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Article

Innovative Fermented Soy Drink with the Sea Buckthorn Syrup and the Probiotics Co-Culture of Lactobacillus Paracasei ssp. Paracasei (L. Casei® 431) and Bifidobacterium Animalis ssp. Lactis (Bb-12®)

by
Nicoleta-Maricica Maftei
1,2,3,
Roxana Elena Goroftei Bogdan
4,
Monica Boev
1,3,*,
Denisa Batîr Marin
1,3,*,
Ana Yndira Ramos-Villarroel
5 and
Alina-Viorica Iancu
3,6,7
1
Department of Pharmaceutical Sciences, Faculty of Medicine and Pharmacy, ‘Dunărea de Jos’ University, 800010 Galati, Romania
2
Clinic Laboratory Department, Clinical Hospital of Children Hospital “Sf. Ioan”, 800487 Galati, Romania
3
Research Centre in the Medical-Pharmaceutical Field, “Dunarea de Jos” University of Galati, 800010 Galati, Romania
4
Clinical Medical Department, Faculty of Medicine and Pharmacy, “Dunărea de Jos” University, 800010 Galați, Romania
5
School of Science of Agro and Environment, University of Oriente, Av. University, Campus the Guaritos, Maturín 6201, Venezuela
6
Medical Laboratory Department, Clinical Hospital of Infectious Diseases “Sf. Cuvioasa Parascheva”, 800179 Galati, Romania
7
Department of Morphological and Functional Sciences, Faculty of Medicine and Pharmacy, “Dunărea de Jos” University, 800010 Galați, Romania
*
Authors to whom correspondence should be addressed.
Fermentation 2023, 9(9), 806; https://doi.org/10.3390/fermentation9090806
Submission received: 30 July 2023 / Revised: 20 August 2023 / Accepted: 29 August 2023 / Published: 31 August 2023
(This article belongs to the Special Issue Quality and Sensory Analysis of Fermented Products)
Browse Figures
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Abstract

:
The area of functional drink is one of the fastest-growing sectors in the world, be it that it is made from plant-based or non-dairy milk. Sea buckthorn syrup is a source of functional ingredients, with a large spectrum of healthy compounds. The study aimed to investigate the suitability of sea buckthorn syrup as a substrate for Lactobacillus paracasei ssp. paracasei (L. casei® 431) and Bifidobacterium animalis ssp. lactis (Bb-12®) development and fermentation in vegetal soy drink and to evaluate the fermented product (at 30 and 37 °C) in terms of bacterial viability, pH, tithable acidity during fermentation and storage period, water holding capacity, antioxidant capacity, total phenolic contents, sensory analysis and in vitro bio-accessibility. During fermentation, a bacterial concentration around of 109–1010 CFU·mL−1 was found in the soy drink with sea buckthorn syrup and L. casei® 431 and Bb-12®. Antioxidant capacity significantly improved after the fermentation of the soy drinks. On the other hand, through the digestibility of the drinks, the bacterial viability significantly decreased for L. casei® 431 and increased for Bb-12®. Further investigation is required on the concentration of sea buckthorn syrup and probiotic encapsulation methods to comprehend the components responsible for the efficient delivery of bacteria across the gastrointestinal tract.

