MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES OPTIMIZING THE FREEZE-DRYING PROCESS FOR KEFIR MILK SNACK BITES Major : BIOTECHNOLOGY Student : PHAM VAN TOAN Student ID : 18126182 Academic year : 2018-2022 Thu Duc City, 09/2023 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES GRADUATE THESIS OPTIMIZING THE FREEZE-DRYING PROCESS FOR KEFIR MILK SNACK BITES Advisor Student CAO THI THANH LOAN, PH. PHAM VAN TOAN Thu Duc City, 09/2023 ACKNOWLEDGEMENT I would like to express my deepest gratitude and appreciation to the following individuals and organizations for their invaluable support and contributions throughout my journey in completing this graduate thesis. First and foremost, I am profoundly thankful to my thesis advisor, Dr. Cao Thi Thanh Loan, for her unwavering guidance, patience, encouragement, and expertise.
Her dedication and mentorship played a pivotal role in shaping the direction ofthis research. My heartfelt thanks go to my family for their endless love, support, and belief in my abilities. Their encouragement sustained me during the challenging phases of this journey. I would also like to thank the participants of this study, express to Mr.
Quoc Thang, Mr. Minh Sang and my friends for their willingness to share their insights and supports. Their contributions are highly valued. I am grateful to the Kefir Home company for their material support, which made it possible to conduct this research.
I would like to acknowledge Nong Lam University for providing the necessary resources, facilities, and opportunities that allowed me to undertake this research. Particularly the administrators of the Faculty of Biological Sciences and all the lecturers, for their invaluable support and resources throughout their studies. They acknowledge that the guidance and teaching they received from these individuals played a critical role in their ability to apply their knowledge, particularly during their thesis work. I would like to thank Dr.
Phan Dang Thai Phuong - academic mentor of class DH18SHC for taking care of me during 5 years of university. This thesis would not have been possible without the collective efforts and support of all those mentioned above. Thank you for being a part of this important milestone in my academic career. AFFIRMATION AND COMMITMENT My name is Pham Van Toan, student ID: 18126182, class: DH18SHC (Phone: 0375525378, email: 18126182@st.vn), Faculty of Biological Sciences, Nong Lam university Ho Chi Minh City.
I declared that all results presented in this graduate thesis were conducted by myself. All the data and information are entirely accurate and unbiased. I fully accept responsibility for these commitments in front of the committee. Thu Duc City, September 7th, 2023 Student’s signature Pham Van Toan 1 ABSTRACT Kefir milk snack bites are gaining popularity as a convenient and healthy dairy- based snack.
However, ensuring the product's quality and shelf-life 1s crucial to meet consumer demands and regulatory requirements. This study aimed to optimize the freeze-drying process for kefir milk snack bites by incorporating food additives (trehalose, maltodextrin, and tapioca starch) to enhance structural stability and preserve beneficial microorganisms. In addition, the freezing temperature (-10 °C, -20 °C, -30 °C) and drying time (12, 18, 24 hrs) were also evaluated to determine the optimal conditions to maintain the quality of the kefir snack. Lactic acid bacteria (LAB) and yeast were isolated from freeze-dried kefir samples to evaluate their viability during the freeze-drying process.
The study demonstrated that freeze-dried kefir snack bites with either 15% trehalose or maltodextrin provided the best structure but trehalose protects more LAB and yeast density. The optimal freeze-drying conditions were determined at -20 °C for 12 hours, resulting in the highest product quality. The freeze-dried kefir products maintained their structure but were decreased 2 logs of LAB density during 28 days of storage in aluminum foil bags at room temperature but remained within an acceptable range for beneficial bacteria. These findings offer valuable insights for developing high-quality, long-lasting kefir milk snack bites, and should be considered for commercial production, and further research on nutritional and food safety aspects is also recommended to ensure the product's overall excellence.
Keywords: Freeze-drying, food additives, hardness, kefir, lactic acid bacteria, trehalose, yeast. il TÓM TẮT Snack kefir sấy khô đang ngày càng phé biến như một loại snack tiện lợi và bổ dưỡng dựa trên sữa. Tuy nhiên, đảm bao chất lượng và tuổi thọ sản phẩm là rất quan trọng dé đáp ứng nhu cầu của người tiêu dùng và yêu cầu của các cơ quan quản lý. Nghiên cứu này nhằm tối ưu hóa quy trình sấy khô bằng đông lạnh cho snack kefir sữa bằng cách sử dụng các chất phụ gia (trehalose, maltodextrin và bột năng) dé cải thiện tính 6n định cấu trúc và bảo tồn vi sinh vật có lợi.
