MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING CAPSTONE PROJECT FOOD TECHNOLOGY PRODUCTION OF ACEROLA-CARROT JUICE FERMENTED WITH WATER KEFIR MICROORGANISMS LECTURER: PhD. VU TRAN KHANH LINH STUDENT: NGUYEN THAO NGOC NGUYEN PHUONG NHU SKL 0 0 8 4 9 0 Ho Chi Minh City, December, 2021 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING DEPARTMENT OF FOOD TECHNOLOGY GRADUATION PROJECT CODE: 2021-17116023 PRODUCTION OF ACEROLA-CARROT JUICE FERMENTED WITH WATER KEFIR MICROORGANISMS NGUYEN THAO NGOC Student ID: 17116023 NGUYEN PHUONG NHU Student ID: 17116107 Supervisor: VU TRAN KHANH LINH, PhD. HO CHI MINH CITY – 12/2021 ACKNOWLEDGEMENTS We wish to express our heartfelt gratitude to our families and all of the individuals that have kindly supported and helped us in this project. First and foremost, we want to show our deepest appreciation to our supervisor, Dr.
Vu Tran Khanh Linh, who has been so conscientious with us throughout this work. This graduation thesis has been a wonderful experience for both of us since we had received her belief, unlimited guidance and encouragement. We also want to give our gratefulness to Dr. Le Ngoc Lieu, from Vietnam National University HCMC International University, who has given us invaluable advice and contribution during the research.
It has been an honor for us to have a chance to work with them. Our sincere thankful also goes to all of the lecturers in the Department of Food Technology, Faculty of Chemical and Food Technology, HCMC University of Technology and Education for giving us limitless support and quality research condition. We also want to show our sincere gratitude to Ms. Hoang Thi Thu and Ms.
Phan My Phuong, from Nurturing Foods Co., Vietnam for their hospitality, compassion and endless support. We are so grateful to be sponsored for the dried water kefir grains, which was the main research material. Furthermore, thanks to them, we also had a chance to use the Alcolyzer for the first time, in the analysis of ethanol content. Special thanks to the juniors at HCMUTE, Nguyen Pham Huyen Phuong, Nguyen Hoang Tan Tai and Phan Nguyen Dai Trang for their dedication during the research.
We are lucky to get to know them, to be their friends and we had a quality time working together. We also would like to acknowledge our colleagues and friends, who we cannot name all. Their support and advice were priceless and we cannot be happier for knowing and working with such amazing people. Ho Chi Minh City, December 23th, 2021 TABLE OF CONTENT LIST OF TABLES.
i LIST OF FIGURES. ii LIST OF ABBREVIATIONS. Microbiota and appearance of water kefir grains. Symbiotic interactions of microorganisms in water kefir fermentation process.
Carbohydrate metabolism of water kefir microorganisms. Benefit of water kefir beverage. Sources of substrates for water kefir fermentation. Vegetable and fruit juice.
Acerola (Malpighia emarginata DC. Chemical composition of acerola. Application of pectinase in acerola juice extraction. Application of acerola juice in fermented foods.
Chemical composition of carrot. Application of carrot juice in fermented foods. MATERIALS AND METHODS. Water kefir grains.
Preparation of acerola juice. Preparation of carrot juice. Preparation of juice water kefir beverage. Experiment 1: Investigate the physicochemical composition of raw materials (acerola fruit and carrot).
Experiment 2: Effects of different acerola/carrot juice ratios (v/v) on the quality of water kefir beverage. Experiment 3: Investigate the effect of replacement of cane sugar with coconut sugar on physicochemical and microbiological properties of the wafer kefir beverage. Statistical data analysis. RESULTS AND DISCUSSION.
Investigate physicochemical composition of raw materials (acerola fruit and carrot). Effects of different acerola/carrot juice ratios (v/v) on the quality of water kefir beverages. Total soluble solids (TSS), pH and titratable acidity (TA). Antioxidant compounds and DPPH radical-scavenging activity.
