Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322
Performing Department
Nutrition Dietetics & Food Sci
Non Technical Summary
Gelatin is commonly used ingredient in the manufacture of ice cream, frozen desserts and yogurt, because it maintains structural integrity and provides desirable mouthfeel. However, use of gelatin in dairy products prohibits their consumption by lacto-vegetarian and Kosher population for being derived from meat. Replacing gelatin with dairy ingredients would cater needs of wider consumer base. Because of gel forming ability at low temperatures (<10oC), micellar casein concentrates (MCC) can potentially be used as a replacement of gelatin in structured dairy. One of the limitations with use of MCC as stabilizer is that it forms gels only at higher concentrations (>15%) making it hard to accommodate with such high protein levels to the dairy products. We hypothesize that by manipulating physico-chemical conditions and adding associative biopolymer, it would be possible to reduce the minimum concentration needed to form a cold gel and increase the gel strength. For achieving this goal, we propose three strategies; 1. modify gel forming conditions by altering pH and introducing calcium chelating agent such as tri-sodium citrate (TSC) and sodium hexametaphosphates (SHMP), 2. addition of an associative biopolymer i.e. kappa-carrageenan and/or low methoxy (LM) pectin; 3. Use of high intensity ultrasound (HIU) waves for manipulating interactions between hydrated casein micelles as well with polysaccharide. Fundamental understanding of cold gelling behavior of MCC will help develop novel plant and dairy (hybrid) food matrices. Findings from this work will help US Dairy and Food Industry to develop diversified and affordable food products to meet specific dietary needs of general population.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall aim of the proposed project is to determine critical factors controlling formation of thermoreversible gel from the concentrated casein micelle solution in the presence of associative polysaccharides (kappa-carrageenan or LM pectin) with or without application of high intensity ultrasound (HIU) treatment. Critical factors for this study include pH, protein concentration, the amount and type of calcium chelating salts and ultrasound treatment.Specific aims of this study areUnderstanding mechanism of cold gel formation from HC-MCC and determining extent of thermoreversibility.Working hypothesis for this objective is that cold gelling behavior of concentrated dispersion of casein micelles depends upon various physico-chemical factors such as pH, amount of colloidal calcium phosphate (CCP) associated with caseins, water holding capacity of casein micelles, and balance of hydrophilic and hydrophobic interactions. Factors that displace CCP from casein micelle would release additional ionic groups that could now engage extra water molecules causing further swelling even at low protein concentrations and reinforce the gel matrix.Optimizing formulation and processing conditions for formation of thermoreversible gels from associative MCC-polysaccharides mixtures.Working hypothesis for this study is that associative mixture of proteins and polysaccharides will form a thermoreversible gel under optimized conditions (ratios, pH and temperature of mixtures) even at low casein concentrations (such as <7%) and at body temperature. The main reason for this behavior will be the change in the particle size of casein micelles and change in protein-water interactions due to presence of polysaccharides.3. Studying the impact of high-intensity ultrasound (HIU) treatment on the cold gelling behavior of protein (caseins) and polysaccharide (kappa carrageenan) associative mixtures (different ratios).Working hypothesis for this objective is that modification of secondary structures of caseins gel due to HIU will improve gel forming characteristics of MCC in the presence or absence of an associative polysaccharide. This will help us to achieve our goal of matching gelling ability of MCC to gelatin gels.The overall goal of this study is to develop fundamental understanding of formation of novel structures from micellar casein concentrate dispersions which can useful in diverse food applications. In this study we do not intend to develop a product or apply the product in a formulation, rather to investigate and establish the potential of MCC solutions for novel applications in the future. These applications will include dairy and non-dairy applications. The long-term goal of this study is to understand the formation of thermoreversible MCC gels that can be used as a novel dairy and non-dairy ingredient for the application in the foods suitable for vegetarians and kosher population. The diversified use of these ingredients will improve overall sustainability of US dairy and food production and processing systems.
