya et al., 2015; Hii, Law, Suzannah, Miswani, & Cloke, 2009). In this study, the contents of total polyphenols and flavonoids in chocolates were highly varied depending on the types of chocolates, as shown in Table 2. The levels of these compounds were significantly high (P≤0.05) in milk and dark chocolates, but much less in white chocolate. The greater quantity of bioactive compounds in chocolates was directly correlated to the bigger portion of cocoa powder in the recipes, whereby the white, milk and dark chocolates contained cocoa powder at 0%, 10% and 50%, respectively. Therefore, the dark chocolate was found to have the highest contents of both component groups (P≤0.05). The antioxidant activities in dark chocolates (DPPH inhibition and FRAP values) were shown to have strong correlation with the contents of total polyphenols and flavonoids (R2=0.8–0.9). These results were similar to the finding of Oliveira, Maciel, Miranda, and Bispo (2011), who showed that the organic cocoa contained high levels of total phenolic, flavonoids, and antioxidant activity (IC50 value) at 196–285 mg GAE/g, 155–274 mg CE/g and 4.2–7.7 µg/ml, respectively, and they also found the positive relationship between total polyphenols and DPPH radical scavenging activity. These results may suggest that the higher contents of total phenols found in chocolates, the greater antioxidant activity the chocolates may contain.
2.5. Stability of probiotics in chocolate during storage To quantify the survival rates of probiotics in all chocolate samples stored at 4 or 25 °C for 60 days, the samples were taken every 10 days and melted at 37 °C for 10 min using a Biohazard Safety Cabinet (Esco Technologies, Inc., Horsham, PA, USA). After that, the samples were diluted with 0.1% (w/v) sterile peptone water (Hi-media) at different concentrations before pour-plating on MRS agar (Hi-media). All plates were anaerobically incubated at 37 °C for 48–72 h in anaerobic system (Anaero Gen™; Oxoid, Wesel, Germany). The number of colonies were counted and expressed as CFU/g. 2.6. Sensory evaluation Sensorial attributions of chocolates and probiotic-supplemented chocolates were evaluated for appearance, color, flavor, texture, and overall acceptability. Fifty untrained panelists who like chocolate (aged 18–35 years) including academic staffs and students from Phranakhon Si Ayutthaya Rajabhat University, Phranakhon Si Ayutthaya province, Thailand were assigned to score the sensorial attributes using the evaluation form of 9-point hedonic scale (9=like extremely, 5=neither like nor dislike, 1=dislike extremely). All chocolates were identified using 3-digit random numbers and placed on plastic dishes before serving. The participants were instructed to rinse their mouths with water every time before tasting the sample (Laličić-Petronijević et al., 2015).
3.2. Microstructures of probiotic powers Fig. 1 shows the microstructures of L. casei 01 and L. acidophilus LA5 powders after spray-drying using skim milk and maltodextrin as supporting materials. There were the clear differences between the size of distribution and the morphology of L. casei 01 (Fig. 1a) and L. acidophilus LA5 (Fig. 1b) powders. As shown by the SEM images, the average sizes of L. casei 01 and L. acidophilus LA5 microcapsules were 8.4–45.9 and 11.6–69.7 µm, respectively. L. casei 01 microcapsules were found to be smaller and had smoother surface, but L. acidophilus LA5 microcapsules were more spherical and had rougher surface. Regarding to the sizes of the probiotic, L. casei 01 cells were short with rod-shape (length ~1 µm), and they were often found in the form of short chains comprising three or four cells; nonetheless, L. acidophilus cells were long with rod-shape (length ~3 µm), and they were often found in pairs or chains of various lengths (Chaikham, Apichartsrangkoon, George et al., 2013). Moreover, Kingwatee et al.
2.7. Survival rates of probiotic cells under in vitro stomach and small intestine conditions The simulated gastric, duodenal and bile solutions were prepared following the procedure of Oomen et al. (2003) (Table 1), and the in vitro gastrointestinal experiments were carried out according to the methods of Chaikham, Apichartsrangkoon, and Worametrachanon et al. (2013) with some modifications. The fresh cells (Section 2.1) were aseptically inoculated into sterile distill water (pH 1.4, 37 °C) at the concentration of ~108 CFU/ml. After that, 5 ml of diluted cells or 39
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Table 1 Compositions of synthetic gastric and small intestinal fluids used for the in vitro digestion (Oomen et al., 2003). Reagents**
Gastric fluid (pH 1.4)
Inorganic substances
NaCl NaH2PO4 KCl CaCl2 NH4Cl HCl (conc.)
2752 mg 266 mg 824 mg 400 mg 306 mg 6.5 ml
NaCl NaHCO3 NaH2PO4 KCl MgCl2 HCl (conc.)
7012 mg 5607 mg 80 mg 564 mg 50 mg 180 µl
NaCl NaHCO3 KCl HCl (conc.)
5259 mg 5785 mg 376 mg 180 µl
Organic substances
Glucose Glucuronic acid Urea Glucosamine hydrochloride
650 mg 20 mg 85 mg 330 mg
Urea
100 mg
Urea
250 mg
Other components
BSA Mucin Pepsin
1000 mg 3000 mg 2500 mg
CaCl2 BSA Pancreatin Lipase
200 mg 1000 mg 9000 mg 1500 mg
CaCl2 BSA Bile
222 mg 1800 mg 30,000 mg
Duodenal fluid (pH 8.1)
Bile fluid (pH 8.1)
** Sodium chloride (NaCl), sodium phosphate monobasic (NaH2PO4), potassium chloride (KCl), ammonium chloride (NH4Cl), hydrochloric acid (HCl conc.), sodium bicarbonate (NaHCO3), magnesium chloride (MgCl2), calcium chloride (CaCl2) and bovine bile were purchased from Sigma-Aldrich. D-Glucose, glucuronic acid, urea, glucosamine hydrochloride, bovine serum albumin (BSA), mucin from bovine submaxillary gland (CAS NO. 84195-52-8, 5 U/mg), pepsin from porcine gastric mucosa (CAS NO. 107185, 0.7 U/mg), pancreatin from porcine pancreas (CAS NO. 107130, 6 U/mg) and lipase from Chromobacterium viscosum (CAS NO. 437707, 2500 U/mg) were obtained from Merck.
3.3. Survival of immobilized probiotics in chocolates during storage
Table 2 Bioactive compounds and antioxidant activities of different chocolate types. Chocolate types
White chocolate Dark chocolate Milk chocolate
Bioactive components
The matrices of white, milk and dark chocolates were supplemented with the excessive amounts of spry-dried L. casei 01 or L. acidophilus LA5 cells. All chocolate samples were stored at 4 and 25 °C for 60 days and then examined for the viable probiotic population (Table 3). The results showed that survivability of probiotics was dependent on the strain of probiotics, type of chocolate, and storage temperature and duration. Even though the initial levels of L. casei 01 and L. acidophilus LA5 in chocolates were approximately 8 log CFU/g, the colony counting results showed that the survival rate of L. casei 01 was higher than that of L. acidophilus LA5 (P≤0.05), regardless of the types of chocolate. Notably, L. casei 01 was found to be able to endure the storage conditions better than L. acidophilus LA5. These results could be also supported by the number of cell loss, which indicating that L. casei 01 exhibited cell loss at levels significantly lower than L. acidophilus LA5 (P≤0.05). Similarly, Chaikham, Apichartsrangkoon, George et al. (2013) and Chaikham (2015) found that L. casei 01 cells were only slightly altered during refrigerating storage in fruit juices and relatively more stable than L. acidophilus LA5. Despite that, LaličićPetronijević et al. (2015) showed that probiotic L. acidophilus NCFM®
Antioxidant activities
Total polyphenols (mg GAE/100 g)
Total flavonoids (mg CE/100 g)
DPPH (µM TE/ g)
FRAP value (µM TE/g)
tracec
tracec
0.6 ± 0.1c
1.2 ± 0.2c
364 ± 2a
49 ± 4a
79 ± 3a
104 ± 4a
94 ± 3b
16 ± 2b
21 ± 2b
48 ± 3b
Means in the same column with the same lowercase letters indicate no significant difference (P > 0.05).
(2015) indicated that different supporting materials could have different effects on the surface morphology, size, solubility and bulk density of probiotic-fruit juice powder.
Fig. 1. SEM images of (a) Lactobacillus casei 01 and (b) Lactobacillus acidophilus LA5 powders.
