A probiotic goats' milk yoghurt was developed containing probiotics (Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp.), and using polymerized whey protein (PWP, 0.4%) and pectin (0.3%) as gellation agents. The yoghurt was analyzed for chemical composition, mold and yeast counts, changes in pH, titratable acidity, and viscosity, and probiotic survivability during storage at 4 Â°C. There was no significant difference in viscosity, however, changes in titratable acidity and pH showed significant difference during storage. Both Lactobacillus casei and Bifidobacterium spp. remained viable and their populations were above 106 CFU g-1 during storage. However, there were no viable counts of Lactobacillus acidophilus by the fourth week. Scanning electron microscopy of goats' milk yoghurt revealed that PWP interacted with casein micelles to form a comprehensive network in the yoghurt gel. The results indicated that PWP may be a novel protein-based thickening agent for improving the consistency of goats' milk
yoghurt and other products alike.
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Key words: goats' milk, yoghurt, probiotic, polymerized whey protein
Goats' milk production ranks after cows and buffalos in the world (Guo, Park, Dixon, Gilmore, & Kindstedt, 2004). Goats' milk products may be used as alternatives for cows' milk products due to fewer allergic reactions (Uysal-Pala, Karagul-Yuceer, Pala, & Savas, 2006). Fermented goats' milk products such as yoghurt and cheese are considered as specialties in the US. However, it is difficult to make goats' milk yoghurt with a consistency comparable to cows' milk yoghurt, which is mainly due to the difference in casein content and its composition (Guo, 2003; Li & Guo, 2006).
The most commonly used methods to improve consistency and/or texture of yoghurt include increase of total solids in the milk and addition of stabilizers such as pectin. Pectins, anionic charged polysaccharides derived from plant cells of fruit, are often used as gelling agents and stabilizers in low-pH food products such as acidified milk drinks and yoghurt (Kazmierski, Wicker, & Corredig, 2003). Improvement of gels can be achieved by use of gelling agents. It has been shown that goats' milk yoghurt firmness and syneresis can be improved by fortification of milk with polymerized whey protein (PWP) (Li & Guo, 2006) and use of microbial transglutaminase (Farnsworth, Li, Hendricks, & Guo, 2006). Vardhanabhuti, Foegeding, McGuffy, Daubert and Swaisgood (2001) defined PWP as "soluble whey protein (WP) aggregates that are formed when heated at a temperature and protein concentration that would normally form a gel but do not due to the low salt condition." More recently, interest in PWP has centered on the formation of WP gels at low temperatures, which are called "cold set" gels (Fitzsimons, Mulvihill, & Morris, 2008). This "cold set" gelation is a two-step process. First, the pH of a WP solution is adjusted sufficiently higher than the isoelectric point of WP to prevent aggregation as a result of electrostatic repulsion between protein aggregates. Then, the solution is heat-treated at a specific temperature and duration (Alting, Hamer, de Kruif, & Visschers, 2003). Upon cooling, the aggregates remain soluble and their properties, such as aggregate size, keep constant for several days (Alting, Hamer, de Kruif, & Visschers, 2000). Second, after minerals are added or pH is lowered to the isoelectric point, the electrostatic repulsion trends to decrease and consequently a cold-set gelation forms (Bryant & McClements, 1998).
Consumers are aware of the health benefits of yoghurt products containing probiotic bacteria (e.g., Lactobacillus acidophilus (L. acidophilus) and bifidobacteria) (Ravula & Shah, 1998). Probiotics have been revealed to have many claimed beneficial effects (e.g., reduction of lactose-intolerance) and therapeutic applications (e.g., alleviation of constipation) in humans (Fuller, 1989). A minimum of 106 CFU g-1 was suggested by Guo (2007) for the total number of probiotic organisms in fermented products to achieve optimal potential therapeutic effects.
The objective of this study was to investigate the effects of PWP on consistency and microstructure of goats' milk yoghurt and to develop a probiotic goats' milk yoghurt using PWP and pectins as gelation agents.
2. Materials and methods
Starter culture, Yo-Fast 10 (Chr. Hansen, Milwaukee, WI, USA), was a blend of strains of Streptococcus thermophilus (S. thermophilus), Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus), L. acidophilus, Bifidobacterium spp., and Lactobacillus casei (L. casei). Whey protein isolate (WPI) (ALACEN 895) was provided by NZMP (Auckland, New Zealand). Low-methoxyl pectin (GENU texturizer type YA-100) was obtained from CP Kelco (Lille Skensved, Denmark). Pasteurized whole goats' milk was gifted from Oak Knoll Dairy (Windsor, VT, USA) and pasteurized whole cows' milk was purchased from commercial source.