Effect of the replacement of cane sugar with coconut sugar on physicochemical and microbiological properties of the wafer kefir beverages (WKB). Enumeration of presumptive microorganisms in water kefir fermentation. Total soluble solids (TSS), pH value and ethanol content .70 LIST OF TABLES Table 2.1 Isolated microorganisms in water kefir grains .2 Homo- and heterofermentative LAB species in water kefir [16] .3 Nutritional content of acerola fruit (per 100 g) [40] .4 Chemical composition of fresh carrot (per 100 g) [49] .1 Analytical methods for physicochemical properties of acerola fruit and carrot .2 Formulation of juice water kefir beverages using cane sugar .3 Physicochemical and microbiological analyzing method of water kefir beverages in experiment 2 .4 Physicochemical and microbiological analyzing method of water kefir beverages in experiment 3 .1 Physicochemical composition of acerola and carrot .2 Color analysis of five different acerola-carrot water kefir samples .1 Physicochemical analysis of ten different acerola-carrot water kefir beverages applied with different sources of sugar .1 Microbiological analysis of ten different acerola-carrot water kefir beverages applied with different sources of sugar. 73 i LIST OF FIGURES Figure 2.1 Manufacturing process of water kefir beverage [15, 18] .2 Appearance of water kefir grains, scale is in cm [20] .3 Interactions of main representative cultivable water kefir isolates [22] .4 Carbohydrate metabolism of LAB [24] .5 Carbohydrate metabolism of yeast [26] .6 Fully ripened Acerola (Malpighia emarginata DC.1 Appearance of water kefir grains in dried form (left) and rehydrated form (right) used in this experiment .2 Propagation of water kefir starter culture .3 Preparation of acerola juice .4 Preparation of carrot juice .5 Preparation of juice water kefir beverage .6 Reaction between reducing sugars and DNS reagent [66] .7 Glucose calibration curve .8 Gallic acid calibration curve .9 Reaction between Ascorbic acid and DCPIP [71] .10 Ascorbic acid calibration curve .11 UV-Vis spectra of anthocyanins in pH 1.5 buffers, and the structures of the flavylium cation (A) and hemiketal forms (B) [73] .1 Gram staining of LAB grown on MRS agar (a) and yeast grown on PDA (b) under oil immersion (100x magnification).2 Viable counts (log CFU/mL) of presumptive (a) LAB and (b) yeast of different samples at 0h and 48h of fermentation.
Results with the same lowercase letters do not differ at p < 0.05 with respect to the 0h and 48h. Results with the same uppercase letters do not differ at p < 0.05 with respect to the samples.3 (a) pH, (b) total soluble solids and (c) titratable acidity of different samples at 0h and 48h of fermentation. Results with the same lowercase letters do not differ at p < 0.05 with respect to the 0h and 48h. Results with the same uppercase letters do not differ at p < 0.05 with respect to the samples.4 Reducing sugars content of different samples at 0h and 48h of fermentation.
Results with the same lowercase letters do not differ at p < 0.05 with respect to the 0h and 48h. Results with the same uppercase letters do not differ at p < 0.05 with respect to the samples.5 (a) Total phenolic content; (b) Ascorbic acid content; (c) Total anthocyanin content and (d) DPPH radical-scavenging activity of different samples at 0h and 48h of fermentation. Results with the same lowercase letters do not differ at p < 0.05 with respect to the 0h and 48h. Results with the same uppercase letters do not differ at p < 0.05 with respect to the samples.6 Ethanol content of different samples at 48h of fermentation.
Results with the same lowercase letters do not differ at p < 0.7 Color difference of different samples. Results with the same lowercase letters do not differ at p < 0.8 Sensory analysis of different samples. Error bars = SD (n=45).9 Physical appearance acerola-carrot juice beverages .10 Viable counts of (a) LAB and (b) yeast for each sample applied with different sugar sources at 48h of fermentation. * symbol indicates statistical significance (p < 0.05) between samples applied with cane sugar and coconut sugar.11 (a) pH and (b) total soluble solids of each sample applied with different sugar sources at 48h of fermentation.