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Table 3 Stability of probiotics in chocolates during storage at 4 and 25 °C for 60 days. Samples
Numbers of survival cells during refrigerated storage (log CFU/g) Day 0
Cell loss(log CFUs)
Day 10
Day 20
Day 30
Day 40
Day 50
Day 60
Storage at 4 °C WLC 6.0 ± 0.6A×108 DLC 6.6 ± 0.5A×108 MLC 6.5 ± 0.3A×108 WLA 5.5 ± 0.4A×108 DLA 5.8 ± 0.4A×108 MLA 5.6 ± 0.3A×108
4.9 ± 0.6A×108 5.5 ± 0.4AB×108 5.2 ± 0.5B×108 3.2 ± 0.2B×108 4.0 ± 0.6B×108 3.5 ± 0.1B×108
2.8 ± 0.3B×108 4.2 ± 0.4B×108 4.0 ± 0.4B×108 1.5 ± 0.4C×108 2.2 ± 0.2C×108 1.8 ± 0.4C×108
1.6 ± 0.5C×108 3.3 ± 0.4C×108 3.0 ± 0.2C×108 8.0 ± 0.6D×107 9.5 ± 0.7D×107 9.1 ± 0.5D×107
8.9 ± 0.6D×107 1.1 ± 0.4D×108 9.6 ± 0.5D×107 5.0 ± 0.4E×107 7.0 ± 0.2E×107 5.8 ± 0.4E×107
4.5 ± 0.4E×107 7.3 ± 0.9E×107 6.1 ± 0.6E×107 8.4 ± 0.3F×106 1.4 ± 0.4F×107 9.1 ± 0.2F×106
8.0 ± 0.6F×106 1.8 ± 0.3F×107 9.0 ± 0.2F×106 2.3 ± 0.5G×106 5.8 ± 0.1G×106 3.4 ± 0.3G×106
1.8b 1.5c 1.8b 2.4a 2.0c 2.2b
Storage at 25 °C WLC 6.0 ± 0.6A×108 DLC 6.6 ± 0.5A×108 MLC 6.5 ± 0.3A×108 WLA 5.5 ± 0.7A×108 DLA 5.8 ± 0.4A×108 MLA 5.6 ± 0.3A×108
6.1 ± 0.4B×106 8.1 ± 0.6B×106 6.5 ± 0.4B×106 9.0 ± 0.8B×105 1.9 ± 0.6B×106 1.1 ± 0.3B×106
8.4 ± 0.3C×104 5.1 ± 0.3C×105 2.2 ± 0.3C×105 2.9 ± 0.7C×103 6.0 ± 0.6C×103 5.1 ± 0.4C×103
1.2 ± 0.5D×103 1.3 ± 0.3D×104 6.1 ± 0.7D×103 2.0 ± 0.6D×102 8.0 ± 0.4D×102 5.2 ± 0.6D×102
1.8 ± 0.2E×102 5.2 ± 0.4E×103 7.1 ± 0.6E×102 < 10E < 10E < 10E
< 10F 5.0 ± 0.8F×102 < 10E < 10E < 10E < 10E
< 10F < 10G < 10E < 10E < 10E < 10E
8.7d 8.8d 8.8d 8.7d 8.8d 8.8d
Means in the same column with the same lowercase letters, and means in the same row with the same uppercase letters indicate no significant difference (P > 0.05). WLC=white chocolate containing immobilized L. casei 01, DLC=dark chocolate containing immobilized L. casei 01, MLC=milk chocolate containing immobilized L. casei 01, WLA=white chocolate containing immobilized L. acidophilus LA5, DLA=dark chocolate containing immobilized L. acidophilus LA5 and MLA=milk chocolate containing immobilized L. acidophilus LA5.
3.4. Sensory evaluation of probiotic-supplemented chocolates
was found to have viability in milk and dark chocolates during the storage at 4 °C for 180 days at levels higher than Bifidobacterium lactis HN019. Regarding to the storage, L. casei 01 cells were found to decline at levels significantly lower than L. acidophilus LA5 cells (P≤0.05). The survivability of probiotic cells was greatly reduced when the chocolates were stored at 25 °C (P≤0.05). Indeed, L. acidophilus LA5 cells embedded in all chocolate matrices were totally disappeared within 40 days of storage at 25 °C, whereas L. casei 01 cells in dark chocolate could maintain their survivability up until 50 days of storage (~2 log CFU/g). On the other hand, during the first 10 days of storage at 4 °C, the survivability of probiotic cells in all chocolate batches reached similar levels (~8 log CFU/g). After 60 days of storage at 4 °C, the survival rates of probiotics in refrigerated chocolate remained rather high at more than 6 log CFU/g. This was complied with the recommended dose for probiotic-supplemented foods as established by Ding and Shah (2008), Chaikham (2015) and Laličić-Petronijević et al. (2015), who suggested that minimum number of live probiotics should be at least 106 CFU/ml or CFU/g before consumption. The similar results were previously described by Żyżelewicz, Nebesny, Motyl, and Libudzisz (2010), Mandal et al. (2013) and Laličić-Petronijević et al. (2015), who investigated the milk and/or dark chocolates enriched with several probiotic bacteria, such as L. acidophilus, L. casei, Lactobacilus paracasei and B. lactis. However, the package for storage could play an important role because oxygen may affect a number of active probiotic cells and thus trigger the reduction of cell survivability over the entire storage period (Chaikham, 2015), in particular the cells on the surface of chocolates (Laličić-Petronijević et al., 2015). Considering the types of chocolates, the probiotic cells could remain highest in dark chocolate (50% cocoa), followed by milk (10% cocoa) and white (0% cocoa) chocolates. The comparable results of chocolates supplemented with probiotics Streptococcus thermophiles MK-10 and Lactobacillus delbrueckii ssp. bulgaricus 151 were reported by Nebesny, Zyzelewicz, Motyl, and Libudzisz (2005). These might be primarily due to the property of dark chocolate that containing higher levels of cocoa and antioxidant compounds and activities than milk and white chocolates (Table 2). The significant levels of antioxidant activities of dark chocolate were able to prevent the probiotic cells from oxygen toxicity and thus improve the survivability of probiotic bacteria (Chaikham, 2015). On the contrary, Laličić-Petronijević et al. (2015) found that the number of living probiotic cells did not significantly differ between milk (27% cocoa) and dark (75% cocoa) chocolates during storage at 4 °C for 180 days.
The chocolates that containing immobilized L. casei 01 cells and storing at 4 °C were used for sensorial characterizations since they had viable cells more than 6 log CFU/g after 60 days of storage (Table 3). The sensory attributes of chocolates and probiotic-supplemented chocolates, including appearance, color, flavor, texture and overall acceptability, were evaluated by 50 untrained panelists, as shown in Table 4. In general, the external presence of chocolates, especially color and appearance, were considered as important factors. For fresh chocolates (day 0, no storage), the similar levels of sensory scores were observed, regardless of whether it was the control or probioticsupplemented chocolate. When comparing between the chocolate matrices, the white chocolate was shown to receive the highest preferences, followed by milk and dark chocolates (P≤0.05). Nonetheless, after 60 days of storage, sensory evaluation scores of all chocolates were found to be significantly reduced when compared to the fresh ones (P≤0.05), especially that of flavor, texture and overall acceptability, although the results showed the acceptable overall liking scores for all sensory attributions. Furthermore, no significant differences (P > 0.05) of sensory evaluation were observed between the control and the probiotic-supplemented chocolates. Thus, for this study, the addition of probiotics to chocolate products did not result in different acceptance when comparing to the control groups. Similarly, Nebesny and Zyzelewicz (2006) observed that the sensory attributions of chocolates supplemented with L. casei and L. paracasei powders were not different from those of the control chocolates. Erdem et al. (2014) also reported that supplementation of probiotic Bacillus indicus HU36, which spray dried with maltrodextrin and lemon fiber, was shown to have no negative effects on consumers’ acceptance of dark chocolate. Furthermore, there were a number of studies that also reported none or only minimal sensory attributions affected by the addition of probiotics in some foods, for instance, chocolate milk (Rouhi, Mohammadi, Mortazavian, & Sarlak, 2015), cheese (Ehsannia & Sanjabi, 2016) and ice-cream (Champagne, Raymond, Guertin, & Bélanger, 2015). 3.5. Survivability of Lactobacillus casei 01 in different chocolate types embedded in simulated gastrointestinal tract The effect of simulated gastrointestinal environments on survivability of L. casei 01, both as the free and the immobilized cells, in chocolates is shown in Table 5. Either free or microencapsulated L. 41
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Table 4 Sensory evaluation of chocolates containing Lactobacillus casei 01 powders stored at 4 °C. Samples
Appearance
Color
Flavor
Texture
Overall acceptability
Day 0 W WLC D DLC M MLC
8.0 ± 0.7a 7.9 ± 0.8a 7.4 ± 0.6b 7.4 ± 0.8b 7.6 ± 0.6ab 7.8 ± 0.6a
7.5 ± 0.9a 7.6 ± 0.6a 7.6 ± 0.5a 7.6 ± 0.5a 7.6 ± 0.5a 7.6 ± 0.6a
7.7 ± 0.7a 7.7 ± 0.7a 7.7 ± 0.5a 7.7 ± 0.4a 7.8 ± 0.5a 7.6 ± 0.5a
7.3 ± 0.8b 7.3 ± 0.7b 7.4 ± 0.8ab 7.5 ± 0.6a 7.3 ± 0.7b 7.4 ± 0.5ab
7.6 ± 0.7a 7.5 ± 0.6a 7.2 ± 0.5b 7.2 ± 0.4b 7.4 ± 0.5ab 7.4 ± 0.5ab
Day 60 W WLC D DLC M MLC
7.4 ± 0.6b 7.3 ± 0.6bc 6.9 ± 0.3d 7.0 ± 0.5cd 7.1 ± 0.5c 7.2 ± 0.5c
7.4 ± 0.6ab 7.5 ± 0.6a 7.0 ± 0.6b 7.0 ± 0.4b 7.1 ± 0.6b 7.2 ± 0.5b
6.2 ± 0.5c 6.2 ± 0.5bc 6.5 ± 0.4b 6.5 ± 0.3b 6.4 ± 0.5b 6.3 ± 0.5bc
6.7 ± 0.2c 6.8 ± 0.4c 6.5 ± 0.4d 6.3 ± 0.5d 6.3 ± 0.3d 6.4 ± 0.5d
6.2 ± 0.5c 6.3 ± 0.6c 6.2 ± 0.4c 6.2 ± 0.3c 6.2 ± 0.4c 6.2 ± 0.6c
Means in the same column with the same lowercase letters indicate no significant difference (P > 0.05). W=white chocolate, WLC=white chocolate containing immobilized L. casei 01, D=dark chocolate, DLC=dark chocolate containing immobilized L. casei 01, M=milk chocolate and MLC=milk chocolate containing immobilized L. casei 01.