1. Introduction

For the normal functioning of the human body, food and nutrition play an important role in everyday life because these factors maintain health and reduce the risk of various diseases [1]. The two terms that describe health are “functional” and “beyond nutrition”; these terms go hand in hand same as the relation between diet and health [2]. During the past years, alongside the improvement in living conditions, but due to the consciousness of consumers as well (referring to the importance of health), the demand for functional foods has exploded as conventional foods can no longer meet the public’s discern for dietary habits [2]. The market size of global functional foods was estimated at USD 280.7 billion in 2021 and from 2022 to 2030, is expected to expand at a compound annual growth rate (CAGR) of 8.5% [3]. Functional foods are considered foods that contain health-promoting ingredients or natural active compounds that exhibit extraordinary effects, such as antioxidant, anti-inflammatory, anti-cancer properties, and other reported health protection benefits [4].
All over the globe, special attention has been drawn to plant-based food, represented by “vegetable milk”. The most popular “vegetable milk” is soy milk because it has multiple health benefits, including its phenolic compounds, isoflavones and soy fatty acids, but also its protein content. A method of preserving and processing soy milk is fermentation because it can improve the bioavailability of nutrients, resistance to oxidation and improve the flavor of the final product [5].
Soy products have witnessed a long development history, in particular in Asia. Over time, owing to the global expansion, varieties of soy products have been developed: tofu, soybean meal, okara, and soy-based beverages [6]. The legendary An Liu is said to have invented soymilk, the simple aqueous extract of whole soybean about 2000 years ago in China [7], and since then, the produce has been used as a base for a wide range of soy-based beverages [6].
During the past few years, lactic acid bacteria (LAB) have been recognized as an innovative starter culture in the food and beverage industry due to their ample endowment to the conservation, texture, flavor, and fermented foods [8]. LABs have been employed for their ability to hydrolyze isoflavones, break down flatulence factors, as well as for their B-vitamin-producing attributes [9], and these attributes of probiotic LABs encourage food and beverage researchers to acquire new insights in the search for novel products for the functional food market [2]. LAB bears the potential of exhibiting several antibacterial elements such as low molecular mass compounds (H2O2, CO2, diacetyl) and high molecular mass compounds like bacteriocins [10,11], elements that effectively cause the inhibition of food-borne pathogens. When fermenting soy-based foods with LAB, it shows antihypertensive effects through the inhibition of angiotensin-converting enzyme (ACE), which is the primary enzyme in the renin-angiotensin system [12]. In the scientific literature, studies indicated that biologically active peptides extracted from the hydrolysates of the soybean protein had ACE-inhibitory activity and the potential to improve hypertension in vivo [13]. Lactobacillus spp. is the most utilized, due to its probiotic effects on human health and because it’s part of the human microbiota as it possesses antioxidant activity. Also, the Lactobacillus spp. can minimize the risk of reactive oxygen species accumulations during the ingestion of food [14]. Through the fermentation process Lactobacillus spp. improves the bioavailability of isoflavones in many soy-based products. The isoflavones are in glucose form (biologically inactive) and not absorbed through the intestinal wall until or unless fermented, in this way, it helps with the digestion of proteins by offering a wider variety of soluble calcium which can improve intestinal health and support the immune system [15]. For a better understanding of intestinal health and support of the immune system, clinical examination and laboratory tests are important for the diagnosis [16,17,18] because they can prove the beneficial effect of probiotics.
In clinical studies within gastrointestinal health and immune function, Bifidobacterium BB-12 has proven its beneficial health effect. Also, bifidobacteria have been shown to improve bowel function and can reduce side effects of antibiotic treatment, such as antibiotic-associated diarrhea. Regarding immune function, clinical studies have shown that bifidobacteria reduces the incidence of acute respiratory tract infection microbiota as well as increases the body’s resistance to common respiratory infections [19]. Regardless of the microorganism used, it is recommended that the minimum daily intake of probiotic viable cells should be between 106 and 107 CFU/g of food product [20].
Sea buckthorn (genus Hippophae L., family Eleagnaceae)- belongs to exceptionally valuable plants that are currently being domesticated and cultivated in orchards, especially in Europe, Canada, and the USA [21]. The importance of sea buckthorn is usually ascribed to higher amounts of antioxidants. Also, a wide range of various positive biological, physiological, and medicinal effects of sea buckthorn was extensively described, such as antioxidative and immunomodulating [22] antibacterial and antiviral effects [23], cardioprotective and antiatherogenic [24], healing effect on acute and chronic wounds [25], anti-inflammatory [26], anticarcinogenic [27], antidiabetic [28], hepatoprotective and dermatological effects [29] and the future risk of developing diabetes, cardiovascular complications, and stroke. Due to these reasons, sea buckthorn berries, seeds, and leaves are widely used for nutraceutical and medicinal purposes.
Due to the increasing number of vegetarian people in the world and consumer concerns regarding an alternative diet to probiotics dairy products with high nutritional value, free of cholesterol and lactose, make it of great interest to researchers now. Thus, because of this, new food matrices as probiotic carriers have been tested. For this reason, the aim of this study was to evaluate the influence of the composition of ingredients of the food matrix and of bacterial culture on innovative drinks during the fermentation of soymilk. To the best of our knowledge, it is the first time that Lactobacillus paracasei ssp. paracacasei and Bifidobacterium animalis subsp. lactis are used in a coculture alongside different concentrations of sea buckthorn syrup in the fermentation of a soymilk food matrix to produce an innovative drink-like food.

