1. Introduction
The consumption of soy-based foods, such as soymilk, tofu, and tempeh, has soared immensely due to heightened awareness of their composition of micronutrients (i.e., B complex vitamins, vitamin K, magnesium, phosphorus), unsaturated fatty acids, dietary fibre, protein, phytosterols, and isoflavones [
1,
2,
3]. Additionally, the adoption of plant-based diets in recent years, such as vegetarianism and veganism, for various health, ethical, religious and/or cultural considerations also played a pivotal role in promoting the increased consumption of soy [
1,
2,
3].
Soybeans have been investigated extensively for their role in mitigating symptoms associated with menopause, hyperlipidaemia, and the risk for various chronic diseases, such as breast cancer and cardiovascular disease (CVD), upon observation of a lower prevalence of these conditions amongst certain cultures wherein soy represents a substantial component of the diet [
1,
2]. For instance, the saponin, fibre, and lecithin components of soy are proposed to exert a hypocholesterolemic effect, which helps minimise CVD-related mortality risk [
1,
2]. Moreover, soymilk, the aqueous extract of soy, has been disclosed as an acceptable substrate for the growth of lactic acid bacteria due to its oligosaccharide, amino acid, and peptide constituents in numerous studies, thereby indicating probiotic carrier potential [
4,
5,
6,
7]. Previous studies also delved into the plethora of benefits that soy-based foods derive from the process of fermentation [
4,
5,
6,
7]. For instance, De Boever et al. supplemented semi-skimmed milk with soygerm powder and incubated the solution with
Lactobacillus reuteri [
8]. The lipid, saponin, and lecithin constituents of the soygerm powder were postulated to preserve viable counts of
L. reuteri observed in the study, even when exposed to deleterious bile salts [
8]. In turn,
L. reuteri promoted conversion of glycosides to bioactive aglycones through its β-glucosidase activity [
3,
9]. Similarly, Marazza et al. observed an improvement in isoflavone bioavailability through fermentation of soymilk with
L. rhamnosus CRL981 [
9]. The fermented soy beverage exhibited better antioxidant potential, nutritional value, and health-promoting effects compared to the unfermented soy [
9]. In addition to promoting the functional properties, fermentation of soy-based foods has been demonstrated to enhance the flavour profile, texture, anti-nutritional constituents (i.e., trypsin inhibitors, phytic acid, and saponin), mineral bioavailability (i.e., zinc, calcium) and digestibility [
2,
10,
11,
12]. Fermentation, particularly with mixed cultures, may also improve the protein content, solubility, and availability of free amino acids [
13,
14].
Viable microorganisms which confer health benefits upon a host when present in adequate amounts are termed probiotics [
15]. A minimum level of 10
6 colony forming units of probiotic bacteria per millilitre (CFU/mL) of the carrier food product are recommended at the time of consumption to claim probiotic potency for optimum therapeutic benefits [
16].
Lactobacillus rhamnosus GR-1 is a facultative anaerobe with probiotic potential, which was used in the production of the first probiotic fermented milk in Africa [
17]. Since then, it has been employed for numerous humanitarian-driven projects in Africa targeted towards individuals living with poverty, malnutrition, and infectious diseases, such as HIV and malaria, and represents the second most extensively studied strain of the
L. rhamnosus species [
7,
17]. Its applications to human health arise from its ability to survive transit and colonise the gastrointestinal and urogenital tracts [
17,
18]. These health benefits range from alleviating intestinal microflora dysfunction, reducing skin rashes, and improving gastrointestinal distress, to enhancing immunity and combatting respiratory tract diseases [
17,
18].
L. rhamnosus GR-1 has also been demonstrated to reduce the incidence of fungal and bacterial infections of the urogenital tract, particularly in collaboration with
L. reuteri RD-14, by more than 50% in one year [
17,
18,
19]. The safety profile of
L. rhamnosus GR-1 was established through its application in previous studies with immunocompromised and/or malnourished patients with HIV and inflammatory bowel diseases [
17]. For instance, Reid and Hekmat explored the effect of probiotic yogurt fortified with
L. rhamnosus GR-1 as an adjunct to the diets of patients living with HIV and AIDS on their productivity, HIV symptoms, and nutrient intake [
17]. The preliminary results indicated improvements in all three primary outcome measures without any harmful side effects [
17].