colon. In addition, Mandal et al. (2013) found that mice which were fed with milk chocolate containing L. casei NCDC 298 and prebiotic inulin were found to have significant increase of colon lactobacilli and decrease of coliforms. Therefore, the findings in this study indicated that chocolates were the excellent carriers for protecting the probiotics from the gastrointestinal environments, which result in the remaining number of survived probiotic cells at levels sufficient to be beneficial for human's health.
casei 01 cells were initially inoculated into simulated stomach compartment at the concentrations between 6.5 ± 0.5 and 6.8 ± 0.5×107 CFU/ml. The number of survival L. casei 01 cells gradually declined when the incubation time increased (P≤0.05), whereby the free cells were less capable of surviving the gastric environment of the simulated stomach and they were eventually eliminated after 1.5 h of incubation. On the contrary, this study showed that the immobilized L. casei 01 in different chocolates were found to have high levels of survivability after being exposed to the gastric environment of simulated stomach for 2 h. These immobilized L. casei 01 cells were subsequently incubated in the simulated small intestinal fluid for another 4 h and they still remained to be viable at ~2 log CFU/ml. This may suggested that either maltodextrin plus skim milk and/or chocolates were beneficial materials for protecting the probiotic cells from the adverse environments (Desmond, Ross, O’Callaghan, Fitzgerald, & Stanton, 2002; Khoramnia et al., 2011; Kingwatee et al., 2015). Several researchers indicated that the survival of probiotic bacteria under in vitro gastrointestinal conditions tended to increase when they were combined with chocolates (da Silva et al., 2012; Possemiers et al., 2010). Yonejima et al. (2015) reported that Lactobacillus brevis ssp. coagulans could survive in gastric juice (pH 2.5) better when being combined with chocolate rather than with commercial beverages (i.e. fruit juices and fermented milks). Correspondingly, Maillard and Landuyt (2008) indicated that chocolates were the great carriers for probiotics Lactobacillus Rosell-52 and Bifidobacterium Rosell-175 during the evaluation using a dynamic human gut model (SHIME reactor). Moreover, they also observed that the probiotic-chocolates could apparently enhanced the growth of beneficial bacteria (i.e. lactobacilli and bifidobacteria) and suppressed some harmful pathogens (i.e. fecal coliforms and clostridia) in the ascending, transverse and descending compartments of the simulated
4. Conclusion In this study, white, milk, and dark chocolates were found to be appropriate carriers for maintaining the number of probiotics during storage and passing through the simulated gastrointestinal conditions. In particular, L. casei 01 cells were found to be able to endure such conditions and survive better than L. acidophilus LA5 cells, and dark chocolate could preserve high levels of viable probiotic cells in all testing conditions. Moreover, 4 °C-storage was found to be suitable for preserving probiotic viability, regardless of chocolate types and probiotic strains. In addition, supplementation of spray-dried probiotics did not affect the sensory attributes of the products. Therefore, these results indicate that all chocolate types have potential to be used as protector matrices for probiotics and may be developed into other novel foods in the future.
Conflict of interest statement No conflicts of interest exist in this study.
Table 5 Viable Lactobacillus casei 01 cells in different chocolate matrices during incubation under anaerobic environment at 37 °C for 6 h in simulated stomach and intestine tract. Treatment conditions
Numbers of survival cells (CFU/ml) during incubation at 37 °C Simulated stomach environment Initial stage
Free cells WLC DLC MLC
aA
Simulated small intestinal environment
0.5 h 7
6.8 ± 0.5 ×10 6.6 ± 0.6aA×107 6.5 ± 0.4aA×107 6.5 ± 0.5aA×107
1h bB
5
4.8 ± 1.1 ×10 6.4 ± 0.7aB×106 6.4 ± 0.7aB×106 6.4 ± 0.8aB×106
1.5 h bC
3
2h bD
9.2 ± 0.6 ×10 7.3 ± 0.5aC×104 7.4 ± 0.8aC×104 7.6 ± 0.6aC×104
2
3.8 ± 0.7 ×10 5.6 ± 0.6aD×103 5.7 ± 0.5aD×103 5.6 ± 0.4aD×103
bE
nd 1.9 ± 0.7aE×103 2.0 ± 0.5aE×103 2.1 ± 0.6aE×103
4h bE
nd 5.9 ± 0.7aF×102 5.5 ± 0.5aF×102 5.7 ± 0.6aF×102
6h ndbE 3.3 ± 0.5aG×102 3.7 ± 0.6aG×102 3.1 ± 0.7aG×102
Means in the same column with the same lowercase letters, and means in the same row with the same uppercase letters indicate no significant difference (P > 0.05). WLC=white chocolate containing immobilized L. casei 01, DLC=dark chocolate containing immobilized L. casei 01 and MLC=milk chocolate containing immobilized L. casei 01. nd=not detected.
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Hii, C. L., Law, C. L., Suzannah, S., Miswani, S., & Cloke, M. (2009). Polyphenols in cocoa (Theobroma cacao L.). Asian. Journal of Food and Agro Industries, 2, 702–722. Khoramnia, A., Abdullah, N., Liew, S. L., Sieo, C. C., Ramasamy, K., & Ho, Y. W. (2011). Enhancement of viability of a probiotic Lactobacillus strain for poultry during freeze-drying and storage using the response surface methodology. Animal Science Journal, 82, 127–135. Kingwatee, N., Apichartsrangkoon, A., Chaikham, P., Worametrachanon, S., Techarung, J., & Pankasemsuk, T. (2015). Spray drying Lactobacillus casei 01 in lychee juice varied carrier materials. LWT – Food Science and Technology, 62, 847–853. Laličić-Petronijević, J., Popov-Raljić, J., Obradović, D., Radulović, Z., Paunović, D., Petrušić, M., & Pezo, L. (2015). Viability of probiotic strains Lactobacillus acidophilus NCFM® and Bifidobacterium lactis HN019 and their impact on sensory and rheological properties of milk and dark chocolates during storage for 180 days. Journal of Functional Foods, 15, 541–550. Maillard, M., & Landuyt, A. (2008). Chocolate: an ideal carrier for probiotics. Agro Food Industry Hi-Tech, 19, 13–15. Mandal, S., Hati, S., Puniya, A. K., Singh, R., & Singh, K. (2013). Development of symbiotic milk chocolate using encapsulated Lactobacillus casei NCDC 298. Journal of Food Processing and Preservation, 37, 1031–1037. Nebesny, E., & Zyzelewicz, D. (2006). Properties of chocolates enriched with viable lactic acid bacteria. Deutsche Lebensmittel-Rundschau, 102, 27–32. Nebesny, E., Zyzelewicz, D., Motyl, I., & Libudzisz, Z. (2005). Properties of sucrose-free chocolates enriched with viable lactic acid bacteria. European Food Research and Technology, 220, 358–362. Oliveira, C. D. S., Maciel, L. F., Miranda, M. S., & Bispo, E. D. S. (2011). Phenolic compounds, flavonoids and antioxidant activity in different cocoa samples from organic and conventional cultivation. British Food Journal, 113, 1094–1102. Oomen, A. G., Rompelberg, C. J. M., Bruil, M. A., Dobbe, C. J. G., Pereboom, D. P. K. H., & Sips, A. J. A. M. (2003). Development of an in vitro digestion model for estimating the bioaccessibility of soil contaminants. Archives of Environmental Contamination and Toxicology, 44, 281–287. Patel, A., Falck, P., Shah, N., Immerzeel, P., Adlercreutz, P., Stålbrand, H. Karlsson, E. N. (2013). Evidence for xylooligosaccharide utilization in Weissella strains isolated from Indian fermented foods and vegetables. FEMS Microbiology Letters, 346, 20–28. Possemiers, S., Marzorati, M., Verstraete, W., & Van de Wiele, T. (2010). Bacteria and chocolate: a successful combination for probiotic delivery. International Journal of Food Microbiology, 141, 97–103. Riveros, B., Ferrer, J., & Bórquez, R. (2009). Spray drying of a vaginal probiotic strain of Lactobacillus acidophilus. Drying Technology: An International Journal, 27, 123–132. Rouhi, M., Mohammadi, R., Mortazavian, A. M., & Sarlak, Z. (2015). Combined effects of replacement of sucrose with D-tagatose and addition of different probiotic strains on quality characteristics of chocolate milk. Dairy Science & Technology, 95, 115–133. Sanders, M. E., Guarner, F., Guerrant, R., Holt, P. R., Quigley, E. M., Sartor, R. B. Mayer, E. A. (2013). An update on the use and investigation of probiotics in health and disease. Gut, 62, 787–796. Šarić, G., Marković, K., Major, N., Krpan, M., Uršulin-Trstenjak, N., Hruškar, M., & Vahčić, N. (2012). Changes of antioxidant activity and phenolic content in acacia and multifloral honey during storage. Food Technology and Biotechnology, 50, 434–441. Sekhon, B. S., & Jairath, S. (2010). Prebiotics, probiotics and synbiotics: an overview. Journal of Pharmaceutical Education and Research, 1, 13–36. de Silva, A. S., Honjoya, E. R., Inay, O. M., de Rezende Costa, M., de Souza, de Santana, E. H. W. Aragon-Alegro, L. C. (2012). Viability of Lactobacillus casei in chocolate flan and its survival to simulated gastrointestinal conditions. Semina: Ciências Agrárias, 33, 3163–3170. Todorovic, V., Redovnikovic, I. R., Todorovic, Z., Jankovic, G., Dodevska, M., & Sobajic, S. (2015). Polyphenols, methylxanthines, and antioxidant capacity of chocolates produced in Serbia. Journal of Food Composition and Analysis, 41, 137–143. Yonejima, Y., Hisa, K., Kawaguchi, M., Ashitani, H., Koyama, T., Usamikrank, Y. Ogawa, J. (2015). Lactic acid bacteria-containing chocolate as a practical probiotic product with increased acid tolerance. Biocatalysis and Agricultural Biotechnology, 4, 773–777. Żyżelewicz, D., Nebesny, E., Motyl, I., & Libudzisz, Z. (2010). Effect of milk chocolate supplementation with lyophilised Lactobacillus cells on its attributes. Czech Journal of Food Sciences, 28, 392–406.