Project Methods
SPECIFIC AIM #1: Understanding mechanism of cold gel formation from HC-MCC and determining extent of thermoreversibility.Overview:In the first specific aim, we will be testing hypothesis that it is possible to form thermoreversible gels by modifying MCC. We further hypothesize calcium chelation, hydrophobic interactions and swelling of micellar structure due to pH modification would improve gelling behavior of the MCC samples. We will use 1% gelatin concentration as a benchmark for various viscoelastic, textural and tribological characteristics. To test above hypotheses, we will be identifying minimum concentration (Cg) of protein needed to form a cold gel in various conditions (pH, TSC/SHMP treatment, calcium content) (Fig. 7). Frozen HC-MCC samples (protein ~23%) used in previous study (Zad Bagher et al., 2021) will be used in this study according to protocol suggested by Lu at al. (2015). Un-treated MCC solutions with varying protein concentration (5-20%) will be first evaluated for cold gelling temperature (CGT) by using combinations of time and temperature sweeps. Gelling behavior and gel characteristics will be studied using small angle oscillatory rheological techniques and texture analysis. Once we will identify lowest possible concentration to form a cold gel, we will then introduce pH (6.0-7.4), TSC (0-50 mM), SHMP (0-100 mM) treatments as a mean to increase gelling temperature and further reduce the minimum gelling concentration needed for forming a cold gel. Liquid samples treated with TSC or with different pH levels will be subjected for in-situ particle size analysis, zeta potential, and colloidal and ionic calcium levels. Ultra-structure of selected samples will be evaluated using transmission electron microscopy (TEM).SPECIFIC AIM #2:Optimizing formulation and processing conditions for formation of thermoreversible gels from associative MCC-polysaccharides mixtures.OVERVIEWOur hypothesis for this phase is that addition of associative polysaccharides to the protein solutions will help minimize concentration needed for forming a thermoreversible gel. Our target protein concentration forming a gel is <7.0%. We further hypothesize that there may be a co-polymerization of both hydrocolloids or a competitive solvation. In both cases, gel strength and minimum protein requirement are going to be improved.Three samples with minimum protein concentration at each treatment i.e. pH, TSC, SHMP will be selected in the specific aim 1. This will be done with respect to minimum concentration (Cg) of protein needed to form a cold gel (at par with 1% gelatin gel) from untreated MCC and samples treated with pH and TSC/SHMP levels. A higher cold gelling temperature (CGT) and thermo reversibility of the gels will be other criteria to short list the samples from the specific aim 1. These samples will then be added with an associative hydrocolloid (kappa-carrageenan) and/or LM pectin at three different levels i.e. 0.01, 0.1 and 0.5% by modifying the method described by Spagnuolo et al., (2005). Preliminary experiments will be conducted to investigate whether kappa-cargeen and/or LM pectin will work better alone or in combination. MCC and kappa-carrageenan/pectin mixture will then be subjected for cooling for cold gel formation and thermoreversibility of cold gels will be tested using the methods described in the specific aim 1. In this specific aim we will target our strategy to further reduce the minimum casein concentration needed to form a thermoreversible gel. To achieve this goal, at each level of kappa-carrageenan concentration, we will use an array of protein concentration ranging 1-10%. Liquid samples will be subjected to particle size and zeta potential analysis using the methods described above. Water binding ability of the new gels will be determined by applying centrifugal force of 20,000 g for 5 min at 4oC. Molecular changes in the proteins (caseins) will be analyzed using Urea-PAGE technique (Lamichhane et al., 2019). From the second objective we will be able to determine whether presence of polysaccharides improved gelling behavior of MCC and it is possible to further reduce the minimum concentration needed for cold gel formation. Ultrastructure analysis will help us to visualize type of structures formed due to interactions between MCC and the polysaccharide.SPECIFIC AIM #3:Studying impact of high intensity ultrasound (HIU) treatment on the cold gelling behavior of protein (caseins) and polysaccharide (kappa carrageenan/pectin) associative mixtures (different ratios).OVERVIEWHypothesis of this phase is HIU treatment may alter the structure of micellar caseins in a way that it may interact more with associative polysaccharide in order to form an elastic, stable thermoreversible gel. In this work too, we will use 1% gelatin as a benchmark testing material for achieving our goal of matching gelling ability of MCC to gelatin gels.HIU treatment will be applied to the samples which exhibited maximum gel strength, elastic properties and gel-sol transition temperature around 37oC great potential for replacing gelatin gels. Based upon the results obtained for specific aim 1 (physical modification) and 2 (associative polymer), treatment conditions (HIU) for this aim will be determined. Objective 3 will be performed in Dr. Martini's laboratory by a master's student. Dr. Martini is an expert in the field of ultrasound treatment and also very familiar with the experimental design described in this proposal.HIU treatment: Three most promising samples from the specific aim 2 will be selected for HIU treatment (20 kHz). Schematics of HIU treatment is shown in the Fig. 8. Samples will be screened based upon the gelling time (shorter), gel strength (high gel strength) and (higher) temperature of cold gelation (CGT). Three levels of sonication (10, 50, 100W) for three different durations (5, 15 and 30 min) will be applied to each sample at three pH levels (6.7, 7.4, and 8.0) and two temperatures (80oC) and gelling temperature. Literature shows, ultrasound treatment of micellar caseins in alkaline environment increases gel strength, similarly high temperature sonication helps with brining out more structural changes in casein micelle structure and its interaction with polysaccharides (Leong et al., 2018; Ragab et al., 2020). Post HIU treatment, pH will be readjusted to 6.7. Temperature will be controlled during ultrasonication using a double walled beaker that allows for temperature control using an external water bath. HIU treated samples will then be loaded on the MCR302 rheometer for studying gelling behavior using protocol developed by Zad Bagher Seighalani et al., (2021). CGT and Gel strength will be determined using multiple wave rheological techniques as described in the specific aim 1 and 2.Fourier transform infrared (FTIR) Analysis: To observe the changes in the secondary structure of caseins due to HIU treatment, FTIR spectroscopic analysis will be conducted on an IRTracer-100 infrared spectrometer (Shimadzu Corporation, Japan) equipped with a diamond ATR crystal (Quest Single Reflection ATR Accessory, Specac Limited, United Kingdom). Samples will be placed on the diamond ATR crystal cell. Total 32 scans with 4 cm-1 resolution will be conducted on each sample. After applying baseline correction, characteristic bands will be interpreted with the software KnowItAll (Biorad Laboratories, Philadelphia, PA, USA).