Traditionally, fermented, dairy-based matrices are used as agents for probiotic delivery, such as yogurt and cheese [
20]. In recent years, due to the growing interest in health eating and sustainable food production practices, plant-based media derived from cereals, fruits, vegetables, and legumes have also been subjected to investigation for their ability to serve as carriers for probiotics as they represent a huge growth potential for the functional food market [
20,
21]. However, despite the growing demand for plant-based milk alternatives, many consumers still prefer cow milk due to its status as a staple food in North America, nutrient content, habitual consumption, better sensory attributes, and health benefits, such as calcium content and promotion of bone and dental health [
22,
23].
Consequently, this investigation was undertaken to elucidate the influence of soy and cow milk-based yogurt blends on the viability of L. rhamnosus GR-1 over 6 h of fermentation and 30 days of refrigerated storage. Additionally, the sensory appeal of these products amongst consumers was evaluated to ensure probiotic fortification does not adversely impact the organoleptic properties of either beverage.
2. Materials and Methods
2.1. MRS Broth and Probiotic Stock Solution Preparation
de Man, Rogosa, and Sharpe (MRS) (EMD Millipore Corporation, Gibbstown, NJ, USA) broth was prepared at a 5.22% weight per volume (w/v) ratio in distilled water. The solution was distributed into 20 mL test tubes and autoclaved at 121 °C for 15 min. Following sterilisation, the MRS broth was refrigerated at 4 °C overnight. L. rhamnosus GR-1 (10% w/v) (Canadian Centre for Human Microbiome and Probiotic Research, Lawson Health Research Institute, London, ON, Canada) was inoculated in the MRS broth under aseptic conditions, incubated anaerobically at 37 °C for 24 h using a GasPak anaerobic system (BD GasPakTM EZ Container System, Becton Dickinson & Co., Sparks, BD, USA), and stored under refrigerated conditions at 4 °C. The preparation of probiotic stock solution in MRS broth was conducted routinely at 10-day intervals to preserve viable counts of L. rhamnosus GR-1 and to allow for proliferation.
2.2. Probiotic Mother Culture Preparation
Partially skimmed cow milk (1% milk fat) (Sealtest® Partly Skimmed Milk, Agropur Dairy Cooperative, Saint-Hubert, QC, Canada), was purchased from Sobeys, London, ON, Canada, covered with aluminium foil, and autoclaved at 121 °C for 15 min. Following sterilisation, the temperature was gradually reduced to 35 °C in a water bath. Subsequently, 2% (w/v) probiotic stock solution was inoculated in the milk, gently stirred to ensure equal distribution of the inoculum, and incubated anaerobically at 38 °C for 6 h using a GasPak anaerobic system (BD GasPakTM EZ Container System, Becton Dickinson & Co., Sparks, BD, USA).
Fermentation and Storage of Probiotic Yogurt
Partially skimmed cow milk (1% milk fat) (Sealtest® Partly Skimmed Milk, Agropur Dairy Cooperative, Saint-Hubert, QC, Canada) and plain, unsweetened soymilk (Earth’s Own Organic So NiceTM Original Soy, Vancouver, BC, Canada) were purchased from Sobeys in London, ON, Canada. Milk treatments were prepared in the following proportions: Treatment 1 contained 100% (w/v) cow milk (control), Treatment 2 contained 75% (w/v) cow milk and 25% (w/v) soymilk, Treatment 3 contained 50% (w/v) cow milk and 50% (w/v) soymilk, and Treatment 4 contained 20% (w/v) cow milk and 75% (w/v) soymilk.
Sucrose (5%
w/
v) was added to all treatments to improve the sensory profile and they were stirred prior to pasteurisation. Each treatment was heated in a water bath, held at a temperature of 85–87 °C for 30 min, and cooled to 40 °C. Thereafter, 3% (
w/
v) plain yogurt starter culture (2% milk fat) (Astro
® Original Balkan Yogourt, Plain, No Gelatin, Parmalat Canada Inc., Toronto, ON, Canada) from Sobeys, London, ON, Canada and 4% (
w/
v) probiotic mother culture were inoculated in each sample under aseptic conditions and stirred well. This probiotic culture concentration has been used in previous studies [
24,
25]. Each treatment was distributed into eight, 30 mL beakers. Beakers 1–4 represented the four fermentation timepoints (0, 2, 4, and 6 h) and beakers 5–8 represented the four refrigerated storage timepoints (1, 10, 20, and 30 days). All beakers were covered using aluminium foil.
Microbial counts and pH measurements were performed every two hours over a total of 6 h of fermentation, and every ten days over 30 days of cold storage. Beaker 1 for each treatment served as the control and it was set aside. Beakers 2, 3, and 4 were incubated at 38 °C for 2, 4, and 6 h, respectively. Following fermentation, these treatments did not undergo refrigeration and were discarded. Beakers 5, 6, 7, and 8 were incubated at 38 °C for 6 h and stored at 4 °C under refrigerated conditions for 1, 10, 20, and 30 days, respectively. All treatments were subjected to two true replications under the same experimental conditions to account for any variability between the results for microbial and pH analyses.