Acknowledgements This research was financially supported by Phranakhon Si Ayutthaya Rajabhat University and Thailand Research Fund (Grant no. TRG5880002). References Andújar, I., Recio, M. C., Giner, R. M., & Ríos, J. L. (2012). Cocoa polyphenols and their potential benefits for human health. Oxidative Medicine and Cellular Longevity. http://dx.doi.org/10.1155/2012/906252. Benzie, I. F. F., & Stain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power; the FRAP assay. Analytical Biochemistry, 239, 70–76. Bianchi, F., Rossi, E. A., Sakamoto, I. K., Adorno, M. A. T., Van de Wiele, T., & Sivieri, K. (2014). Beneficial effects of fermented vegetal beverages on human gastrointestinal microbial ecosystem in a simulator. Food Research International, 64, 43–52. Chaikham, P. (2015). Stability of probiotics encapsulated with Thai herbal extracts in fruit juices and yoghurt during refrigerated storage. Food Bioscience, 12, 61–66. Chaikham, P., & Apichartsrangkoon, A. (2012). Comparison of dynamic viscoelastic and physicochemical properties of pressurised and pasteurised longan juice with xanthan addition. Food Chemistry, 134, 2194–2200. Chaikham, P., & Apichartsrangkoon, A. (2013). Effects of encapsulated Lactobacillus acidophilus along with pasteurized longan juice on the colon microbiota residing in a dynamic simulator of the human intestinal microbial ecosystem. Applied Microbiology and Biotechnology, 98, 485–495. Chaikham, P., Apichartsrangkoon, A., Jirarattanarangsri, W., & Van de Wiele, T. (2012). Influence of encapsulated probiotics combined with pressurized longan juice on colon microflora and their metabolic activities on the exposure to simulated dynamic gastrointestinal tract. Food Research International, 49, 133–142. Chaikham, P., Apichartsrangkoon, A., George, T., & Jirarattanarangsri, W. (2013). Efficacy of polymer coating of probiotic beads suspended in pressurized and pasteurized longan juices on the exposure to simulated gastrointestinal environment. International Journal of Food Science and Nutrition, 64, 862–869. Chaikham, P., Apichartsrangkoon, A., Worametrachanon, S., Supraditareporn, W., Chokiatirote, E., & Van der Wiele, T. (2013). Activities of free and encapsulated Lactobacillus acidophilus LA5 or Lactobacillus casei 01 in processed longan juices on exposure to simulated gastrointestinal tract. Journal of the Science of Food and Agriculture, 93, 2229–2238. Champagne, C. P., Raymond, Y., Guertin, N., & Bélanger, G. (2015). Effects of storage conditions, microencapsulation and inclusion in chocolate particles on the stability of probiotic bacteria in ice cream. International Dairy Journal, 47, 109–117. Desmond, C., Ross, R. P., O’Callaghan, E., Fitzgerald, G., & Stanton, C. (2002). Improved survival of Lactobacillus paracasei NFBC 338 in spray-dried powders containing gum acacia. Journal of Applied Microbiology, 93, 1003–1011. Ding, W. K., & Shah, N. P. (2008). Survival of free and microencapsulated probiotic bacteria in orange and apple juices. International Food Research Journal, 15, 219–232. Ehsannia, S., & Sanjabi, M. R. (2016). Quality characterization of processed cheese inoculated by Bacillus coagulans during cold storage: compositional and sensorial attributes and probiotic microorganism viability. Journal of Food Processing and Preservation, 40, 667–674. Erdem, Ö., Gültekin-Özgüven, M., Berktas, I., Erşan, S., Tuna, H. E., Karadağ, A. Cutting, S. M. (2014). Development of a novel synbiotic dark chocolate enriched with Bacillus indicus HU36, maltodextrin and lemon fiber: Optimization by response surface methodology. LWT – Food Science and Technology, 56, 187–193. FAO/WHO. (2001). Report of a joint FAO/WHO expert consultation one valuation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Argentina: Cordoba 1–4 October 2001. Fooks, L. J., & Gibson, G. R. (2002). Probiotics as modulators of the gut flora. British Journal of Nutrition, 88, 39–49. Fuller, R. (Ed.). (1992). Probiotics: the scientific basis . London: Chapman Hall. Gadhiya, D., Patel, A., & Prajapati, J. B. (2015). Current trend and future prospective of functional probiotic milk chocolates and related products – a review. Czech Journal of Food Sciences, 33, 295–301.
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Survival of immobilized probiotics in chocolate during storage and with an in vitro gastrointestinal model ⁎
Varongsiri Kemsawasda, Pittaya Chaikhamb, , Paweena Rattanasenab a b
Institute of Nutrition, Mahidol University, Nakorn Pathom Campus, Nakorn Pathom 73170, Thailand Faculty of Science and Technology, Phranakhon Si Ayutthaya Rajabhat University, Phranakhon Si Ayutthaya 13000, Thailand
A R T I C L E I N F O
A BS T RAC T
Keywords: Lactobacillus casei 01 Lactobacillus acidophilus LA5 White chocolate Milk chocolate Dark chocolate
Probiotics are the bacteria that can provide health benefits to the consumers and they are suitable to be added to a variety of foods. In this research, viability of immobilized potential probiotics, including Lactobacillus casei 01 and Lactobacillus acidophilus LA5, in three different types of chocolate (white, milk and dark) were studied during storage and with an in vitro gastrointestinal model. The sensory attributes of probiotic-chocolates were also evaluated. Both cultures were found to be viable up to 60 days of storage at 4 °C ( > 6 log CFU/g) which were sufficient to potentially provide health benefits to the consumer. As a carrier, chocolates also may protect probiotics in both the stomach and small intestine environments. The overall sensory results suggested that the probiotic powders had no significant effect on sensory attributes. After 60 days storage, significant decreases of the overall liking scores in all the chocolate samples were observed. Therefore, this study may be useful for the future development of probiotic-supplemented chocolates as foods with health benefits for the consumers.