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2.2. Preparation of polymerized whey protein (PWP)
WPI powder was dissolved in cold purified water and held at 4 Â°C overnight. The (10%, w/v) WP dispersion was adjusted to pH 7.0 with 0.1 M sodium hydroxide at 21 Â°C. It was heated at 85 Â°C for 30 min in a water bath and was cooled rapidly to room temperature in ice-water with agitation.
2.3. Preliminary trials for optimization of manufacture technology using PWP and pectin as gelation agents
To determine optimal consistency, goats' milk yoghurt with different contents of WP, pectin, PWP and mixture of PWP and pectin in different ratios were studied (see Table 1). Yo-Fast 10 starter culture (0.02%, w/w) was added to goats' milk at 43 Â°C. WP, pectin, PWP, and mixture of PWP and pectin were added and incubated at 43 Â°C for 4.5 h. Where pectin was added to milk, it was then heated to 80 Â°C, dissolved completely and then cooled down to 43 Â°C. Cows' milk yoghurt samples with similar formulations to the above were also prepared as control. Three trials of each experiment were carried out.
Viscosity of all samples was measured using a Brookfield viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) and expressed in mPa.s. Viscosity measurements were made for 30 seconds at 100 rpm.
In order to determine water-holding capacity of the yoghurt, syneresis of both goats' and cows' milk yoghurts with PWP content of 0, 0.2, 0.4, 0.6 (%, w/w) was determined by a centrifugation procedure (Keogh & O'Kennedy, 1998) with modifications according to Li and Guo (2006). 200 g of yoghurt (Y) samples were fermented in centrifuge cups and centrifuged at 640 Ã- g for 10 min at 4 Â°C. The separated whey (W) was poured out to a preweighted beaker and weighed. Three trials were carried out. The syneresis was calculated using the formula below:
Syneresis (%) = (W/Y) Ã- 100% (1)
2.4. Preparation of samples
Based on the results of preliminary studies, the goats' milk yoghurt was prepared using a combination of pectin (0.3%, w/w) and PWP (0.4%, w/w). Again, cows' milk yoghurt was prepared as a control. Cold milk and pectin were heated to 80 Â°C to dissolve the pectin, then cooled down to 43 Â°C and PWP and Yo-Fast 10 yoghurt starter (0.02%, w/w) were added. The mix was incubated at 43 Â°C for 4.5 h and stored at 4 Â°C before testing. Three batches of samples were prepared on different days for chemical analysis, shelf-life tests and survivability of probiotics during storage at 4 Â°C.
2.5. Chemical analysis
The yoghurt samples were analyzed for total solids, protein, fat, and ash contents using standard AOAC procedures (AOAC, 2002). Total solids content was determined by forced air oven drying. Protein content was assayed by Kjeldahl method. Fat content was determined by Soxhlet method. Ash content was measured by dry-ashing using a muffle furnace. The content of carbohydrate was determined by the difference of total solids minus other solid components as described by Guzman-Gonzalez, Morais, Ramos and Amigo (1999). Minerals were determined from an ash in nitric acid solution from the ash samples using an atomic absorption flame emission spectrophotometer (AA-6200 Series, Shimadzu, Kyoto, Japan). All values reported were the mean of three measurements.
2.6. Shelf-life tests and survivability of probiotics
The values of pH, titratable acidity (TA) and viscosity and enumeration of probiotics were determined weekly for 12 weeks, while mold and yeast counts were evaluated every two weeks for 12 weeks for both goats' and cows' milk yoghurts. The measurements of pH, TA and viscosity were carried out at 21 Â± 2 Â°C. The value of pH was determined using a pH meter (IQ Scientific Instruments Inc., San Diego, CA, USA). TA was measured by titrating a sample (9 grams), diluted with 18 ml water, with 0.1 M sodium hydroxide using phenolphthalein as an indicator. Viscosity was measured using a Brookfield viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) as described above.
Probiotic survivability was quantified according to the procedures of Walsh, Ross, Hendricks and Guo (2010). Enumeration of L. acidophilus and L. casei was done using the spread plate method on MRS-IM agar. Bifidobacterium spp. was enumerated using the pour plate method on MRS-IM agar. L. acidophilus plates were incubated at 37 Â°C and L. casei plates were incubated at 20 Â°C. Bifidobacterium spp. plates were incubated anaerobically at 37 Â°C. Mold and yeast counts were carried out using Yeast and Mold Petrifilm plates (3MTM PetrifilmTM, St. Paul, MN, USA). The plates were stored at 21 Â°C for 5 days.