2.3. Microbial Analysis
An enumeration of viable colonies of L. rhamnosus GR-1 was conducted for all treatments following the four fermentation (0, 2, 4, and 6 h) and refrigerated storage (1, 10, 20, and 30 days) timepoints. Each sample (11% w/v) was subjected to serial, 10-fold dilutions in sterile saline solution (0.85% w/v) to produce five dilution factors: 10−1, 10−3, 10−5, 10−6, and 10−7. 0.1 mL aliquots of each sample were transferred to two selective MRS agar plates per dilution factor. These MRS plates were prepared using 5.22% (w/v) MRS (EMD Millipore Corporation, Gibbstown, NJ, USA), 1.5% (w/v) agar (EMD Millipore Corporation, Gibbstown, NJ, USA), and 0.0015% (w/v) fusidic acid (Enzo Life Sciences, Farmingdale, NY, USA). The plates were incubated anaerobically at 37 °C for 24 h using a GasPak anaerobic system (BD GasPakTM EZ Container System, Becton Dickinson & Co., Sparks, BD, USA). Following incubation, bacterial colonies were recorded in the form of CFU/mL. An average microbial count in CFU/mL was determined for the two replications for each sample.
2.4. pH Analysis
pH was measured for all treatments following the four fermentation (0, 2, 4, and 6 h) and refrigerated storage (1, 10, 20, and 30 days) timepoints by means of a calibrated pH meter (VWR® SymphonyTM B10P pH Meter, VMR International, Radnor, PA, USA). An average value for the pH was determined for each sample through the two true replications of the experiment.
2.5. Sensory Evaluation
One hundred and twenty untrained panellists, consisting of seven males and 113 females between the ages of 18–55 years, participated in a sensory evaluation held at the Sensory Lab of the Academic Pavilion at Brescia University College, London, ON, Canada. Nine invalid and/or incomplete responses were excluded, bringing the total count to 111 responses. All interested participants were required to provide written consent and were excluded if they were allergic and/or intolerant to soy and/or cow milk, unable to provide consent, pregnant, and/or undergoing chemotherapy. Additionally, participants who had diabetes, required a translator, or those who did not satisfy the age requirement were also excluded. Approval for the sensory evaluation was granted by the Health Sciences Research Ethics Board at Western University.
Eligible panellists were guided to well-lit sensory booths and provided with trays, which consisted of four portion cups representing each of the four treatments, a CAD 10 Tim Horton’s gift card as an honorarium, and a sensory evaluation questionnaire. Treatments 1–4 were prepared as detailed in
Section 2.2, incubated at 38 °C for 6 h, and refrigerated at 4 °C for four days. All treatments were stirred prior to serving to obtain the drinkable format of a yogurt beverage and portioned in uniform, 30 mL disposable cups, which did not impart any flavour to the treatments and were large enough to allow for retasting.
Participants were instructed to limit contact with their neighbours, taste the treatments from left to right, and to cleanse the palette with water between tastings to eliminate any residual flavours. Participants indicated their ratings for the appearance, flavour, texture, and overall acceptability for each sample using a nine-point hedonic scale ranging from 1 (dislike extremely) to 9 (like extremely). Participants were also asked to indicate the sample(s) they are most and least likely to purchase, their consumption patterns of probiotic yogurt, and their views about incorporating soymilk in yogurt.
2.6. Statistical Analysis
Statistical analyses were conducted using IBM SPSS Statistics 25.0 (IBM Corporation, Armonk, NY, USA). The data expressed are average values from the two replicate determinations and are presented in the form of mean ± standard deviation (SD). A one-way repeated measures analysis of variance (RMANOVA) was used to analyse the effect of fermentation and refrigerated storage on viability of L. rhamnosus GR-1 and pH between and within treatments over time, as well as differences in mean hedonic scale ratings for appearance, flavour, texture, and overall acceptability between treatments for all participants. A post-hoc Fisher’s least significance difference (LSD) test was used to conduct pair-wise comparisons of means when a significance (p < 0.05) was detected.