1. Introduction Presently, the worldwide market of functional food products with health benefits, especially those supplemented with probiotics, has been promptly expanding. In accordance with a joint report of Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO), the definition of probiotics is “live microorganisms which, when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2001). Sufficient amounts of viable and active probiotics should successfully reach the intestines for improving the microbial balance in the intestine environments, and this is greatly beneficial to the consumers (Chaikham, Apichartsrangkoon, Jirarattanarangsri & Van de Wiele, 2012; Fooks & Gibson, 2002; Fuller, 1992; Sekhon & Jairath, 2010). The health benefits of probiotics have previously been reported viz. preventing the infectious diarrhea, lowering the levels of blood cholesterol, controlling the intestinal infections, reducing the symptoms of lactose intolerance, enhancing some types of immune responses and functioning as antitumor/anticancer agents (Fuller, 1992; Patel et al., 2013; Sanders et al., 2013). Probiotics can be found in fermented dairy products, particularly yogurts and cheeses, and also some non-dairy products, which are currently receiving attention by people in all age groups. Some probiotics, especially Lactobacillus casei 01 and Lactobacillus acidophilus LA5, were suggested to have positive effects on human colon
⁎
microbiota because they could enhance short-chain fatty acids formation and increase beneficial colon bacteria, including lactobacilli and bifidobacteria (Chaikham et al., 2012). At the same time, certain harmful bacteria, such as fecal coliforms and clostridia, were noticeably suppressed (Chaikham & Apichartsrangkoon, 2013; Chaikham et al., 2012). Similar outcomes were observed by Bianchi et al. (2014) who investigated the fermented vegetal beverages (synbiotic aqueous extracts of quinoa and soy) enriched with L. casei 01. Therefore, it is interesting to evaluate the supplementation of both probiotics in other novel food products with health benefits. As food matrix, chocolate has been proposed to be supplemented with probiotics and it may be developed into different future products (Gadhiya, Patel, & Prajapati, 2015). Notably, chocolate was found to contain various bioactive compounds, such as polyphenols and flavonoids (i.e. catechin, epicatechin and procyanidin), both of which possessing high levels of antioxidant activities (Todorovic et al., 2015). Possemiers, Marzorati, Verstraete, and Van de Wiele (2010), Mandal, Hati, Puniya, Singh, and Singh (2013) and Laličić-Petronijević et al. (2015) reported that chocolate was an ideal carrier for protecting the probiotic cells during shelf storage and for enhancing their ability to tolerate the adverse environments of gastrointestinal tract. Moreover, several factors, including water activity (aw), oxygen tension and temperature were found to play the essential roles in forming probiotic-chocolates (Kingwatee et al., 2015). There are a variety of techniques, for example, encapsulation by spray-drying, which have been used for extending the
Corresponding author. E-mail address: [email protected] (P. Chaikham).
http://dx.doi.org/10.1016/j.fbio.2016.09.001 Received 25 February 2016; Received in revised form 10 September 2016; Accepted 14 September 2016 Available online 15 September 2016 2212-4292/ © 2016 Elsevier Ltd. All rights reserved.
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milk chocolate containing 10% (w/w) cocoa powder (Jiangsu Linzhi Shanyang Group Co., Ltd., Jiangsu, China), 49% (w/w) cocoa butter, 30% (w/w) sucrose, 10% (w/w) skim milk powder or probioticsupplemented skim milk powders, 0.9% (w/w) soy lecithin and 0.1% (w/w) ethyl vanillin; and (iii) dark chocolate containing 50% (w/w) cocoa powder, 9% (w/w) cocoa butter, 30% (w/w) sucrose, 10% (w/w) skim milk powder or probiotic-supplemented skim milk powders, 0.9% (w/w) soy lecithin and 0.1% (w/w) ethyl vanillin. All ingredients, except probiotic-supplemented skim milk powders, were mixed, heated, thoroughly stirred, immediately cooled down to ~35 °C and finally inoculated with 10% (w/w) probiotic-supplemented skim milk powders. After that, the chocolates and probiotic-chocolate mixtures were transferred to 2×2×0.5 cm plastic containers and kept in a refrigerator for 20 min. The final products were packed in aluminum foil with 0.2 mm thickness (Diamond, Zhangjiang, China) and then stored at 4 or 25 °C for 60 days.
viability and activity of these microbes in products during storage (Riveros, Ferrer, & Bórquez, 2009). However, the knowledge of supplementing the spray-dried probiotic cells into chocolate is still quite limited. Therefore, this research aimed to evaluate the survival rates of immobilized probiotics, L. casei 01 and L. acidophilus LA5 that were incorporated in different chocolate matrices (white, milk and dark) during storage and with an in vitro gastrointestinal model. The sensorial attributions of these probiotic-supplemented chocolates were also evaluated using 50 untrained volunteers. 2. Materials and methods 2.1. Activation and immobilization of probiotic cultures Briefly, freeze-dried L. casei 01 (Lot NO. 3230647) and L. acidophilus LA5 (Lot NO. 3251532) which obtained from Chr. Hansen (Hørsholm, Denmark) were rehydrated, activated and anaerobically incubated in de Man Rogosa and Sharp (MRS) broth (Hi-media, Mumbai, India) at 37 °C for 16 and 18 h, respectively, to reach their stationary phases (Chaikham, Apichartsrangkoon, George, & Jirarattanarangsri, 2013). The activated cells were then harvested by centrifugation at 4000g and 4 °C for 20 min (Centrifuge model Rotina 46 R, Tuttlingen, Germany), followed by washing twice using sterile 0.85% (w/v) saline water (Hi-media). After that, the cell pellets were diluted using deionized water (RCI Labscan, Bangkok, Thailand) to adjust the bacterial population at ~1012 CFU/ml. The skim milk (CP Meiji, Saraburi, Thailand) was processed using a ultra-high temperature (UHT) method and mixed with 20% (w/v) maltodextrin (10.5 DE: Ingredient Center, Bangkok, Thailand), then heated at 80 °C for 10 min before being cooled down to 35 °C. Both of probiotic cultures were separately inoculated into the skim milk mixture to obtain the cell concentration of ~1010 CFU/ml and subsequently dried into powders using a spray-dryer (JCM Engineering Concept, Bangkok, Thailand). Drying conditions for this experiment was set at 30 °C of feeding temperature, 1 l/h of feeding rate, 15 psi of atomizing pressure, and 170 °C of hot-air-inlet temperature to generate 90 °C outlet temperature. The powders were vacuum-sealed in laminated bags (nylon plus polyethylene: Siam Pack, Chiang Mai, Thailand) and stored in a refrigerator overnight before preparation into the probiotic-supplemented chocolates. The plate counts of survival L. casei 01 and L. acidophilus LA5 cells after spray-drying were 6.6 ± 1.2×1010 and 8.1 ± 1.6×1010 CFU/g, respectively.
2.4. Determination of antioxidant properties of chocolates 2.4.1. Extraction of samples The chocolates were extracted according to the method of Todorovic et al. (2015). First, chocolate samples were ground using a coffee grinder (Bosch, Farmington Hills, MI, USA). Two g of each ground sample were extracted three times using 10 ml n-hexane (Merck, Darmstadt, Germany) for eliminating the lipids from the samples. The defatted samples were then air-dried overnight to evaporate the n-hexane residue and then extracted twice using 5 ml of a mixture of 70% (v/v) acetone (Merck), 29.8% (v/v) distilled water (RCI Labscan) and 0.2% (v/v) acetic acid (Merck) for 30 min using a ultrasonic bath (FALC, Treviglio, Italy). After that, the mixtures were centrifuged at 4000g for 15 min. The supernatants of the extracts were used for determinations of bioactive components and antioxidant activities by the following methods. 2.4.2. Total polyphenols The levels of total polyphenol contents of the chocolate extracts (Section 2.4.1) were spectrophotometrically determined using FolinCiocalteu method (Chaikham & Apichartsrangkoon, 2012). Each supernatants of the extracts (0.5 ml) were thoroughly mixed with 10 ml of absolute ethanol (Merck), 2.5 ml of 10% Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA), and 2 ml of saturated sodium carbonate solution (Ajax, Sydney, Australia), and finally incubated at 25 °C for 2 h. The mixtures were measured for absorbance at 765 nm using a Perkin Elmer UV WINLAB spectrophotometer (Perkin Elmer, Waltham, MA, USA). Gallic acid (Sigma-Aldrich) was used as standard reagent. The regression line between absorbance (y) and gallic acid content (x) was y=0.006x+0.018. The results were expressed as mg gallic acid equivalent per 100 g of sample (mg GAE/ 100 g).
2.2. Scanning electron microscopic (SEM) images A scanning electron microscope (Hitachi S-3000 N, Hitachi HighTechnologies Co., Ltd., Tokyo, Japan) was used to examine the microstructure characteristics of both immobilized probiotics. The powdered probiotics were placed on to the SEM sample holders and coated with 10 nm of gold particles using a Hitachi E1010 ion sputter (Hitachi Science Systems Co., Ltd., Tokyo, Japan). The external structures of gold-coated samples were then examined at an accelerating voltage of 15 kV (Kingwatee et al., 2015).
2.4.3. Total flavonoids The levels of total flavonoids of the chocolate extracts (Section 2.4.1) were determined following the modified method of Šarić et al. (2012). In brief, 2 ml of each extracts were thoroughly mixed with 4 ml of deionized water and 2 ml of 5% (w/v) sodium nitrite (Ajax) and allowed to react for 6 min. Two ml of 10% (w/v) aluminum chloride (Sigma-Aldrich) were added to each mixtures followed by shaking for 6 min and adding of 12 ml of 1 M sodium hydroxide (Merck). The mixtures were spectrophotometrically measured for absorbance at 510 nm. Catechin (Sigma-Aldrich) was used as standard reagent. The standard curve was plotted and the regression line between absorbance (y) and catechin content (x) was y=0.003x+0.120. The results were expressed as mg catechin equivalent per 100 g sample (mg CE/100 g).