2. Materials and Methods

2.1. Materials

2.1.1. Soymilk

In this experiment, the soy milk used (Dr. Oetker, Curtea de Arges, Romania) is a sterilized product produced by the Romanian company Inedit. The product contains 1.1% proteins, 0.0% sugars, and 1.9% lipids, according to the information provided on the product label.
According to the manufacturer, the qualities of the soy milk used in this study are:
-
the product obtained directly from NMG soybeans (95% soy content).
-
it contains natural ingredients and flavors.
-
it does not contain preservatives or dyes.
-
it does not contain lactose.

2.1.2. Sea Buckthorn Syrup

Alongside the soymilk, in these experiments, sea buckthorn syrup, which is available for purchase at the Plafar market in Galati Romania, was used. The pH was 3.10, and the soluble solids content of untreated syrup sample was 6° Brix. All samples used in the study were obtained from a single syrup batch.

2.1.3. Probiotic Lactic Acid Bacteria

The probiotics Lactobacillus paracasei ssp. paracacasei (L. casei® 431) and Bifidobacterium animalis subsp. lactis (Bb-12®) was provided by Chr. Hansen, Hørsholm, Denmark, as a freeze-dried commercial starter. The strains were maintained as frozen stocks in 50% glycerol and stored at −80 °C.

2.1.4. Preparation of Fermented Products

The soy-based drinks were prepared in triplicate by adding 5.0% (v/v), 10.0% (v/v), 15.0% (v/v), and 20.0% (v/v) sea buckthorn syrup to the soymilk. Volumes of 100 mL mixtures were then allotted into 250 mL flasks and inoculated with Lactobacillus paracasei ssp. paracacasei and Bifidobacterium animalis subsp. lactis and fermented at 30 and 37 °C for 12 h. The drinks were inoculated with 0.1 mL each of inocula of lactic acid bacteria and bifidobacteria. In these experiments, the initial population of each organism in the soymilk was 5 log CFU·mL−1. To determine the cell concentration, a UV-Vis Jenway 6506 spectrophotometer (Bibby Scientific Ltd., Staffordshire, UK) was used to determine the optical density (OD) of a cell suspension in sterile saline solution (0.85% NaCl w/v) at a wavelength of 600 nm. A standard curve was constructed, giving the relationship between the number of bacteria and optical density. The linear equation was found by fitting a straight line. This equation was used to find out the approximate amount of cell suspension to be used for preparing the product. At the end of the fermentation, the samples were stored for 14 days at 4 °C ± 1 °C. The fermented samples were collected at 0, 2, 4, 6, 8, and 12 h for microbiological and chemical analysis. Also, the storage samples were analyzed for microbiological and chemical analysis (pH, titratable acidity, and water holding capacity) during the whole storage time.