4. Discussion
The primary aim of the present study was to propose a new layout for a fermented, soy and cow milk-based functional food and to determine the potential of this formulation to serve as a suitable substrate for the growth and proliferation of
L. rhamnosus GR-1. This option is postulated to provide an alternative medium for probiotic delivery, thereby making the functional food market accessible to a wider segment of the population, such as to individuals who are seeking lower cholesterol, saturated fat, and lactose alternatives to purely cow milk-based probiotic yogurt, as well as in lower income countries where cow milk is costly and legumes are more readily accessible [
2].
Despite the growing interest in plant-based foods such as soy, the sensory appeal of soymilk is low amongst most consumers due to its beany flavour, which is attributed to the presence of unsaturated fatty acids and lipoxygenases that give rise to volatile compounds (hexanal and pentanal) [
2,
27,
28]. Additionally, some individuals report abdominal discomfort, diarrhoea, and flatulence following soybean consumption due to its oligosaccharides (raffinose and stachyose), which are indigestible in the human intestinal tract [
11,
21,
28]. Other organoleptic properties, such as a brownish colour, chalky texture, and thin mouthfeel, also promote limited acceptance of soy-based foods [
11]. Thus, our secondary objective was to conduct a sensory evaluation to elucidate whether this new soy and cow-milk based formulation offers considerable appeal to consumers.
During 6 h of fermentation, all treatments containing soymilk (i.e., Treatments 2–4) supported the growth of
L. rhamnosus GR-1 and allowed viable counts of at least 10
8 CFU/mL to be achieved and maintained over 30 days of cold storage at 4 °C. This value surpassed the minimum recommended microbial load of 10
6 CFU/mL for probiotic effect [
16]. The results of Treatments 2–4 were comparable to that of the control (Treatment 1), thereby exhibiting the incorporation of soymilk and durations of fermentation and storage did not induce a loss in bacterial viability. Previous studies also demonstrated successful inoculation of
L. rhamnosus GR-1 in non-dairy matrices, such as fruit and vegetable juices and fermented rice pudding to determine their suitability as carriers of probiotics [
24,
25,
29,
30]. Mean viable microbial loads of at least 10
7–10
9 CFU/mL were reached in all cases [
24,
25,
29,
30].
An investigation by Rostami et al. yielded similar results, wherein cow milk-based yogurt formulations supplemented with either 20%, 40%, or 60% soymilk allowed for the microbial counts of
L. acidophilus,
L. casei, and yogurt starter culture (i.e.,
L. delbrueckii ssp. bulgaricus and
Streptococcus thermophilus) to be comparable to the counts of these strains in 100% cow milk-based yogurt [
31]. When analysing the growth of either
L. rhamnosus GG or
L. johnsonii La-1 in conjunction with yogurt starter culture in soy yogurt, Farnworth et al. found the microbial load of these probiotic bacteria to be 5 and 3 times better in the soy yogurt compared to cow milk yogurt, respectively, and
L. rhamnosus GG exhibited a better growth potential in the soy yogurt compared to
L. johnsonii La-1 [
4]. Likewise, Liu et al. introduced a probiotic soy cheese fortified with
L. rhamnosus 6013 [
32]. The microbial count of the probiotic strain surpassed the minimum level of 10
6 CFU/mL for therapeutic effect, reaching 10
8–10
9 CFU/mL after 6 h of fermentation, and an increase in acidity of the product was recorded after 30 days of cold storage at 10 °C [
32]. The levels of stachyose and raffinose were also significantly reduced (
p < 0.05) due to metabolism by the probiotic bacteria [
32].
Previous studies reported an improvement in nutritional value through fortification of dairy-based milk with soymilk [
33,
34]. Nurliyani et al. developed kefir using varying combinations of soy and goat milk and found the microbial counts of lactic acid bacteria to be comparable between the mixture and goat milk alone [
33]. Additionally, the 50–50 combination of soy and goat milk contained higher levels of an omega-9 fatty acid, oleic acid, compared to 100% goat milk, and lower concentrations of certain saturated fatty acids, specifically caproic, heptadecanoic, and behenic acids [
33]. Ghoneem et al., who developed bioyogurt using various blends of buffalo, cow, and soymilk with ABT-5 culture (consisting of
Streptococcus thermophiles,
L. acidophilus, and
Bifidobacterium BB-12), also reported significant improvements (
p < 0.05) in linoleic and α-linolenic acid concentrations and a decrease in the level of saturated fatty acids upon replacement of 25% buffalo or cow milk with soymilk [
34].
