Evaluation of the bioprotectivity of Lactobacillus binary/ternary cultures in yogurt

Abstract The attempts toward addition of biocontrol agents in dairy products have gained popularity. Here, we worked on analysing the antifungal activity of binary and ternary combinations of three Lactic Acid Bacteria (LAB) against five spoilage yeasts in yogurt. The yogurt samples were characterized in terms of pH, acidity, WHC, textural parameters, viscosity, survivability and antifungal activity of LAB and sensorial properties during cold storage. The results showed that the inoculation of LAB in yogurt gave rise in significant reduction of pH throughout cold storage while titrable acidity and WHC decreased (p < .05). Inoculation of LAB resulted in significant increase in hardness and adhesiveness while springiness remained constant. On the other hand, apparent viscosity of all samples experienced a profound increase up to the 10th day of storage followed by a reduction trend for the rest of storage period. Analysis of inhibitory activity of LAB showed an efficient barrier against all five yeasts, in which the most activity was recorded for Lactobacillus reuteri followed by Lactobacillus acidophilus. On the other hand, the most resistance yeast was Kluyveromyces marxianus followed by Rhodotorula mucilaginosa. Sensorial analysis revealed that addition of LAB in yogurt brought about a profound improvement in textural quality of samples. Inoculation of LAB cultures in yogurt at 5% (v/v) not only could improve the physicochemical and sensorial properties of yogurt, but also could introduce a strategy toward substituting of chemical preservatives with biocontrol agents.

Using of LAB in dairy products can increase the shelf-life and overall quality of this products and also can respond to the society's need for chemical-free, less processed, and safe products on the other hand. The aim of this study was to assess the antifungal activity of binary and ternary combinations of Lactobacillus helveticus, Lactobacillus reuteri and Lactobacillus acidiphilus in yogurt followed by investigating the product in terms of antifungal activity against five common yeasts in yogurt (Debaryomyces hansenii, Rhodotorula mucilaginosa, Kluyveromyces marxianus, Kluyveromyces lactis, and Yarrowia lipolytica), LAB population, water holding capacity (WHC), viscosity, texture, and sensory properties.

| Antifungal activity assays
In this study, the overlay and the agar well diffusion methods were applied to assess antifungal activity. The overlay method was performed using MRS agar plates on which the LAB were inoculated as two 2-cm-long lines and incubated at 30°C for 48 hr in anaerobic jars. The plates were then overlaid with 9 ml of PDA and 1 ml of yeast. The plates were then incubated aerobically at 30°C for 48 hr.
Then, the clear zones of inhibition around the bacterial streaks were examined and the area of the zones were scored (Magnusson & Schnürer, 2001).
For the microdilution method, active isolates were inoculated in a 100 ml conical flask containing MRS broth and incubated at 30 ± 2°C for 48 hr. After that, the suspension was centrifuged (12,500 g, 10 min, 4°C) and the supernatant was collected and filtered through 0.22 μm membrane filters. 190 μl of the isolate supernatants and 10 μl of the conidial suspensions were dispensed in 96 wells. All the experimental plates were incubated at 30 ± 2°C for 72 hr. Fungal growth was measured at 600 nm using a microplate reader. Here, the growth of the fungi in the control was considered 100% growth. Based on the percentage of the fungal growth, inhibition was calculated. The sample free of the fermentative metabolites was considered as control (Ilavenil et al., 2015).

| Yogurt preparation
The yogurt samples were prepared using milk with 1.5% fat, 8.1% no fat milk solids, and a pH value of 6.63. After heat treatment (85°C, 30 min), the milk was rapidly cooled down to 45°C before adding 0.05% w/v of the thermophilic yoghurt culture (

| Determination of pH and acidity
The pH values of the samples were measured using a digital pH meter (HANNA, pH 211) at 27°C. For determination of the titratable acidity (TA), a certain amount of each sample was mixed with 10 ml of hot distilled water and titrated with NaOH 0.1 N in the presence of 0.5% phenolphthalein indicator (Li et al., 2013).

| Water holding capacity
The weight of an empty centrifuge tube was recorded, and then, 15 ml of each sample was poured into it, after that the entire weight was recorded. The sample was centrifuged (Anting, LXJ-IIB) at 4,000 g for 20 min, and the supernatant was drained. The centrifuge tube was inverted for 10 min, and its weight was measured. WHC (%) was calculated using equation (1): where W and W 0 are the weight of the precipitate and the weight of the yogurt, respectively (Amal, Eman, & Nahla, 2016). Eventually, the yogurt samples viscosity was assessed at shear rates ranging from 14.4 to 296/s at 25°C and the apparent viscosity was reported at a shear rate of 51.5/s (Morris, 1994).