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2.7. Microstructure analysis by scanning electron microscopy (SEM)
Microstructure of the yoghurt samples made with 0.4% PWP, 0.3% pectin and mixture of 0.4% PWP and 0.3% pectin was examined using SEM according to the procedures of Walsh et al. (2010). Samples were prepared by embedding the yoghurt samples into agar cubes and were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and post fixed in 1.0% osmium tetroxide followed by three rinses in diluted (50 mM) cacodylate buffer (pH 7.2). After dehydration using a ethanol dehydration series, the samples were fixed on aluminum SEM stubs and sputter coated with 3 nm of Au/Pd (80/20) alloy and evaluated using a scanning electron microscope (FEI Quanta 200F MKII, Eindhoven, The Netherlands) operated at 5 KV. Micrographs were taken at different magnifications and these were marked on each photograph.
2.8. Statistical analysis
The data were analysed using a 2-way repeated measures ANOVA. A Bonferoni post-test compared each individual week with all others, to determine where significant differences occurred.
3. Results and discussion
3.1. Preliminary trial results
The viscosity of the yoghurt samples with different levels of WP, PWP, pectin and mixture of PWP and pectin in various ratios were investigated (Fig. 1). The results showed that yoghurt fortified with mixture of PWP and pectin had higher viscosity than that of those with single thickening agent and the viscosity of yoghurt containing WP was the lowest. Native whey proteins exhibit much lower viscosity than PWP and have not traditionally been utilized as a thickening agent due to their small molecular size and approximately spherical shapes. However, PWP has a much larger effective hydrodynamic volume than native globular proteins. Heating WP solutions under controlled conditions forms soluble WP polymers of high molecular weight, resulting in an increase in viscosity (Vardhanabhuti & Foegeding, 1999). Pectin is often added to yoghurt to improve consistency (increase viscosity) and reduce syneresis (Everett & McLeod, 2005). Although PWP and pectin increased yoghurt viscosity individually, the texture of the goats' milk yoghurt was not greatly improved. The textural defects in the yoghurt include weak body, whey separation and gumminess. The addition of a mixture of PWP and pectin resulted in a desirable texture of yoghurt with increased viscosity and mouth feeling. As evidenced by syneresis analysis (Fig. 2), goats' milk yoghurt fortified with 0.4% PWP showed minimal syneresis. Visual inspection showed that the consistency of the yoghurt with 0.4% PWP and 0.3% pectin was the most desirable, being fairly firm and very little whey separation.
Whey separation refers to the appearance of serum on a gel surface, such as on the top of a set yoghurt during storage, which is caused by shrinkage of the gel (syneresis), leading to whey separation (Lucey, 2002). There were significant differences between goats' and cows' milk yoghurts (P < 0.001) and between the PWP levels (P < 0.001) for syneresis (Fig. 2). For cows' milk yoghurt there was no significant difference of syneresis across the levels of PWP (P > 0.05). However, syneresis of goats' milk changed significantly depending on the levels of PWP. The differences occurred between PWP levels of 0% and 0.4%, 0.6% (P < 0.001), 0.2% and 0.4%, 0.6% (P < 0.01) for goats' milk yoghurt. The interaction was also significant (P < 0.001) indicating that the rate of changes of syneresis was different for both yoghurts. When 0.4% PWP was added to the goats' milk yoghurt the syneresis was the least (Fig. 2). The results were similar with the findings of Britten (2002) and Li and Guo (2006) showing that incorparation of
PWP can decrease syneresis of yoghurt.
Low solids content, high incubation temperature, excessive WP to casein ratio, and physical mishandling of the product are common attributes to the occurrence of syneresis in yoghurt (Lucey, 2004). As a fermented milk product formed by gradual acidification with a lactic starter, yoghurt may have whey separation or syneresis with a change of temperature or physical impact (Li & Guo, 2006). WP gels have high capacity to hold water in their matrix and goats' milk with added PWP has a greater capability to immobilize the aqueous phase, therefore decreasing the susceptibility to syneresis in the yoghurt gel network (Li & Guo, 2006; Sullivan, Khan, & Eissa, 2008). Goats' milk yoghurt with PWP (0.4% w/w) resulted in minimal syneresis. However, the syneresis increased when extra PWP was added to goats' milk yoghurt suggesting excessive use of PWP has a negative impact on syneresis.
3.2. Chemical composition
Gross composition and mineral contents of goats' and cows' milk yoghurts are shown in Tables 2. Chemical composition of yoghurt varies depending on the type of milk used, type of yoghurt manufactured, and fortification methods, etc. (Farnsworth et al., 2006). There were significant differences between the yoghurts for zinc (P < 0.01), magnesium (P < 0.01), potassium (P < 0.001), total solids (P < 0.05), carbohydrates (P < 0.05) and ash (P < 0.01). The levels of total solids, protein, carbohydrates and sodium in goats' milk yoghurt were lower than those in cows' milk yoghurt. However, goats' milk yoghurt had higher levels of fat, ash, zinc, magnesium, calcium, and potassium.