Upon evaluation of the pH profile of the treatments in the present study during fermentation and cold storage, formulations containing soymilk (i.e., Treatment 2–4) experienced a significant increase (
p < 0.05) in acidity. While the decline in pH was steeper in the soy-based formulations compared to Treatment 1, the final pH values of all treatments were similar at each point during fermentation and storage. This observation is similar to that noted by Farnworth et al., who compared the growth of the yogurt starter culture in soy yogurt with its growth in cow milk yogurt [
4]. The steeper rise in acidity of the soy yogurt was attributed to the lower buffering capacity of soy compared to cow milk due to its different protein composition and the physicochemical properties of these proteins [
4,
6]. Champagne et al.’s results were also consistent with the present study as they reported a greater acidification rate in soy yogurt inoculated with
L. rhamnosus R0011 compared to cow milk yogurt [
35].
However, Ghoneem et al. reported different observations, wherein a bioyogurt formulation containing soymilk alone had a lower titratable acidity compared to buffalo and cow milk, and a mixture of soymilk with either beverage had an even lower titratable acidity level [
34]. Similarly, Osman and Razig evaluated the quality attributes of four soymilk and cow milk formulations fermented using yogurt starter culture [
32]. Interestingly, the 25% soymilk and 75% cow milk blend produced the lowest pH value of 3.3 during fermentation, followed by the 50–50 mixture, and finally the 75% soymilk and 25% cow milk blend [
36].
Consistent with our results during cold storage, Osman and Razig also noted an increase in acidity during the 20-day storage period [
36]. A similar observation of a drop in pH during cold storage was described by Mondragón-Bernal et al. in synbiotic soy yogurt formulation consisting of
L. rhamnosus LR32 and standard probiotic inoculum [
37]. pH values in the range of 5.5 to 6.0 are conducive to the proliferation of strains belonging to the
Lactobacillus genus; however, they can sustain pH values as low as 3.7–4.3 [
16]. Thus, while a steep decline in pH (i.e., lower buffering capacity) may be concerning due to the potential adverse effects on microbial counts, the observation in the present study was not detrimental as bacterial viability was maintained during fermentation and storage [
15,
20].
Through the sensory evaluation, it was determined that panellists had a greater preference for the texture and appearance of Treatment 1, while scores for the flavour and overall acceptability of the treatments were comparable and in the “neither like nor dislike” to “like slightly” range of the hedonic scale. Similarly, Osman and Raziq reported a 75% cow milk and 25% soymilk blend attained the best scores for appearance, flavour, texture, and overall acceptability compared to treatments with a higher proportion of soymilk [
36]. Organoleptic properties are the main setback for the adoption of soy-based products and, typically, consumers prefer cow milk yogurt over soy yogurt due to its colour and texture [
28]. Incorporation of natural and synthetic flavouring agents, fruit pulps, fructooligosaccharides, and essences may help attenuate the undesirable taste, while enhancing the nutrient profile of the yogurt [
6,
38,
39]. Additionally, response surface technology, as demonstrated by Kumar and Mishra through the development of mango soy fortified yogurt, may also help mask the beany taste of soymilk [
40].
While soy and cow milk share similar nutrient profiles compared to other plant-based milk alternatives, using a combination of soy and cow milk to produce a functional food integrates the favourable characteristics of both foods [
2,
3,
39]. For instance, the final product supplies an extra boost in protein quality, including sulphur-containing amino acids, methionine, cysteine, and cystin, which are present in limited quantities in soymilk [
2,
3,
39]. Further, the isoflavone, B vitamin, soluble fibre, magnesium, riboflavin, niacin, and calcium content of the mixture is improved [
2,
3,
39,
40,
41,
42]. The fat and protein content of cow milk, as well as its buffering capacity, can be leveraged to fuel bacterial metabolism, survival, and proliferation [
20]. Additionally, the less desirable features of the individual components are minimised, such as the poor palatability of soymilk and high lactose, caloric, carbohydrate, saturated fat, and cholesterol content of cow milk [
41]. In combination, these factors allow for a nutritionally similar alternative to both cow milk and soymilk to be produced with distinct nutritional advantages, allowing consumers to derive a range of unique health benefits [
40,
42,
43].
Future research should explore the retention of the functional efficacy of
L. rhamnosus GR-1 in this new matrix through simulated gastric and intestinal juice models, and whether the viability observed during fermentation and storage is adequately preserved in the gastrointestinal tract for the therapeutic effects of
L. rhamnosus GR-1 to be acquired [
43]. Additionally, the incorporation of prebiotics, such as inulin, should be investigated for their added support to
L. rhamnosus GR-1 during transit through the gastrointestinal tract, as well as the effect of inulin on the viscosity of soy yogurt [
44]. Finally, the potential role of the oligosaccharides, which are found in soymilk, as prebiotics for
L. rhamnosus GR-1 warrants investigation [
44].