* symbol indicates statistical significance (p < 0.05) between samples applied with cane sugar and coconut sugar.12 Ethanol content of each sample applied with different sugar sources at 48h of fermentation. * symbol indicates statistical significance (p < 0.05) between samples applied with cane sugar and coconut sugar.13 Reducing sugars content of each sample applied with different sugar sources at 0h and 48h of fermentation. * symbol indicates statistical significance (p < 0.05) between samples applied with cane sugar and coconut sugar.1 Sensory evaluation questionnaire .1 Reducing sugar analysis results of fresh acerola and carrot. 74 iv LIST OF ABBREVIATIONS 1.
AAB: Acetic Acid Bacteria 2. AAE: Ascorbic Acid Equivalents 3. ANOVA: Analysis of Variance 4. AOAC: Association of Official Analytical Chemist 5.
ATP: Adenosine triphosphate 6. CFU: Colony Forming per Unit 7. DCPIP: 2,6-Dichlorophenol indophenol 8. DNS: 3,5-dinitrosalicylic acid 9.
DPPH: 2,2-diphenyl-1-picrylhydrazyl 10. GAE: Gallic Acid Equivalents 11. LAB: Lactic Acid Bacteria 12. MRS: De Man, Rogosa and Sharpe 13.
NAD+: Nicotinamide adenine dinucleotide 14. PDA: Potato Dextrose Agar 15. SD: Standard Deviation 16. TAC: Total Anthocyanin Content 17.
TCVN: Tiêu Chuẩn Việt Nam 18. TPC: Total Phenolic Content 19. TSS: Total Soluble Solids 20. WKB: Water Kefir Beverage v ABSTRACT Water kefir is a non-dairy probiotic beverage fermented by microbial multispecies of water kefir grains wherein the key microorganisms are lactic acid bacteria (LAB) and yeast.
Several studies have mainly focused on the isolation and identification of water kefir starter cultures. However, there is limited information on the combination of fruit and vegetable juices in water kefir fermentation. Therefore, this study aimed to focus on the production of acerola-carrot juice fermented with water kefir grains to evaluate the effect of different acerola/carrot juice ratios on the quality of the products. The fermentation media were adjusted to pH 4.5 and 12 oBrix by the addition of cane or coconut sugar.
The fermentation was performed at 25 oC for 48 hours. The beverages were analyzed for microbiological and physicochemical properties at 0 and 48 hours of fermentation, while sensory evaluation was conducted on the final beverages. Results showed that pH value and total soluble solids decreased after fermentation, while titratable acidity and ethanol content witnessed an opposite trend. The highest growth of microorganisms was obtained in the sample fermented with 100% acerola juice, with cell counts of LAB and yeast was of 8.48 log CFU/mL and 7.37 log CFU/mL, respectively.
Only the sample with 100% of carrot juice fermented with cane sugar showed an increase in reducing sugars content after 48h of fermentation. All of the samples also showed a significant increase in radical-scavenging activity after fermentation, demonstrating a positive effect of water kefir fermentation on phytonutrients in the final products. Changes in color attribute were considered unnoticeable at the human perception scale. Regarding sensory analysis, samples with 50:50 and 25:75 of acerola/carrot juices were highly evaluated in flavor and overall acceptability.
The results also showed that all of the water kefir drinks fermented with coconut sugar had higher microbial loads and ethanol content than those with cane sugar after fermentation. Therefore, the outcomes of this research provide strong evidence that a new type of fermented acerola- carrot beverage can be produced using water kefir grains as a suitable starter along with the supporting substrates of cane sugar or coconut sugar. Rationale The ongoing COVID-19 pandemic has had a profound impact on people's health concerns, as well as their eating and drinking habits. Thus, the demand for healthier products like probiotic foods has also increased since they are supposed to promote the composition of the gut microbiota and overall health [1].
Traditionally, probiotics have been found in yogurt and other dairy-based fermented products. However, high fat and cholesterol content and lactose intolerance have limited the consumption of these products. Besides, another issue limiting dairy product intake is the rise in the number of vegans.