Journal of food science and technology(Iran)

Journal of food science and technology(Iran)

Application of microencapsulated Lactobacillus rhamnosus ATCC 7469 with milk protein concentrated in the production of functional orange juice and investigation of its qualitative characteristics

Document Type : Original Article

Authors
Fereydoun Nabizadeh 1
Mahmoud Rezazadeh Bari 2
Saber Amiri 3
Hossein Abdollah 4
Seyed Alireza Banihashemi 5
1 Graduate M.Sc., Food Science and Technology, Saba Instituted of Higher Education, Urmia, Iran
2 Professor, Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran
3 Associated Professor, Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran
4 Assistant Professor, Department of Polymer Engineering, Faculty of Engineering, Urmia University, Urmia, Iran
5 Ph.D. Candidate, Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran
10.48311/fsct.2026.84092.0
Abstract
Microencapsulation technology is a novel approach to enhancing bacterial resistance against environmental, chemical, and gastrointestinal stresses. In this study, milk protein concentrate (MPC) was used as a wall material due to its strong biological and protective properties. The aim was to evaluate the effect of this material, combined with transglutaminase enzyme, on the survival of Lactobacillus rhamnosus in the acidic environment of orange juice. Four samples were prepared: S1 (plain orange juice), S2 (protein + bacteria), S3 (protein + bacteria + enzyme), and S4 (protein + enzyme). During storage, samples were analysed for bacterial viability, antioxidant activity, pH, titratable acidity, vitamin C, reducing sugar, total phenolic content, color, FTIR spectra, and molecular weight. The results showed that sample S3 exhibited the best performance with a viability of 5.5×10⁷ CFU/mL, antioxidant activity of 68.5%, and acidity of 4.4%, along with a pH of 3.6. In contrast, sample S4 showed the lowest viability (3.8×10⁷ CFU/mL) and vitamin C content (32.3 mg/L). Sample S2 had the highest total phenolic content (14.7 mg GAE/mL) and the lowest reducing sugar (9.07 mg/mL). During storage, vitamin C, reducing sugars, and phenolics decreased, whereas acidity and antioxidant activity increased. Samples containing bacteria showed higher vitamin C and acidity, while those with the enzyme exhibited higher reducing sugar content. FTIR analysis confirmed the presence of carbonyl, methyl, and amine functional groups. Sample S2 showed decreased molecular weight and increased solubility, indicating effective interactions between bacteria and protein.
Keywords
Microencapsulation
Lactobacillus rhamnosus
Milk protein concentrate
Viability
Orange juice
Subjects

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[2]    Jia H, Li Y, Tian Y, Li N, Zheng M, Zhang W, et al. Recent advances in electrospray encapsulation of probiotics: influencing factors, natural polymers and emerging technologies. Critical Reviews in Food Science and Nutrition. 2024:1-18.

[3]    Bu W, Mcclements DJ, Zhang Z, Zhang R, Jin Z, Chen L. Encapsulation method of probiotic embedded delivery system and its application in food. Food Hydrocolloids. 2025;159:110625.

[4]    Zolfaghari H, Khezerlou A, Banihashemi SA, Tavassoli M, Ehsani A. Review on bio-detoxification of aflatoxins based on lactic acid bacteria: mechanism and applications. Journal of microbiology, biotechnology and food sciences. 2023;13(1):e9424-e.

[5]    Amin MR, Biswas AP, Tasnim M, Islam MN, Azam MS. Probiotics and their Applications in Functional Foods: A Health Perspective. Applied Food Research. 2025:101193.

[6]    Khezerlou A, Zolfaghari H, Banihashemi SA, Forghani S, Ehsani A. Plant gums as the functional compounds for edible films and coatings in the food industry: A review. Polymers for Advanced Technologies. 2021;32(6):2306-26.

[7]    Afzal A, Afzaal M, Saeed F, Shah YA, Raza MA, Khan MH, et al. Milk protein based encapsulation of probiotics and other food material: comprehensive review. International Journal of Food Properties. 2024;27(1):245-62.

[8]    Velazquez-Dominguez A, Hiolle M, Abdallah M, Delaplace G, Peixoto PP. Transglutaminase cross-linking on dairy proteins: Functionalities, patents, and commercial uses. International Dairy Journal. 2023;143:105688.

