The Interaction Effect of Mixing Starter Cultures on Homemade Natural Yogurt ’ s pH and Viscosity

Dairy yogurts are common food products consumed by people all over the world. Due to the simple process, many people have made their own natural yogurt at home. The fermentation due to the starter culture causes the textural properties of dairy yogurt. However, the literature is surprisingly scarce on the topic of starter culture interactions in the development of textural properties of dairy yogurt. This study investigated the interaction effect of three common starter cultures, Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophiles, on the viscosity of homemade yogurt. Using Design Expert software, a 10-run mixture model experiment was designed to examine the textural properties developed by single or multiple inoculation of these starter cultures. All yogurt formulations reached the isoelectric point of milk and had pHs in the range 3.97 to 4.32. Yogurt formulations with L. acidophilus and S. thermophilus resulted in viscosities which were similar to commercial yogurt viscosity (1.77 Pa·s), while L. bulgaricus resulted in yogurt with a lower viscosity. Based on the mixture model, L. acidophilus had most influence on the yogurt viscosity, followed by S. thermophilus and L. bulgaricus. In conclusion, L. acidophilus can be used as a single starter culture or combined with other starter cultures to develop high viscosity homemade yogurt. A Combination of S. thermophilus and L. acidphilus can also be used to develop high viscosity yogurts. However, L. bulgaricus should not be inoculated alone or become a dominant ratio in multiple starter culture inoculation as it will decrease the overall homemade yogurt viscosity.


Introduction
Yogurt is one of the basic fermented foods consumed worldwide.It is produced by fermenting bacteria in milk at appropriate temperatures (incubating temperature) and time.Yogurt's development is said to originate from the nomads in the Middle East (Tamime & Robin-son, 1999).The fermenting bacteria are now marketed as starter culture products (Mazzoli, Bosco, Mizrahi, Bayer, & Pessione, 2014).Each starter culture will coagulate the substrate where it is inoculated.The coagulation is due to accumulation of lactic acid that converts casein into lumps of micelles (Lee & Lucey, 2010).Lactobacillus acidophilus, Lactobacillus bulgari-cus and Steptococcus thermophilus are some of the most utilized starter cultures in the production of dairy products (Delorme, 2008).Both L. acidophilus and L. bulgaricus possess proteolytic capability, that is the capable of hydrolyzing food proteins to peptides and amino acids to contribute to texture, taste and aroma of fermented products (McSweeney & Sousa, 2000).
The key role of S. thermophilus is to breakdown lactose to lactic acid, and therefore reduce pH to preserve the product.S. thermophilus also produces secondary metabolites, such as exopolysaccharides and flavoured compounds (Broadbent, McMahon, Welker, Oberg, & Moineau, 2003).These probiotic starter cultures are now available for public use and sold in specialised shops for food ingredients.Thus, the general public can easily produce homemade natural yogurt for personal consumption (Fisberg & Machado, 2015).However, homemade natural yogurts are usually made without preservatives, unlike most commercial yogurts (O'Rell & Chandan, 2013;Smith, 2015).The literature regarding homemade natural yogurt is surprisingly scarce.Thus, this study aims to contribute towards the body of literature by investigating the interaction effect of mixing starter cultures on homemade natural yogurt's pH and viscosity.

Yogurt Preparation
Three common yogurt starter cultures were used: Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus.All three starter cultures were obtained from Custom Probiotics (Custom Probiotics Inc., California) in powder form.Pasteurized milk fresh from a local dairy farm in Sabak Bernam, Selangor, under the Farm Fresh Milk brand was obtained.100 ml of the dairy milk was poured into respective cups, before it was inoculated with approximately 1 g of starter cultures.Table 1 shows the mixture design that was used to make the 10 yogurt formulations, where each yogurt contains a different ratio of the 3 selected probiotic strains.The inoculation volume was based on the weight of the single starter culture powder or cumulative of multiple starter culture powders.For example, formulation 7 required one third of all three starter cultures.Therefore, 0.33 g of all three starter cultures was inoculated into the dairy milk.After inoculation with starter cultures, the milk samples were homogenized using an orbital shaker (MS Major Science, MS-NOR-30, USA) for 15 minutes at 100 rpm.The mixture design was a simplex Centroid design, and it was developed on Design-Expert ® Version 6.9 (State-Ease, Inc. Minneapolis, 2003).
All homemade yogurts were incubated at 37 ºC for 24 hours via a Trio Yogurt Maker (Trio Kaden (M) Sdn Bhd, TYM-7, Malaysia).All yogurt samples were analyzed within a maximum of 3 days to ensure freshness.

Yogurt pH Measurement
Yogurt pH measurements were made using a benchtop pH meter (Mettler Toledo Inc., Parkway, USA).The pH-meter was calibrated before it was used for yogurt measurement.The yogurt samples were measured immediately after the incubation period (Sadler & Murphy, 2010).

Yogurt Viscosity Measurement
A rotational viscometer (Brookfield, model DV II, USA) was used to measure the viscosity of the dairy yogurts.Approximately 50 ml of yogurt in a 50-ml beaker was used as a sample during measurement.The viscosity measurements were made at room temperature (27 ºC) using a Brookfield LV spindle no. 4 at 10 r.p.m..

pH of Yogurt
lowest yogurt pH of the ten formulations (Table 2).Dual starter inoculation containing L. bulgaricus (formulations 4 and 6) also had low pH values when compared to the other yogurt formulations.This is probably one of the reasons why the dairy industry utilises multiple inoculations when producing commercial yogurt (Leroy & De Vuyst, 2004).