3.3. Changes in pH, TA and viscosity during storage
There was no significant difference (P > 0.05) in pH between goats' and cows' milk yoghurts (Fig. 3A). There was, however, a significant difference between the weeks (P < 0.001) for both yoghurts, especially during the first a few weeks for goats' milk yoghurt (P < 0.01) and cows' milk yoghurt (P < 0.05). The interaction was also significant (P < 0.05) indicating that the rate of pH changes was different for both yoghurts. The pH values decreased from 4.23 Â± 0.05% to 4.08 Â± 0.03%, and from 4.23 Â± 0.02% to 4.12 Â± 0.02% for goats' and cows' milk yoghurts over the 12 weeks, respectively.
The differences in TA were significant between goats' and cows' milk yoghurts (P < 0.05) and between the weeks (P < 0.001). TA increased significantly in the first two weeks, however, there were no statistically significant changes from week 2 onwards for both yoghurts (Fig. 3B). The interaction was not significant (P > 0.05) indicating that the rate of TA changes was not different for both yoghurts. TA increased from 0.86 Â± 0.02% to 0.90 Â± 0.01%, and from 0.83 Â± 0.01% to 0.91 Â± 0.01% for goats' and cows' milk yoghurts, respectively, during the 12-week storage.
The levels of viscosity were shown to be significantly different between goats' and cows' milk yoghurts (P < 0.05) (Fig. 4). There was not, however, a significant difference between the weeks (P > 0.05) for each yoghurt type. For goats' milk yoghurt there was no significant difference between all 12 weeks (P > 0.05). The differences occurred between week 1 and 3, 7, 8, 11, 12 (P < 0.05) for cows' milk yoghurt. The interaction was not significant (P > 0.05) showing that the rate of changes was the same for both yoghurts. The viscosity value showed a significant decrease from week 1 to week 3. The viscosity dropped until week 3 followed by a rate of change that was not significant for cows' milk yoghurt.
The pH decreased and TA increased upon 12-week storage for both yoghurts, which was probably caused by the starter cultures which utilize lactose as a substrate and convert it into lactic acid during fermentation of milk. Lactic acid bacteria can produce lactic acid even during storage which was the principal cause of lowering of the pH (Kailasapathy, 2006).
3.4. Survivability of probiotics during storage
The population of L. acidophilus was above 106 CFU g-1 for the initial three weeks in goats' milk yoghurt and it remained above this level for the first six weeks for cows' milk yoghurt. Results showed a steep decline after the third and sixth weeks and became too low to count by the fourth and the seventh weeks for goats' and cows' milk yoghurts, respectively (Fig. 5A). The counts of L. acidophilus were shown to be significantly different between goats' and cows' milk yoghurts (P < 0.001) and between the weeks (P < 0.001). The interaction was also significant (P < 0.001) indicating that the rate of survival was different in the two yoghurts.
There were significant differences between goats' and cows' milk yoghurts (P < 0.001) and between weeks (P < 0.001) for Bifidobacterium spp. (Fig. 5B). For goats' milk yoghurt the difference was significant between week 3 and 12 (P < 0.05). The differences occurred between week 1 and 2 (P < 0.01), week 2 and 4-12 (P < 0.01), week 3 and 10-12 (P < 0.05) for cows' milk yoghurt. The interaction was also significant (P < 0.001) meaning that the rate of survival was different for each type. Changes in Bifidobacterium spp. counts showed a gradual decline for each type during storage, but was still well above the level of 106 CFU g-1 required for therapeutic effects over the 12-week shelf life.
In the case of L. casei the difference was significant between goats' and cows' milk yoghurts (P < 0.001) and between the weeks (P < 0.001). The differences occurred between week 1 and weeks 9-12, week 2 and weeks 3-12, week 3 and weeks 9-12 (P < 0.05) for goats' milk yoghurt (Fig. 5C). However there was no significant changes between all 12 weeks (P > 0.05) for cows' milk yoghurt. The interaction was also significant (P < 0.001) indicating that the rate of L. casei decline was different for each type. Like Bifidobacterium spp., L. casei counts remained viable at above 106 CFU g-1 during the 12-week storage.