[9]    Hazervazifeh A, Rezazadeh A, Banihashemi A, Ghasempour Z, Kia EM. Pulsed microwave radiation for extraction of betalains from red beetroot: Engineering and technological aspects. Process Biochemistry. 2023;134:121-30.

[10] Maleki O, Khosrowshahi Asl A, Alizadeh Khaledabad M, Amiri S. Production and characterization of synbiotic ice cream using microencapsulation and cryopreservation of Lactobacillus rhamnosus in whey protein/bio-cellulose/inulin composite microcapsules. Journal of Food Measurement and Characterization. 2023;17(4):3909-17.

[11] Dikmetas DN, Acar EG, Ceylan FD, İlkadım F, Özer H, Karbancioglu-Guler F. Functional fermented fruit juice production and characterization by using water kefir grains. Journal of Food Science and Technology. 2025:1-14.

[12] Randazzo CL, Pitino I, Licciardello F, Muratore G, Caggia C. Survival of Lactobacillus rhamnosus probiotic strains in peach jam during storage at different temperatures. Food Science and Technology. 2013;33:652-9.

[13] Shao Y, Gao S, Guo H, Zhang H. Influence of culture conditions and preconditioning on survival of Lactobacillus delbrueckii subspecies bulgaricus ND02 during lyophilization. Journal of Dairy Science. 2014;97(3):1270-80.

[14] Pérez M, Dominguez-López I, Lamuela-Raventós RM. The chemistry behind the folin–ciocalteu method for the estimation of (poly) phenol content in food: Total phenolic intake in a mediterranean dietary pattern. Journal of agricultural and food chemistry. 2023;71(46):17543-53.

[15] Yamauchi M, Kitamura Y, Nagano H, Kawatsu J, Gotoh H. DPPH measurements and structure—Activity relationship studies on the antioxidant capacity of phenols. Antioxidants. 2024;13(3):309.

[16] Borba VS, Lemos AC, Rodrigues MHP, Gropelli VM, Cerqueira MBR, Badiale-Furlong E. Miniaturization of spectrophotometric methods to determine reducing sugars, total starch and soluble proteins in wheat-based products. Food Analytical Methods. 2022;15(10):2756-66.

[17] Gruska RM, Kunicka-Styczyńska A, Jaśkiewicz A, Baryga A, Brzeziński S, Świącik B. Fourier transform mid-infrared spectroscopy (FT-MIR) as a method of identifying contaminants in sugar beet production process—Case studies. Molecules. 2023;28(14):5559.

[18] Tian H, Zhao Q, He Z, Wang Z, Qin F, Zeng M, Chen J. Effects of molecular weight and degree of esterification of soluble soybean polysaccharide on the stability of casein under acidic conditions. Foods. 2021;10(3):686.

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[20] Silalahi J, Nadarason D, Silalahi YC. The effect of storage condition on antioxidant activity of probiotics in yogurt drinks. Asian Journal of Pharmaceutical and Clinical Research. 2018;11(12):280-3.

[21] La Torre C, Fazio A, Caputo P, Plastina P, Caroleo MC, Cannataro R, Cione E. Effects of long-term storage on radical scavenging properties and phenolic content of kombucha from black tea. Molecules. 2021;26(18):5474.

[22] Shahmoradi Z, Khaledabad MA, Amiri S. Effect of co-encapsulation of Lactobacillus acidophilus LA5 and selenium in hydrogelated matrix of basil seed mucilage/sodium caseinate on properties of set yogurt. Food Bioscience. 2023;55:103039.

[23] Neofytos DD, Gregersen SB, Corredig M. FTIR Imaging as a Tool for Studying Protein Dynamics and Interactions at the Oil/Water Interface with Spatial Resolution: A Single Droplet Analysis Approach. Langmuir. 2025;41(21):12858-72.

[24] Kaur G, Panigrahi C, Agarwal S, Khuntia A, Sahoo M. Recent trends and advancements in nanoemulsions: Production methods, functional properties, applications in food sector, safety and toxicological effects. Food Physics. 2024;1:100024.

[25] Manga MS, Higgins L, Kumar AA, Lobel BT, York DW, Cayre OJ. Exploring effects of polymeric stabiliser molecular weight and concentration on emulsion production via stirred cell membrane emulsification. Polymer chemistry. 2023;14(45):5049-59.