2.3. Preparation of probiotic-chocolates Three different types of chocolate viz. white, milk and dark chocolates were mixed with probiotic powders (Section 2.1) following the method of Laličić-Petronijević et al. (2015) with some modifications. These chocolates were prepared as following: (i) white chocolate containing 59% (w/w) cocoa butter (Foodchem International Corporation, Shanghai, China), 30% (w/w) sucrose (Mitr Phol, Bangkok, Thailand), 10% (w/w) skim milk powder or probioticsupplemented skim milk powders, 0.9% (w/w) soy lecithin (Modernist Pantry, Portsmouth, NH, USA) and 0.1% (w/w) ethyl vanillin (Chemipan Corporation Co., Ltd., Bangkok, Thailand); (ii) 38
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5 g of probiotic-supplemented chocolates were mixed with 25 ml of gastric solution (pH 1.4, 37 °C) in sterile glass bottles and then anaerobically incubated at 37 °C. After 2 h of incubation, 25 ml of simulated small intestinal solution (a mixture of 15 ml of duodenal solution and 10 ml of bile solution, pH 8.1, 37 °C) were added to the prepared bottles and they were then incubated for 4 h. For enumeration of survival cells, 1 ml of each incubated fluids was taken to be diluted with 9 ml of 0.2 M sterile phosphate buffer (pH 7; Merck) and used for plate count assay for viable cells using MRS agar. The results were expressed as CFU/ml.
2.4.4. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity The levels of DPPH radical scavenging activity of the chocolate extracts (Section 2.4.1) were determined by the method of Chaikham and Apichartsrangkoon (2012) with some modifications. The extract (0.5 ml) was well-mixed with 4.5 ml of 0.5 μM DPPH (Fluka, Buchs, Switzerland) solution (diluted using methanol) (Merck), then incubated at room temperature for 1 h and finally measured for the absorbance at 517 nm. Trolox (Sigma-Aldrich) was used as standard reagent and the DPPH radical inhibition was calculated from a calibration curve plotted between absorbance (y) and Trolox content (x). The regression line was y=147.1x–11.0. The results were expressed as μM Trolox equivalent/g sample (μM TE/g).
2.8. Statistical data analysis All data were shown as the averages of six replicates (n=6) with standard deviations. Analysis of variance (ANOVA) was carried out using a SPSS program version 15.0 (SPSS Inc., Chicago, IL, USA). Duncan's multiple range tests were used to examine the significant differences among the means of treatments (P≤0.05).
2.4.5. Ferric reducing antioxidant power Ferric reducing antioxidant power (FRAP) values of the chocolate extracts were assessed following the modified method of Benzie and Stain (1996). Two ml of each extracts (Section 2.4.1) were mixed with 8 ml of deionized water and 3 ml of FRAP reagent [a mixture of 300 mM sodium acetate buffer (pH 3.6; Sigma-Aldrich)], 10 mM 2,4,6tripyridyls-triazine solution (Sigma-Aldrich) and 20 mM ferric chloride hexahydrate solution (Merck) at a ratio of 10:1:1 and then incubated at 37 °C for 1 h in the dark. The mixtures were measured for absorbance at 593 nm. The FRAP values were calculated from a calibration curve (y=1.35x−0.03; where y is absorbance and x is Trolox content) and expressed as μM TE/g.
3. Results and discussion 3.1. Bioactive components and antioxidant capacity of chocolates Several studies have reported that cocoa possessed high levels of polyphenols and flavonoids, which were shown to have beneficial effects towards human's health, in particular prevention of cardiovascular disease and cancer (Andújar, Recio, Giner, & Ríos, 2012; Gadhiya et al., 2015; Hii, Law, Suzannah, Miswani, & Cloke, 2009). In this study, the contents of total polyphenols and flavonoids in chocolates were highly varied depending on the types of chocolates, as shown in Table 2. The levels of these compounds were significantly high (P≤0.05) in milk and dark chocolates, but much less in white chocolate. The greater quantity of bioactive compounds in chocolates was directly correlated to the bigger portion of cocoa powder in the recipes, whereby the white, milk and dark chocolates contained cocoa powder at 0%, 10% and 50%, respectively. Therefore, the dark chocolate was found to have the highest contents of both component groups (P≤0.05). The antioxidant activities in dark chocolates (DPPH inhibition and FRAP values) were shown to have strong correlation with the contents of total polyphenols and flavonoids (R2=0.8–0.9). These results were similar to the finding of Oliveira, Maciel, Miranda, and Bispo (2011), who showed that the organic cocoa contained high levels of total phenolic, flavonoids, and antioxidant activity (IC50 value) at 196–285 mg GAE/g, 155–274 mg CE/g and 4.2–7.7 µg/ml, respectively, and they also found the positive relationship between total polyphenols and DPPH radical scavenging activity. These results may suggest that the higher contents of total phenols found in chocolates, the greater antioxidant activity the chocolates may contain.
2.5. Stability of probiotics in chocolate during storage To quantify the survival rates of probiotics in all chocolate samples stored at 4 or 25 °C for 60 days, the samples were taken every 10 days and melted at 37 °C for 10 min using a Biohazard Safety Cabinet (Esco Technologies, Inc., Horsham, PA, USA). After that, the samples were diluted with 0.1% (w/v) sterile peptone water (Hi-media) at different concentrations before pour-plating on MRS agar (Hi-media). All plates were anaerobically incubated at 37 °C for 48–72 h in anaerobic system (Anaero Gen™; Oxoid, Wesel, Germany). The number of colonies were counted and expressed as CFU/g. 2.6. Sensory evaluation Sensorial attributions of chocolates and probiotic-supplemented chocolates were evaluated for appearance, color, flavor, texture, and overall acceptability. Fifty untrained panelists who like chocolate (aged 18–35 years) including academic staffs and students from Phranakhon Si Ayutthaya Rajabhat University, Phranakhon Si Ayutthaya province, Thailand were assigned to score the sensorial attributes using the evaluation form of 9-point hedonic scale (9=like extremely, 5=neither like nor dislike, 1=dislike extremely). All chocolates were identified using 3-digit random numbers and placed on plastic dishes before serving. The participants were instructed to rinse their mouths with water every time before tasting the sample (Laličić-Petronijević et al., 2015).
3.2. Microstructures of probiotic powers Fig. 1 shows the microstructures of L. casei 01 and L. acidophilus LA5 powders after spray-drying using skim milk and maltodextrin as supporting materials. There were the clear differences between the size of distribution and the morphology of L. casei 01 (Fig. 1a) and L. acidophilus LA5 (Fig. 1b) powders. As shown by the SEM images, the average sizes of L. casei 01 and L. acidophilus LA5 microcapsules were 8.4–45.9 and 11.6–69.7 µm, respectively. L. casei 01 microcapsules were found to be smaller and had smoother surface, but L. acidophilus LA5 microcapsules were more spherical and had rougher surface. Regarding to the sizes of the probiotic, L. casei 01 cells were short with rod-shape (length ~1 µm), and they were often found in the form of short chains comprising three or four cells; nonetheless, L. acidophilus cells were long with rod-shape (length ~3 µm), and they were often found in pairs or chains of various lengths (Chaikham, Apichartsrangkoon, George et al., 2013). Moreover, Kingwatee et al.
2.7. Survival rates of probiotic cells under in vitro stomach and small intestine conditions The simulated gastric, duodenal and bile solutions were prepared following the procedure of Oomen et al. (2003) (Table 1), and the in vitro gastrointestinal experiments were carried out according to the methods of Chaikham, Apichartsrangkoon, and Worametrachanon et al. (2013) with some modifications. The fresh cells (Section 2.1) were aseptically inoculated into sterile distill water (pH 1.4, 37 °C) at the concentration of ~108 CFU/ml. After that, 5 ml of diluted cells or 39
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Table 1 Compositions of synthetic gastric and small intestinal fluids used for the in vitro digestion (Oomen et al., 2003). Reagents**
Gastric fluid (pH 1.4)
Inorganic substances
NaCl NaH2PO4 KCl CaCl2 NH4Cl HCl (conc.)
2752 mg 266 mg 824 mg 400 mg 306 mg 6.5 ml
NaCl NaHCO3 NaH2PO4 KCl MgCl2 HCl (conc.)
7012 mg 5607 mg 80 mg 564 mg 50 mg 180 µl
NaCl NaHCO3 KCl HCl (conc.)
5259 mg 5785 mg 376 mg 180 µl
Organic substances
Glucose Glucuronic acid Urea Glucosamine hydrochloride
650 mg 20 mg 85 mg 330 mg
Urea
100 mg
Urea
250 mg
Other components
BSA Mucin Pepsin
1000 mg 3000 mg 2500 mg
CaCl2 BSA Pancreatin Lipase
200 mg 1000 mg 9000 mg 1500 mg
CaCl2 BSA Bile
222 mg 1800 mg 30,000 mg
Duodenal fluid (pH 8.1)
Bile fluid (pH 8.1)
** Sodium chloride (NaCl), sodium phosphate monobasic (NaH2PO4), potassium chloride (KCl), ammonium chloride (NH4Cl), hydrochloric acid (HCl conc.), sodium bicarbonate (NaHCO3), magnesium chloride (MgCl2), calcium chloride (CaCl2) and bovine bile were purchased from Sigma-Aldrich. D-Glucose, glucuronic acid, urea, glucosamine hydrochloride, bovine serum albumin (BSA), mucin from bovine submaxillary gland (CAS NO. 84195-52-8, 5 U/mg), pepsin from porcine gastric mucosa (CAS NO. 107185, 0.7 U/mg), pancreatin from porcine pancreas (CAS NO. 107130, 6 U/mg) and lipase from Chromobacterium viscosum (CAS NO. 437707, 2500 U/mg) were obtained from Merck.