Decrease in pH of the yoghurt and accumulation of organic acids and other compounds which are caused by bacterial growth and fermentation may be responsible for the reduced viability of probiotics (Hood & Zoitola, 1988; Shah & Jelen, 1990). Hydrogen peroxide produced by L. bulgaricus during the manufacture and storage of yoghurt was also claimed to inhibit the viability of L. acidophilus (Gilliland & Speck, 1977). The viability of L. acidophilus in goats' milk yoghurt was lower than that in cows' milk yoghurt (Fig. 5A). Figs. 5A and 5B show a better viability of Bifidobacterium spp. than that of L. acidophilus during storage. S. thermophilus could be beneficial for growth and survival of Bifidobacterium spp. as an oxygen scavenger creating an anaerobic environment (Lourens-Hattingh & Viljoen, 2001).
3.5. Mold and yeast
There was no mold or yeast detected in both goats' and cows' milk yoghurts at any time throughout 12 weeks of the study indicating that the 12-week shelf life was not limited by yeasts or molds. One of important factors determining the shelf life of yoghurt is the time the product remains safe to eat. In yoghurt products, yeasts and molds which tolerate low pH are principal spoilage organisms due to contamination in the processing operations (MacBean, 2009).
The microstructures of goats' milk yoghurt with 0.3% pectin (A), 0.4% PWP (B) and a mixture of 0.3% pectin and 0.4% PWP (C) are shown in Fig. 6. The SEM micrograph revealed that the casein micelles appeared relatively uniformly distributed and were relatively similar in size (Fig. 6A). Figs. 6B and 6C showed that the appearance of casein micelles were less defined. These differences were probably due to the interactions between casein micelles and PWP through mainly hydrophobic interaction leading to the formation of casein-PWP complexes.
Casein micelles play the major role in acid coagulation of milk. When the isoelectric point of casein micelles (pH 4.6) is approached a reduction occurs in surface charge (zeta potential) from the originally high net negative charges in milk to near no net charge. In addition, solubilization of colloidal calcium phosphate (CCP) which is an integral part of casein micelles also occurs during acidification (Lucey, 2004). The solubilization causes a disorganization of the micelles and a reorganization of the micellar subunits. Consequently, hydrophobic interactions increase, which results in the formation of a three dimensional network of casein micelles linked together in chains, clusters and strands (Phadungath, 2005). In goats' milk yoghurt, the gel is weak and less consistent compared with cows' milk yoghurt due to the difference in casein content and composition (e.g., low level of Î±s1-casein) (Guo, 2003; Li & Guo, 2006).
The bacterially acidified cold-set gelation of prepolymerized whey proteins may be a novel method to improve the texture and water-binding property of fermented dairy foods, such as yoghurt (Li & Guo, 2006). Cold-set gelation requires an initial preheat step to denature whey proteins, followed by lowering the pH to reduce the electrostatic repulsion between aggregates, and subsequently promotes aggregation (Bryant & McClements, 1998). When whey proteins are preheated alone they combine to form aggregates mainly linked by disulphide bonds and hydrophobic interactions. The aggregates remain soluble rather than precipitated and gelation at room or refrigerated temperatures (Alting et al., 2000). When these aggregates or polymerized whey proteins were added to the milk they interacted with casein micelles, probably through hydrophobic interactions, as the pH was decreased. Upon acidification of the milk an instantaneous gel was formed consisting of a mixture of casein micelles and WP aggregates (Schorsch, Wilkins, Jones, & Norton, 2001). Improvement of milk gel texture can also be achieved by adding polysaccharides such as pectins (Turgeon & Beaulieu, 2001). The mechanism of pectin stabilization of acidified casein micelles may be both explained from the adsorption of pectin onto the surface of casein micelles via electrostatic interactions and from the electrostatic repulsion between casein micelles caused by pectin conferring a net negative charge to the casein micelles (Lucey, Tamehana, Singh, & Munro, 1999). The balance between aggregation or gelation of casein, casein-PWP complex and so-called demixing effect by the addition of pectin favoring repulsive casein-pectin interactions eventually determined the microstructure of the gel.
SEM analysis for the microstructure of goats' milk yoghurt showed that when PWP was added to the yoghurt a relatively more comprehensive network was formed thus resulting in improved consistency and water-holding capacity of the goats' milk yoghurt.
PWP appears to be a suitable thickening agent for goats' milk yoghurt to improve its consistency and syneresis. The survival rates for L. casei and Bifidobacterium spp. were good and remained viable counts at above 106 CFU g-1 over the 12-week storage. Microstructure analysis indicated that PWP may interact with casein micelles to form a comprehensive protein network in the goats' milk gel matrix. Findings from this study demonstrated that PWP as a co-gellation agent may be useful for making quality goats' milk yoghurt and other similar fermented products.