[26] Dickinson E. Colloids in food: ingredients, structure, and stability. Annual review of food science and technology. 2015;6(1):211-33.

[27] Masiá C, Geppel A, Jensen PE, Buldo P. Effect of Lactobacillus rhamnosus on physicochemical properties of fermented plant-based raw materials. Foods. 2021;10(3):573.

[28] Kupikowska-Stobba B, Domagała J, Kasprzak MM. Critical review of techniques for food emulsion characterization. Applied Sciences. 2024;14(3):1069.

[29] Du J, Song Y, Li X, Liu N, An X, Qi J. Dynamic changes and correlation analysis of polysaccharide content and color parameters in Glycyrrhiza stems and leaves during fermentation. Fermentation. 2023;9(10):900.

[30] Ababaf K, Jooyandeh H, Nasehi B. Effect of transglutaminase enzyme treatment on the physicochemical and microbial properties of synbiotic soy yogurt. Food Research Journal. 2020;30(3):189-201.

[1]    [1] Bhutto RA, Mahar H, Khanal S, Wang M, Iqbal S, Fan Y, Yi J. Recent trends in co-encapsulation of probiotics with prebiotics and their applications in the food industry. Trends in Food Science & Technology. 2025;156:104829.
[2]    Jia H, Li Y, Tian Y, Li N, Zheng M, Zhang W, et al. Recent advances in electrospray encapsulation of probiotics: influencing factors, natural polymers and emerging technologies. Critical Reviews in Food Science and Nutrition. 2024:1-18.
[3]    Bu W, Mcclements DJ, Zhang Z, Zhang R, Jin Z, Chen L. Encapsulation method of probiotic embedded delivery system and its application in food. Food Hydrocolloids. 2025;159:110625.
[4]    Zolfaghari H, Khezerlou A, Banihashemi SA, Tavassoli M, Ehsani A. Review on bio-detoxification of aflatoxins based on lactic acid bacteria: mechanism and applications. Journal of microbiology, biotechnology and food sciences. 2023;13(1):e9424-e.
[5]    Amin MR, Biswas AP, Tasnim M, Islam MN, Azam MS. Probiotics and their Applications in Functional Foods: A Health Perspective. Applied Food Research. 2025:101193.
[6]    Khezerlou A, Zolfaghari H, Banihashemi SA, Forghani S, Ehsani A. Plant gums as the functional compounds for edible films and coatings in the food industry: A review. Polymers for Advanced Technologies. 2021;32(6):2306-26.
[7]    Afzal A, Afzaal M, Saeed F, Shah YA, Raza MA, Khan MH, et al. Milk protein based encapsulation of probiotics and other food material: comprehensive review. International Journal of Food Properties. 2024;27(1):245-62.
[8]    Velazquez-Dominguez A, Hiolle M, Abdallah M, Delaplace G, Peixoto PP. Transglutaminase cross-linking on dairy proteins: Functionalities, patents, and commercial uses. International Dairy Journal. 2023;143:105688.
[9]    Hazervazifeh A, Rezazadeh A, Banihashemi A, Ghasempour Z, Kia EM. Pulsed microwave radiation for extraction of betalains from red beetroot: Engineering and technological aspects. Process Biochemistry. 2023;134:121-30.
[10] Maleki O, Khosrowshahi Asl A, Alizadeh Khaledabad M, Amiri S. Production and characterization of synbiotic ice cream using microencapsulation and cryopreservation of Lactobacillus rhamnosus in whey protein/bio-cellulose/inulin composite microcapsules. Journal of Food Measurement and Characterization. 2023;17(4):3909-17.
[11] Dikmetas DN, Acar EG, Ceylan FD, İlkadım F, Özer H, Karbancioglu-Guler F. Functional fermented fruit juice production and characterization by using water kefir grains. Journal of Food Science and Technology. 2025:1-14.
[12] Randazzo CL, Pitino I, Licciardello F, Muratore G, Caggia C. Survival of Lactobacillus rhamnosus probiotic strains in peach jam during storage at different temperatures. Food Science and Technology. 2013;33:652-9.
[13] Shao Y, Gao S, Guo H, Zhang H. Influence of culture conditions and preconditioning on survival of Lactobacillus delbrueckii subspecies bulgaricus ND02 during lyophilization. Journal of Dairy Science. 2014;97(3):1270-80.