3.3. Survival of immobilized probiotics in chocolates during storage
Table 2 Bioactive compounds and antioxidant activities of different chocolate types. Chocolate types
White chocolate Dark chocolate Milk chocolate
Bioactive components
The matrices of white, milk and dark chocolates were supplemented with the excessive amounts of spry-dried L. casei 01 or L. acidophilus LA5 cells. All chocolate samples were stored at 4 and 25 °C for 60 days and then examined for the viable probiotic population (Table 3). The results showed that survivability of probiotics was dependent on the strain of probiotics, type of chocolate, and storage temperature and duration. Even though the initial levels of L. casei 01 and L. acidophilus LA5 in chocolates were approximately 8 log CFU/g, the colony counting results showed that the survival rate of L. casei 01 was higher than that of L. acidophilus LA5 (P≤0.05), regardless of the types of chocolate. Notably, L. casei 01 was found to be able to endure the storage conditions better than L. acidophilus LA5. These results could be also supported by the number of cell loss, which indicating that L. casei 01 exhibited cell loss at levels significantly lower than L. acidophilus LA5 (P≤0.05). Similarly, Chaikham, Apichartsrangkoon, George et al. (2013) and Chaikham (2015) found that L. casei 01 cells were only slightly altered during refrigerating storage in fruit juices and relatively more stable than L. acidophilus LA5. Despite that, LaličićPetronijević et al. (2015) showed that probiotic L. acidophilus NCFM®
Antioxidant activities
Total polyphenols (mg GAE/100 g)
Total flavonoids (mg CE/100 g)
DPPH (µM TE/ g)
FRAP value (µM TE/g)
tracec
tracec
0.6 ± 0.1c
1.2 ± 0.2c
364 ± 2a
49 ± 4a
79 ± 3a
104 ± 4a
94 ± 3b
16 ± 2b
21 ± 2b
48 ± 3b
Means in the same column with the same lowercase letters indicate no significant difference (P > 0.05).
(2015) indicated that different supporting materials could have different effects on the surface morphology, size, solubility and bulk density of probiotic-fruit juice powder.
Fig. 1. SEM images of (a) Lactobacillus casei 01 and (b) Lactobacillus acidophilus LA5 powders.
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Table 3 Stability of probiotics in chocolates during storage at 4 and 25 °C for 60 days. Samples
Numbers of survival cells during refrigerated storage (log CFU/g) Day 0
Cell loss(log CFUs)
Day 10
Day 20
Day 30
Day 40
Day 50
Day 60
Storage at 4 °C WLC 6.0 ± 0.6A×108 DLC 6.6 ± 0.5A×108 MLC 6.5 ± 0.3A×108 WLA 5.5 ± 0.4A×108 DLA 5.8 ± 0.4A×108 MLA 5.6 ± 0.3A×108
4.9 ± 0.6A×108 5.5 ± 0.4AB×108 5.2 ± 0.5B×108 3.2 ± 0.2B×108 4.0 ± 0.6B×108 3.5 ± 0.1B×108
2.8 ± 0.3B×108 4.2 ± 0.4B×108 4.0 ± 0.4B×108 1.5 ± 0.4C×108 2.2 ± 0.2C×108 1.8 ± 0.4C×108
1.6 ± 0.5C×108 3.3 ± 0.4C×108 3.0 ± 0.2C×108 8.0 ± 0.6D×107 9.5 ± 0.7D×107 9.1 ± 0.5D×107
8.9 ± 0.6D×107 1.1 ± 0.4D×108 9.6 ± 0.5D×107 5.0 ± 0.4E×107 7.0 ± 0.2E×107 5.8 ± 0.4E×107
4.5 ± 0.4E×107 7.3 ± 0.9E×107 6.1 ± 0.6E×107 8.4 ± 0.3F×106 1.4 ± 0.4F×107 9.1 ± 0.2F×106
8.0 ± 0.6F×106 1.8 ± 0.3F×107 9.0 ± 0.2F×106 2.3 ± 0.5G×106 5.8 ± 0.1G×106 3.4 ± 0.3G×106
1.8b 1.5c 1.8b 2.4a 2.0c 2.2b
Storage at 25 °C WLC 6.0 ± 0.6A×108 DLC 6.6 ± 0.5A×108 MLC 6.5 ± 0.3A×108 WLA 5.5 ± 0.7A×108 DLA 5.8 ± 0.4A×108 MLA 5.6 ± 0.3A×108
6.1 ± 0.4B×106 8.1 ± 0.6B×106 6.5 ± 0.4B×106 9.0 ± 0.8B×105 1.9 ± 0.6B×106 1.1 ± 0.3B×106
8.4 ± 0.3C×104 5.1 ± 0.3C×105 2.2 ± 0.3C×105 2.9 ± 0.7C×103 6.0 ± 0.6C×103 5.1 ± 0.4C×103
1.2 ± 0.5D×103 1.3 ± 0.3D×104 6.1 ± 0.7D×103 2.0 ± 0.6D×102 8.0 ± 0.4D×102 5.2 ± 0.6D×102
1.8 ± 0.2E×102 5.2 ± 0.4E×103 7.1 ± 0.6E×102 < 10E < 10E < 10E
< 10F 5.0 ± 0.8F×102 < 10E < 10E < 10E < 10E
< 10F < 10G < 10E < 10E < 10E < 10E
8.7d 8.8d 8.8d 8.7d 8.8d 8.8d
Means in the same column with the same lowercase letters, and means in the same row with the same uppercase letters indicate no significant difference (P > 0.05). WLC=white chocolate containing immobilized L. casei 01, DLC=dark chocolate containing immobilized L. casei 01, MLC=milk chocolate containing immobilized L. casei 01, WLA=white chocolate containing immobilized L. acidophilus LA5, DLA=dark chocolate containing immobilized L. acidophilus LA5 and MLA=milk chocolate containing immobilized L. acidophilus LA5.
3.4. Sensory evaluation of probiotic-supplemented chocolates
was found to have viability in milk and dark chocolates during the storage at 4 °C for 180 days at levels higher than Bifidobacterium lactis HN019. Regarding to the storage, L. casei 01 cells were found to decline at levels significantly lower than L. acidophilus LA5 cells (P≤0.05). The survivability of probiotic cells was greatly reduced when the chocolates were stored at 25 °C (P≤0.05). Indeed, L. acidophilus LA5 cells embedded in all chocolate matrices were totally disappeared within 40 days of storage at 25 °C, whereas L. casei 01 cells in dark chocolate could maintain their survivability up until 50 days of storage (~2 log CFU/g). On the other hand, during the first 10 days of storage at 4 °C, the survivability of probiotic cells in all chocolate batches reached similar levels (~8 log CFU/g). After 60 days of storage at 4 °C, the survival rates of probiotics in refrigerated chocolate remained rather high at more than 6 log CFU/g. This was complied with the recommended dose for probiotic-supplemented foods as established by Ding and Shah (2008), Chaikham (2015) and Laličić-Petronijević et al. (2015), who suggested that minimum number of live probiotics should be at least 106 CFU/ml or CFU/g before consumption. The similar results were previously described by Żyżelewicz, Nebesny, Motyl, and Libudzisz (2010), Mandal et al. (2013) and Laličić-Petronijević et al. (2015), who investigated the milk and/or dark chocolates enriched with several probiotic bacteria, such as L. acidophilus, L. casei, Lactobacilus paracasei and B. lactis. However, the package for storage could play an important role because oxygen may affect a number of active probiotic cells and thus trigger the reduction of cell survivability over the entire storage period (Chaikham, 2015), in particular the cells on the surface of chocolates (Laličić-Petronijević et al., 2015). Considering the types of chocolates, the probiotic cells could remain highest in dark chocolate (50% cocoa), followed by milk (10% cocoa) and white (0% cocoa) chocolates. The comparable results of chocolates supplemented with probiotics Streptococcus thermophiles MK-10 and Lactobacillus delbrueckii ssp. bulgaricus 151 were reported by Nebesny, Zyzelewicz, Motyl, and Libudzisz (2005). These might be primarily due to the property of dark chocolate that containing higher levels of cocoa and antioxidant compounds and activities than milk and white chocolates (Table 2). The significant levels of antioxidant activities of dark chocolate were able to prevent the probiotic cells from oxygen toxicity and thus improve the survivability of probiotic bacteria (Chaikham, 2015). On the contrary, Laličić-Petronijević et al. (2015) found that the number of living probiotic cells did not significantly differ between milk (27% cocoa) and dark (75% cocoa) chocolates during storage at 4 °C for 180 days.