| Texture profile analysis
The yogurt samples were stored at 20°C for 10 min followed by being analyzed using a Brookfield texture analyzer (CT V1.5 Texture Analyzer; Brookfield) equipped with a cylindrical probe, 20 mm in diameter. The TPA measurements were conducted at 2 mm/s penetration speed up to a depth of 20 mm. The textural parameters, including hardness (g), adhesiveness (mj), cohesiveness, springiness, gumminess (g), and chewiness (mj), were studied for each sample.

| Enumeration of LAB in yogurt
One gram of each yogurt sample was diluted with 99 ml of Ringer solution. Subsequent 10-fold serial dilutions were made with ringer, and 0.1 ml of the diluted samples was spread on MRS agar. After anaerobic incubation at 37°C for 48-72 hr, and enumeration, the number of LAB was calculated as CFU/g. This method was repeated on days 10, 20, and 30 by the initial cell count on day 0.

| Sensory analysis
A trained panel of 15 assessors evaluated the samples in terms of texture, color, and taste. A 100-point scale was used to evaluate the sensory properties of the yogurt samples.

| Water holding capacity
The gel network of yogurt is characterized by its dynamic nature affecting by proteins and calcium salt, which chemical and process variables may have sensible influence on it (Ziarno & Zaręba, 2019).
The results of variations in WHC of yogurt samples during storage were shown in Table 2. For all LAB added yogurt, WHC was increased significantly with time (p < .05), while for control sample, the change was nonsignificant (p > .05). The interactions occur during storage periods in terms of acid production due to the LAB growth bring about the casein particles to hold water molecules more efficiently, resulted in syneresis decrementation (Öztürk & Öner, 1999).
The proteins WHC incrementing due to decrease in pH in parallel to LAB growth give rise to increase yogurt curd stability (Öztürk & Öner, 1999).

| Survivability of LAB in yogurt sample
The survivability of LAB as a function of storage time is shown in Table 3, in which the viable counts of LAB in the yogurt samples of control, 2, 3, and 23 were changed insignificantly (p > .05), while the viable counts in the yogurt samples 1, 13, and 123 were significantly decreased (p < .05). Ziarno and Zaręba (2019)

| Antifungal activity
The results of assessment the antifungal activity of LAB by two approaches of overlay and microdilution are shown in Tables 4 and 5.
The antifungal activity of L. reuteri in both methods was higher than other LAB followed by L. acidophilus. The higher antifungal activity of L. reuteri could attribute to its ability toward producing dedicate active compound, named reuterin (Vimont, Fernandez, Ahmed, Fortin, & Fliss, 2019). Based on the overlay method, two yeasts, R. mucilaginosa and K. marxianus, had higher spoilage potential than others so no inhibitory was observed in the presence of L. helveticus for both of them. In agreement with our findings, Magnusson and Schnürer (2001) showed that Lactobacillus coryniformis could induce a weak suppression against K. marxianus and D. hansenii. It is reported that the co-culture of L. helveticus and K. marxianus was applied in the process of producing some dairy and nondairy products such as fermentation of cheese whey and sourdough, due to their synergistic effect on each other (Banu & Aprodu, 2012;Plessas, Bosnea, et al., 2008;Plessas, Fisher, et al., 2008). Accordingly, it is predictable that no inhibitory zone will be observed in this case. Similarly, the growth of A, B Significant differences among the means in each column (p < .05).   (Zotta et al., 2018).

TA B L E 3 The survivability of Lactic
The differences observing between the results of LAB antifungal activity by two approaches (Tables 4 and 5) could explain relying on the work conducting by Magnusson, Ström, Roos, Sjögren, and Schnürer (2003) who reported that the extent of antifungal compounds production by LAB was profoundly depended on their growth environment. Similarly, Leyva Salas et al. (2017) illustrated that the medium at which antifungal activity of LAB was assessed had a decision influence toward producing active metabolites. In this regard, Delavenne et al. (2013) and Le Lay et al. (2016) showed that some of LAB just were metabolically active in vitro and were ineffective in product. of yogurt are profoundly affected by process parameters and fermentation variables such as acid production (Ziarno & Zaręba, 2019).