[14] Pérez M, Dominguez-López I, Lamuela-Raventós RM. The chemistry behind the folin–ciocalteu method for the estimation of (poly) phenol content in food: Total phenolic intake in a mediterranean dietary pattern. Journal of agricultural and food chemistry. 2023;71(46):17543-53.
[15] Yamauchi M, Kitamura Y, Nagano H, Kawatsu J, Gotoh H. DPPH measurements and structure—Activity relationship studies on the antioxidant capacity of phenols. Antioxidants. 2024;13(3):309.
[16] Borba VS, Lemos AC, Rodrigues MHP, Gropelli VM, Cerqueira MBR, Badiale-Furlong E. Miniaturization of spectrophotometric methods to determine reducing sugars, total starch and soluble proteins in wheat-based products. Food Analytical Methods. 2022;15(10):2756-66.
[17] Gruska RM, Kunicka-Styczyńska A, Jaśkiewicz A, Baryga A, Brzeziński S, Świącik B. Fourier transform mid-infrared spectroscopy (FT-MIR) as a method of identifying contaminants in sugar beet production process—Case studies. Molecules. 2023;28(14):5559.
[18] Tian H, Zhao Q, He Z, Wang Z, Qin F, Zeng M, Chen J. Effects of molecular weight and degree of esterification of soluble soybean polysaccharide on the stability of casein under acidic conditions. Foods. 2021;10(3):686.
[19] Kardas M, Kiciak A, Szynal K, Sitkiewicz B, Staśkiewicz-Bartecka W, Bielaszka A. Assessment of the color of orange juice in the context of dietitians’ food preferences. Frontiers in Nutrition. 2024;10:1328795. Journal of food science and technology(Iran).14(67):273-65.
[20] Silalahi J, Nadarason D, Silalahi YC. The effect of storage condition on antioxidant activity of probiotics in yogurt drinks. Asian Journal of Pharmaceutical and Clinical Research. 2018;11(12):280-3.
[21] La Torre C, Fazio A, Caputo P, Plastina P, Caroleo MC, Cannataro R, Cione E. Effects of long-term storage on radical scavenging properties and phenolic content of kombucha from black tea. Molecules. 2021;26(18):5474.
[22] Shahmoradi Z, Khaledabad MA, Amiri S. Effect of co-encapsulation of Lactobacillus acidophilus LA5 and selenium in hydrogelated matrix of basil seed mucilage/sodium caseinate on properties of set yogurt. Food Bioscience. 2023;55:103039.
[23] Neofytos DD, Gregersen SB, Corredig M. FTIR Imaging as a Tool for Studying Protein Dynamics and Interactions at the Oil/Water Interface with Spatial Resolution: A Single Droplet Analysis Approach. Langmuir. 2025;41(21):12858-72.
[24] Kaur G, Panigrahi C, Agarwal S, Khuntia A, Sahoo M. Recent trends and advancements in nanoemulsions: Production methods, functional properties, applications in food sector, safety and toxicological effects. Food Physics. 2024;1:100024.
[25] Manga MS, Higgins L, Kumar AA, Lobel BT, York DW, Cayre OJ. Exploring effects of polymeric stabiliser molecular weight and concentration on emulsion production via stirred cell membrane emulsification. Polymer chemistry. 2023;14(45):5049-59.
[26] Dickinson E. Colloids in food: ingredients, structure, and stability. Annual review of food science and technology. 2015;6(1):211-33.
[27] Masiá C, Geppel A, Jensen PE, Buldo P. Effect of Lactobacillus rhamnosus on physicochemical properties of fermented plant-based raw materials. Foods. 2021;10(3):573.
[28] Kupikowska-Stobba B, Domagała J, Kasprzak MM. Critical review of techniques for food emulsion characterization. Applied Sciences. 2024;14(3):1069.
[29] Du J, Song Y, Li X, Liu N, An X, Qi J. Dynamic changes and correlation analysis of polysaccharide content and color parameters in Glycyrrhiza stems and leaves during fermentation. Fermentation. 2023;9(10):900.
[30] Ababaf K, Jooyandeh H, Nasehi B. Effect of transglutaminase enzyme treatment on the physicochemical and microbial properties of synbiotic soy yogurt. Food Research Journal. 2020;30(3):189-201.