The chocolates that containing immobilized L. casei 01 cells and storing at 4 °C were used for sensorial characterizations since they had viable cells more than 6 log CFU/g after 60 days of storage (Table 3). The sensory attributes of chocolates and probiotic-supplemented chocolates, including appearance, color, flavor, texture and overall acceptability, were evaluated by 50 untrained panelists, as shown in Table 4. In general, the external presence of chocolates, especially color and appearance, were considered as important factors. For fresh chocolates (day 0, no storage), the similar levels of sensory scores were observed, regardless of whether it was the control or probioticsupplemented chocolate. When comparing between the chocolate matrices, the white chocolate was shown to receive the highest preferences, followed by milk and dark chocolates (P≤0.05). Nonetheless, after 60 days of storage, sensory evaluation scores of all chocolates were found to be significantly reduced when compared to the fresh ones (P≤0.05), especially that of flavor, texture and overall acceptability, although the results showed the acceptable overall liking scores for all sensory attributions. Furthermore, no significant differences (P > 0.05) of sensory evaluation were observed between the control and the probiotic-supplemented chocolates. Thus, for this study, the addition of probiotics to chocolate products did not result in different acceptance when comparing to the control groups. Similarly, Nebesny and Zyzelewicz (2006) observed that the sensory attributions of chocolates supplemented with L. casei and L. paracasei powders were not different from those of the control chocolates. Erdem et al. (2014) also reported that supplementation of probiotic Bacillus indicus HU36, which spray dried with maltrodextrin and lemon fiber, was shown to have no negative effects on consumers’ acceptance of dark chocolate. Furthermore, there were a number of studies that also reported none or only minimal sensory attributions affected by the addition of probiotics in some foods, for instance, chocolate milk (Rouhi, Mohammadi, Mortazavian, & Sarlak, 2015), cheese (Ehsannia & Sanjabi, 2016) and ice-cream (Champagne, Raymond, Guertin, & Bélanger, 2015). 3.5. Survivability of Lactobacillus casei 01 in different chocolate types embedded in simulated gastrointestinal tract The effect of simulated gastrointestinal environments on survivability of L. casei 01, both as the free and the immobilized cells, in chocolates is shown in Table 5. Either free or microencapsulated L. 41
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Table 4 Sensory evaluation of chocolates containing Lactobacillus casei 01 powders stored at 4 °C. Samples
Appearance
Color
Flavor
Texture
Overall acceptability
Day 0 W WLC D DLC M MLC
8.0 ± 0.7a 7.9 ± 0.8a 7.4 ± 0.6b 7.4 ± 0.8b 7.6 ± 0.6ab 7.8 ± 0.6a
7.5 ± 0.9a 7.6 ± 0.6a 7.6 ± 0.5a 7.6 ± 0.5a 7.6 ± 0.5a 7.6 ± 0.6a
7.7 ± 0.7a 7.7 ± 0.7a 7.7 ± 0.5a 7.7 ± 0.4a 7.8 ± 0.5a 7.6 ± 0.5a
7.3 ± 0.8b 7.3 ± 0.7b 7.4 ± 0.8ab 7.5 ± 0.6a 7.3 ± 0.7b 7.4 ± 0.5ab
7.6 ± 0.7a 7.5 ± 0.6a 7.2 ± 0.5b 7.2 ± 0.4b 7.4 ± 0.5ab 7.4 ± 0.5ab
Day 60 W WLC D DLC M MLC
7.4 ± 0.6b 7.3 ± 0.6bc 6.9 ± 0.3d 7.0 ± 0.5cd 7.1 ± 0.5c 7.2 ± 0.5c
7.4 ± 0.6ab 7.5 ± 0.6a 7.0 ± 0.6b 7.0 ± 0.4b 7.1 ± 0.6b 7.2 ± 0.5b
6.2 ± 0.5c 6.2 ± 0.5bc 6.5 ± 0.4b 6.5 ± 0.3b 6.4 ± 0.5b 6.3 ± 0.5bc
6.7 ± 0.2c 6.8 ± 0.4c 6.5 ± 0.4d 6.3 ± 0.5d 6.3 ± 0.3d 6.4 ± 0.5d
6.2 ± 0.5c 6.3 ± 0.6c 6.2 ± 0.4c 6.2 ± 0.3c 6.2 ± 0.4c 6.2 ± 0.6c
Means in the same column with the same lowercase letters indicate no significant difference (P > 0.05). W=white chocolate, WLC=white chocolate containing immobilized L. casei 01, D=dark chocolate, DLC=dark chocolate containing immobilized L. casei 01, M=milk chocolate and MLC=milk chocolate containing immobilized L. casei 01.
colon. In addition, Mandal et al. (2013) found that mice which were fed with milk chocolate containing L. casei NCDC 298 and prebiotic inulin were found to have significant increase of colon lactobacilli and decrease of coliforms. Therefore, the findings in this study indicated that chocolates were the excellent carriers for protecting the probiotics from the gastrointestinal environments, which result in the remaining number of survived probiotic cells at levels sufficient to be beneficial for human's health.
casei 01 cells were initially inoculated into simulated stomach compartment at the concentrations between 6.5 ± 0.5 and 6.8 ± 0.5×107 CFU/ml. The number of survival L. casei 01 cells gradually declined when the incubation time increased (P≤0.05), whereby the free cells were less capable of surviving the gastric environment of the simulated stomach and they were eventually eliminated after 1.5 h of incubation. On the contrary, this study showed that the immobilized L. casei 01 in different chocolates were found to have high levels of survivability after being exposed to the gastric environment of simulated stomach for 2 h. These immobilized L. casei 01 cells were subsequently incubated in the simulated small intestinal fluid for another 4 h and they still remained to be viable at ~2 log CFU/ml. This may suggested that either maltodextrin plus skim milk and/or chocolates were beneficial materials for protecting the probiotic cells from the adverse environments (Desmond, Ross, O’Callaghan, Fitzgerald, & Stanton, 2002; Khoramnia et al., 2011; Kingwatee et al., 2015). Several researchers indicated that the survival of probiotic bacteria under in vitro gastrointestinal conditions tended to increase when they were combined with chocolates (da Silva et al., 2012; Possemiers et al., 2010). Yonejima et al. (2015) reported that Lactobacillus brevis ssp. coagulans could survive in gastric juice (pH 2.5) better when being combined with chocolate rather than with commercial beverages (i.e. fruit juices and fermented milks). Correspondingly, Maillard and Landuyt (2008) indicated that chocolates were the great carriers for probiotics Lactobacillus Rosell-52 and Bifidobacterium Rosell-175 during the evaluation using a dynamic human gut model (SHIME reactor). Moreover, they also observed that the probiotic-chocolates could apparently enhanced the growth of beneficial bacteria (i.e. lactobacilli and bifidobacteria) and suppressed some harmful pathogens (i.e. fecal coliforms and clostridia) in the ascending, transverse and descending compartments of the simulated
4. Conclusion In this study, white, milk, and dark chocolates were found to be appropriate carriers for maintaining the number of probiotics during storage and passing through the simulated gastrointestinal conditions. In particular, L. casei 01 cells were found to be able to endure such conditions and survive better than L. acidophilus LA5 cells, and dark chocolate could preserve high levels of viable probiotic cells in all testing conditions. Moreover, 4 °C-storage was found to be suitable for preserving probiotic viability, regardless of chocolate types and probiotic strains. In addition, supplementation of spray-dried probiotics did not affect the sensory attributes of the products. Therefore, these results indicate that all chocolate types have potential to be used as protector matrices for probiotics and may be developed into other novel foods in the future.
Conflict of interest statement No conflicts of interest exist in this study.
Table 5 Viable Lactobacillus casei 01 cells in different chocolate matrices during incubation under anaerobic environment at 37 °C for 6 h in simulated stomach and intestine tract. Treatment conditions
Numbers of survival cells (CFU/ml) during incubation at 37 °C Simulated stomach environment Initial stage
Free cells WLC DLC MLC
aA
Simulated small intestinal environment
0.5 h 7
6.8 ± 0.5 ×10 6.6 ± 0.6aA×107 6.5 ± 0.4aA×107 6.5 ± 0.5aA×107
1h bB
5
4.8 ± 1.1 ×10 6.4 ± 0.7aB×106 6.4 ± 0.7aB×106 6.4 ± 0.8aB×106
1.5 h bC
3
2h bD
9.2 ± 0.6 ×10 7.3 ± 0.5aC×104 7.4 ± 0.8aC×104 7.6 ± 0.6aC×104
2
3.8 ± 0.7 ×10 5.6 ± 0.6aD×103 5.7 ± 0.5aD×103 5.6 ± 0.4aD×103
bE
nd 1.9 ± 0.7aE×103 2.0 ± 0.5aE×103 2.1 ± 0.6aE×103
4h bE
nd 5.9 ± 0.7aF×102 5.5 ± 0.5aF×102 5.7 ± 0.6aF×102
6h ndbE 3.3 ± 0.5aG×102 3.7 ± 0.6aG×102 3.1 ± 0.7aG×102
Means in the same column with the same lowercase letters, and means in the same row with the same uppercase letters indicate no significant difference (P > 0.05). WLC=white chocolate containing immobilized L. casei 01, DLC=dark chocolate containing immobilized L. casei 01 and MLC=milk chocolate containing immobilized L. casei 01. nd=not detected.
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