| Texture profile analysis
Addition of LAB resulted in significant increase in hardness and adhesiveness. The increase in hardness of yogurt during storage can related to the pH reduction caused by LAB activity give rise to change of casein electric charge (Harwalkar & Kalab, 1986). During the storage period, as a result of producing acids followed by pH reduction, the surface charge of casein increase gives rise to yogurt gel to be more rigid. Several researchers reported similar results toward increasing the yogurt hardness by time (Sah, Vasiljevic, McKechnie, & Donkor, 2016). Mani-López, Palou, and López-Malo (2014) reported that the firmness of yogurts containing L. delbrueckii ssp. bulgaricus and L. reuteri or L. acidophilus increased during 35 days of storage. Penna, Gurram, and Barbosa-Cánovas (2006)  defined as the ability of yogurt gel network to recover after the first deformation, is related to the protein-protein bonding, that itself is affected by the rate in which the colloidal phosphates released from casein micelles. These reactions is governed by the acid production rate by starter culture (Sah et al., 2016). The same pattern was observed in terms of gumminess and chewiness. It is reported that starter culture had a meaningful impact on gumminess and chewiness of yogurt (Penna et al., 2006). Adhesiveness is strongly linked to firmness (Hilali et al., 2011). Mani-López et al. (2014) reported that yogurts with L. delbrueckii ssp. bulgaricus and L. reuteri or L. casei increased adhesiveness during storage, and adhesiveness was significantly different between products during storage, at which larger firmness values were generally associated with low adhesion values.

| Viscosity
The apparent viscosity of yogurt samples is shown in Table 7. A similar trend was observed in all samples, in which a significant increase was recorded in day 10 followed by reduction up to the end of storage. Several researchers attributed these changes to the acidity of medium, in which a firmer network was appeared as a result of acid coagulation at low pH (Beal, Skokanova, Latrille, Martin, & Corrieu, 1999;Garcia-Garibay & Marshall, 1991). It is reported that the factors affecting fermentation pathway and duration could influence the viscosity of final product (Penna et al., 2006). In this study, inoculation of LAB at different ratios resulted in producing different acids with different rates and concentrations, bringing about the apparent viscosity to be a culture dependent feature. In this regard, Beal et al. (1999) reported that the viscosity of yogurt is a function of strain association, temperature, and final pH. In contrary, Li et al. (2013) represented results toward the effectless of inclusion L. casei on the apparent viscosity of yogurt samples during the refrigerated storage period. This opposed results can interpret relying on the difference in type and concentration as well as the interactions occurred between binary and ternary cultures.

| Sensory analysis
The impact of LAB cultures on the sensorial characteristics of the yogurt samples was shown in Figure 2. At a glance, the sensorial quality of all LAB included yogurt samples was significantly improved during 30 days of cold storage. The most changes in sensorial scores were come after taste characteristic (Figure 2a). The taste score of LAB-included yogurt samples was significantly increased during 30 days of storage. These observations could interpret relying on the ability of LAB toward producing several taste related compounds. LAB conduct the biochemical processes including glycolysis, proteolysis, and lipolysis resulting in formation of aldehydes, ketones, acids, alcohols, esters, aromatic hydrocarbons, heterocyclic, furans, and sulfur compounds (Chen et al., 2017). It is reported that inclusion of L. helveticus in fermented soybean milk had no significant influence on the sensory quality during 21 days cold storage (Bian et al., 2016). Similarly a report launched by Li et al. (2013) Hekmat and Reid (2006) reported that panelists did not recognize texture or flavor differences among probiotic and nonprobiotic samples; therefore, probiotic yogurts can be modified using different culture mixtures without sensory complaints.

| CON CLUS ION
The bioprotectivity of three LAB, namely L. reuteri, L. helveticus, and L. acidophilus, in the inoculation level of 5% (v/v) in forms of binary and ternary combinations in yogurt was assessed against five spoilage yeasts, namely D. hansenii, R. mucilaginosa, K. marxianus, K. lactis, and Y. lipolytica. The further LAB added yogurt analysis showed several significant changes including pH reduction, WHC incremention, improvement sensory quality and textural properties, maybe due to the acid accumulation as a result of LAB growth throughout of storage. The investigated LAB had sensible inhibitory against all five yeasts. Our study provided information toward using binary and ternary cultures of three LAB as an efficient route in terms of introducing a biocontrol system in dairy products for simultaneous growth inhibition of a variety of yeasts.

CO N FLI C T O F I NTE R E S T
None.

E TH I C A L A PPROVA L
This article does not contain any studies with human or animal subjects.