Ngoài ra, nhiệt độ đông lạnh (-10 °C, -20 °C, -30 °C) và thời gian say khô (12, 18, 24 giờ) cũng được đánh giá dé xác định điều kiện tối ưu dé duy trì chất lượng của snack kefir. Vi khuẩn lactic acid (LAB) và nam men đã được phân lập từ các mẫu kefr sấy khô dé đánh giá khả năng sống sót của chúng trong quá trình sấy khô. Nghiên cứu đã chứng minh rang snack kefir sấy đông khô với 15% trehalose hoặc maltodextrin cung cấp cấu trúc tốt nhất, nhưng trehalose bảo vệ mật độ LAB và nam men tốt hơn. Điều kiện tối ưu của quá trình sấy đông khô được xác định là đông lạnh tại -20 °C và sấy khô trong 12 giờ, dẫn đến chất lượng sản phẩm cao nhất.
Snack kefir say khô duy trì cau trúc của mình nhưng mật độ LAB giảm khoảng 2 log trong vòng 28 ngày lưu trữ trong túi nhôm ở nhiệt độ phòng (25 + 1 °C), nhưng vẫn nằm trong khoảng chấp nhận được cho vi khuẩn có lợi. Những kết quả này cung cap thông tin hữu ích dé phát triển snack kefir sữa chất lượng cao, bền vững và nên được xem xét cho việc sản xuất thương mai và nghiên cứu thêm về các chỉ tiêu đinh dưỡng và an toàn thực phẩm khác. Từ khoá: độ cứng, kefir, nam men, phụ gia thực phẩm, sấy đông khô, trehalose, vi khuẩn lactic acid. IV CONTENTS Page.
1 AFFIRMATION AND COMMITMENT .:c:c::cescecescesseseeseeseeseeseeeceseseeceseeeaseneees il ABS TRACT aonunanunans aman om nee ern een 1H 772.ÔÔÔÔỎ V LIST OF ABBREVIATIONS. 22 1212121121121111 211111211 81101101121 e2 vill LIST (OF TABLE wrccescssacsassnsrasmnnconsssesssusuecanteass acumen ccna meme amaaeear eam maaeeaN 1x LIST 0138516101100 ẺẺẺẻẻốẻẻốẻẺẻẻẻẻẻẻ X 0. Problenn SIa[GTIIGTIbsssosistisetblioigrtSiiTSEIEESGSIESEIEBINAEEGIDBGGISGESESNGEHRSERSRlSfESegsSitsm 1 LBs OD] GV oiroergberstinineODDEEiSS-EETUlRGPEstotiRudtrradiptpsgdbStsrdosingiÐlg3 oer reeset 2 Ve Sig CGIIIETTEỗoossnissebssbbisbttttbsstiseotggtias6nddosbstbrtsilsDtsiiggstBSD8LG0E00830504285EDIJBDESiiESDDHGILGSĐSSEH2HHHg01400. LITERATURE RE VIE W sscsevssnwesscevessnsinsesconeonsoncenscsmvensexvesonssnesunescamusteuusses 3 2.
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8 DA, PUCEZS IVINS TDHG GEN cac scsice cnc va sevasitah aniusindna 330B. 12 2505: LẠITUWDNISEBT(GHEsrazbstotorrorsiltiiezrraeltbsgoolý/23gzxppzs:psirfoslparlilbygosgszasgiibostosgipe@sitatteGbBsseienisi 12 c0ke CWA OSS Ï sgrrgoisoisiEtEDEHENRUANHIAIEGRSIEBENBHGNEGENNEEIEAESRIIGIDESSEENDENHEGIAEGNGISIGSIEREENGIDSIGMERS 13 Chapter 3. AND METHODS sczeseseseaeeasisosssaeiesronosaee 14 Sila Lie atid; lô6ấtióRR GÍ THe SH seesessesdeseooinsseGiotoishBLSBSEUDS. 14 SA Material Said tm Cth OS TƯ ỚỚỚớỚợẢ xa ốc 14 31/51 VIRECELAssxssssuzexessisieSbsxbiibiiusokssisdd2.
Freeze-drying DFOC€SS. Experimental design sasssspsssesssisessgg515505610845339584/95165143551S01603939455E53836345555585139855458 17 3. Technique of Spreading on Plate for microbial ©OUH. Analyze the hardness of the snack bite.
Determine the moisture content of the snack bIfe. RESULTS AND DISCUSSIƠN. Lactic acid bacteria (LAT).egšt-and;THO sssassessssbieas640166S5101G001833018 moma ROE Es 25 4. Evaluated the effects of food additives on the freeze-drying process and quality of i0.
Structure evaluation and shape observations of freeze-dried kefir. Moisture Content and texture hardness .-- -- 5-52 +++xs+ssreeerrrerrrrrrrrrree 28 vi 4:2:3› Viability Of HHGfOOT0NH1SHIS sussddrbsos Big 0g 1800 0018141040150001814G013410)00003308903401888 29 4. Optimizing the Freeze-Drying Process. Structural and shape Obs€rVAfIOTIS.- ---- 5 5+ 32x sen 32 SÀN ii.