Viscosity of Yogurt
Table 3 shows the viscosity of the 10 yogurt formulations which were within the range 2.307 Pa•s to 1.058 Pa•s.According to Lee and Lucey (2010), gelation occurs when pH is just above the dairy milk isoelectric point (pH 5.2) and the observed viscosities are indicative of gelation in all 10 formulations.Normally, commercial yogurt contains multiple starter cultures and has an average viscosity of 1.77 Pa•s (Ares et al., 2007).However, not all commercial yogurts declare which starter cultures are used.Several yogurt formulations are near or exceed the commercial yogurt viscosity (formulations 3, 5, 6 and 10) (Table 3).Yogurt formulations with L. acidophilus have the highest viscosity (formulations 1 and 8), followed by those with S. thermophilus.When L. acidophilus and S. thermophilus are combined (formulation 5), the yogurt viscosity increases above that of S. thermophilus alone.When L. acidophilus and L. bulgaricus were combined (for-mulation 4), the resulting yogurt had a higher viscosity than the yogurt with a single L. bulgaricus inoculation.L. acidophilus seems to have high viscosity capability.This was shown even in multiple starter culture yogurt formulations, for example, in formulation 8 where L. acidophilus was the dominant starter culture ratio.Conversely, L. bulgaricus seems to decrease yogurt viscosity whenever it is used to inoculate.Yogurt formulations with dominant L. bulgaricus starter culture ratio have lower viscosities than yogurt formulations without L. bulgaricus inoculations (formulation 6 and 9).variations in the models cannot be accounted for by random error.The F-value obtained was less than 0.05 (0.0109).The R 2 for yogurt was 0.6334.The yogurts also displayed statistically insignificant lack-offit, thus indicating that the experimental data was satisfactorily accepted for model fitting even though the R 2 of the dairy yogurt is less than 0.75, it can be used with confidence (Henika, 1982;Yusnita, Aida, Maskat, & Aminah, 2007).

Model System
According to the Design Expert software, there were interactions between binary and tertiary components.However, these interactions were not significant and were removed in order to develop a more robust model using the significant single components.
The canonical Scheffe's equations for the dairy yogurt viscosities were obtained from the software.The models were analyzed as follows.The positive (+) sign in the equation meant that the response values increased with an increase of the variables, whether it is linear (A, B, C), binary (AB, AC, BC) or ternary combination (AC (A-C)).There is an inverse meaning when a negative (-) sign is shown on the variables.The canonical Scheffe's equation for dairy yogurt viscosity is shown in equation 1: (1) where A is L. acidophilus, B is L. bulgaricus and C is S. thermophilus.In equation 1, the single inoculation of L. acidophilus had the highest influence on the homemade dairy yogurt viscosity followed by S. thermophilus and L. bulgaricus.L. acidohpilus has the highest mathematical weigh (+0.222).This is because L. acidophilus has the ability to convert 23 % of lactose into lactic acid.The accumulating lactic acid then contributes to the viscosity of the yogurt (Anjum et al., 2014).Formulations 1 and 8 of the homemade dairy yogurts validate this statement.The second highest was S. thermophilus (+0.15282).S. thermophilus has the ability to breakdown lactose into glucose and galactose.S. thermophilus then proceeds to digest glucose monomers for growth (Broadbent et al., 2003;Drouault, Anba, & Corthier, 2002).Almost all formulations that contain S. thermophilus had high viscosity, especially when paired with L. acidophilus (formulation 8 and 10).This is because L. acidophilus also benefited from the breakdown of lactose, since it utilizes galactose in its growth (Noh & Gilliland, 1993).L. bulgaricus had the lowest influence in dairy yogurt viscosity (+0.12236).Although L. bulgaricus was able to breakdown peptides into free amino acids that can contribute to viscosity and sensory quality (McSweeney & Sousa, 2000), it relies on other starter cultures for its growth.L. bulgaricus requires galactose or glucose for its metabolism but it cannot breakdown the lactose in order to feed itself (Nguyen et al., 2012;Zourari, Accolas, & Desmazeaud, 1992).This is an example of why dairy yogurt requires an emulsifier in its formulation (O'Rell & Chandan, 2013;Smith, 2015).

Conclusions
There are significant differences in the texture of homemade dairy yogurts inoculated with different ratios of starter culture.All homemade yogurt formulations reached the isoelectric point of milk and had pHs in the range 3.97 to 4.32.The most viscous homemade yogurt (2.3 Pa•s) came from a multiple inoculation formulation with L. acidophilus as the dominant ratio.When results are fitted into a Scheffe's canonical special quadratic equation for three components, the model shows that L. acidophilus brings the most positive influence to increase homemade yogurt viscosity.This is followed by S. thermophilus and L. bulgaricus.In conclusion, L. acidophilus can be used as a single starter culture or combined IJFS October 2017 Volume 6 pages 152-158 with other starter cultures to develop high viscosity homemade natural yogurts.A combination of S. thermophilus and L. acidophilus can also be used to develop high viscosity homemade yogurts.However, L. bulgaricus should not be inoculated alone or become the dominant ratio in a multiple starter culture inoculation since it will decrease the overall viscosity of homemade yogurt.

Table 1 :
Mixture experiment design of multiple species inoculation

Table 3 :
Viscosity of ten dairy yogurt formulations

Table 4 :
Coefficient estimates, model significance, regression coefficient and lack-of-fit values for Dairy yogurt viscosity