Viability of mICTOOTBATN1SINS. Determination ofthe shelf life of the freeze-drying kefir snaeks. LEXIUIES HA LOTHỆŠÃösssssnnniseiide s06 t0n3063099553606939404915E43993308S83499S950NSSSSEE43935003.3 Viability GiT110fEGGTFBBIHRHHEonsesseeooiseioibseisetePiistcdbb2sdg8BS050508.GSH4GI2ĐSEEHSP 39 Chapter 5. CONCLUSION AND SUGGESTION .cccececeeseeseeceeceeeeeeeeeeneeeeeesteaees 41 S0 MCGTEIIS TOTS: 5 nan seneinsith ốốốố ốc CC 4] 5.
sácececsaiionnsbiosii165114616360943995653514393395855859893894389303935918SXS5N433333053803355553838 47 Vil LIST OF ABBREVIATIONS CFU : Colony-forming unit DRBC : Dichloran-rose bengal chloramphenicol FAO : Agriculture Organization of the United Nations LAB : Lactic acid bacteria MRS : Man, Rogosa and Sharpe SD : Standard deviations WHO : World Health Organization of the United Nations vill LIST OF TABLE Page Table 3. Mixing recipe for snack producfIOH. The formulations were set up under different dry time and freeze (eniperalite CONGMONS:.<5 252s oe eee ee eee ee ee eee eee, 19 Table 4. The hardness and moisture of freeze-dried kefir products.
Yeast density (CFU/mg) of freeze-dried kefir products after freeze- 6227. The influence of temperature and drying time on the hardness (g) of freeze- dfL6d KEHF SIPUCHIEE sasnerseeiaoeniinsiDDL00810181180386330118898548365535033K011908SE434S8480105858360/0848590888/0Ể0 34 Table 4. The effect of temperature and drying time on the yeast density (CFU/g) of PES TEAC TEM, GEE scscescesnssannssisnncsaswaansics sas uancawae sxssucas sence 315629388 Sh2HE4182:G883140673605981064935586g8858 35 Table 4. The effect of temperature and drying time on the LAB density (CFU/g) of ANEEZE- CELE IES LIT.
sneneisnnssoinnnnsitenonsineiinniassnnninesienasidnaieninaslonainsnseinnnnitenalians nebtinenanindt npeaeainnnannsnanan 36 IX LIST OF FIGURES Page Figure 2. Different fermentation pathways follow from the main microbial contained in kefir and kefir grains. Biological properties, nutritional value, and macro- and micronutrient CONPOSILON Of KENT wsememmncscmamnemcavenmeemnenemne eee erm 6 Figure 2. Lactic acid bacteria.
Cryo-SEM of Lactobacillus delbrueckii ssp. bulgaricus CFL1, frozen in a TiTGGGHIWGITTIRHTNobisnoieebbbiG Go tenioSaliesbgfoBqusisgidbioiohlgeusEo:Rbssgbio3i8oz8g33sEpcRglgigssstsssiSenl 10 Figure 3. ETIệE 6 DIV Ei cua ga hán ngà H1 g4101415. Flowchart of snacks produ€tION.
Snack bites hardness measurement by fruit hardness tester. LAB resolves CaCOs3 in MRS medium.-- cece cece ceeteeeeeeeees 24 ETipure:4:2; Gram=positive’ bactertascacsscmpe eae ne RRO ES 24 Figure 4. The catalase reaction. Colony of yeast in DRBC.
Yeast morphology observed under a m1ICrOSCOD€. The appearance of fresh kefir after freeze-drying when removed from the SIIIIfET TH) l6]: ee etter eee nna ee een ner eee ance ee ene See ee ee ene Eee 26 Figure 4. The structure and shape of kefir snack bites when removed from the milbte(oii (og16(0) (0 Reece eee eee eee ee ee ee ee ee 27 Figure 4. LAB density after freeze drying.
Freeze-dried kefir (T15) under different drying conditions. Preserve kefir dried using aluminum foil bags. Texture hardness on 28 store days. Yeast density (CFU/g) after 28 store days.
LAB density (CFU/g) after 28 store days. Problem statement In today's fast-paced lifestyle, consumers increasingly demand nutritious and convenient snack options that align with their health-conscious choices. Kefir milk snack bites have emerged as a promising product, combining the goodness of probiotics and the delectable taste of kefir milk, making them an attractive choice for health- conscious individuals seeking on-the-go sustenance. Freeze-drying has been identified as an effective preservation technique for kefir milk snack bites, allowing for extended shelf life while preserving the product's nutritional value and sensory attributes.
However, the current freeze-drying protocols for kefir milk snack bites present challenges that hinder widespread adoption and commercial success. Recent studies have highlighted several critical issues concerning the freeze- drying process for kefir milk snack bites. Despite its potential benefits, the existing protocols may not adequately preserve the product's quality attributes, leading to inconsistencies in texture, taste, and appearance. Consumers' acceptance of these snack bites depends on their sensory appeal and any negative impact on these attributes may hamper market acceptance and commercial viability.
Moreover, kefir milk snack bites are often formulated with live probiotic cultures that confer numerous health benefits, including improved gut health and immune function. However, during the freeze-drying process, the viability of these beneficial microorganisms may be compromised, reducing the product's overall health-promoting potential.