2.1.5. Analytical Methodology

pH Measurement: pH of samples was measured through a calibrated Mettler Toledo 2000 pH Meter potentiometer. Replicates were prepared, and the acquisition of pH values was made in aseptic conditions (into the laminar flux chamber with calibrated and sanitized potentiometer with alcohol 70% and sterilized-deionized water).
Titratable acidity Measurement: titratable acidity (g of acid/100 mL) was determined by titration of a 10.0 mL aliquot with NaOH 0.01 M solution using 1% phenolphthalein as an indicator.
Water holding capacity (WHC) Measurement: WHC was determined through the centrifugation procedure-approximately 10 g of drink was transferred into a 20 mL glass tube and was centrifuged at 5.433× g for 10 min at 200 °C and a Micro Universal 320 R centrifuge (Hettich Zentrifugen, Germany) was used (modified method of [30]). The WHC was estimated as the percentage of the released whey over the initial beverage weight:
Water   holding   capacity ,   % = w e i g h t   o f   s u p e r n a t a n t w e i g h t   o f   d r i n k × 100
Counts culture medium: The counts of potential probiotics were determined by serial decimal dilutions with 0.1% (w/v) peptone water (Merck), which were subsequently plated on MRS agar (Merck) for lactobacilli.
Bifidobacteria were enumerated using MRS-NNLP agar. MRS-NNLP agar consisted of MRS agar (Sigma-Aldrich, St. Louis, MO, USA) supplemented with nalidixic acid −15 mg·L−1, neomycin sulphate −100 mg·L−1, lithium chloride −3.0 g·L −1, paromomycin sulphate −200 mg·L−1. The plates were incubated in anaerobic conditions using a jar Anaerocult® A kit, anaerobic incubator, Merck KGaA, Darmstadt, Germany, for 48 h, at 37 °C. Plates containing 25–250 colonies were selected, and CFU mL−1 fermented product was recorded. All plate counts were carried out in triplicates.
Total phenolic compounds Measurement: were determined by the colorimetric method described by [31] using the Folin-Ciocalteu reagent. An amount of 0.5 mL of the drink was mixed with 0.5 mL of Folin-Ciocalteu reagent and 10 mL of saturated Na2CO3 solution, and samples were kept at room temperature for 1 h. After this time, the drinks were filtered (0.2 µm membrane), and the absorbance was measured using a UV-Vis Jenway 6506 spectrophotometer (Bibby Scientific Ltd., Staffordshire, UK) at λ = 725 nm. Concentrations were determined by comparing the absorbance of the samples with a calibration curve built with 0, 100, 250, 500, and 1000 mg gallic acid/100 mL (Scharlau Chemie, SA, Barcelona, Spain). Results were expressed as mg of gallic acid per 100 mL of drink.
Antioxidant activity Measurement: The free radical scavenging activity of all fermented samples was determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method [32]. Aliquots of 0.1 mL drink were mixed with: 3.9 mL of methanolic DPPH (0.025 g/L) and 0.090 mL of distilled water and the homogenate was shaken vigorously and kept in darkness for 30 min. Absorption of all the samples was measured using a UV-Vis Jenway 6506 spectrophotometer at λ = 515 nm, against a blank of methanol without DPPH. Results were expressed as % of inhibition of the radical DPPH, which can be related to the decrease in absorbance with respect to the control value, DPPH initial absorption value.
In vitro gastrointestinal digestion: The in vitro gastrointestinal digestion was carried out in two sequential phases: gastric and intestinal digestion.
Gastric digestion: it was performed in vitro simulated gastric juice (SGJ) as per the procedure of the USP [33], National Formulary: 2.0 g NaCl, 3.2 g pepsin and 3.0 mL concentrated HCl diluted to 1 L and adjusting the pH to 2.0 with concentrated HCl or sterile 0.1 mol L−1 NaOH.
Intestinal digestion: by suspending pancreatin USP (P-1500) in sterile sodium chloride solution (0.5% w/v), stimulated intestinal juices (SIJ) were prepared to a final concentration of 1 g L−1, adjusting the pH to 8.0 with sterile 0.1 mol L−1 NaOH and with 4.5% bile salts (Oxoid, Merck, Germany). Both solutions were filtered through a 0.22 μm membrane with the purpose of sterilization. As to replicate the temperature of the human body and the peristaltic bowel movements the experiment was carried out in a shaker incubator at 37 °C. Briefly, in a flask, 0.2 mL from each type of beverage was taken, added, and mixed with 10 mL of SGJ and incubated for 5, 30, 60, and 120 min and 60, 90, and 120 min, respectively in SIJ at 37 °C. Throughout each step, the shaking was kept at 50 rpm. After the completion of the process, the samples were evaluated for the count of bacteria by pour plate techniques (in MRS agar by anaerobic incubation at 37 °C for 3 days). All samples were analyzed in triplicate.
Sensory analysis: Sensory analysis of the mixed soy milk and sea buckthorn syrup containing Lactobacillus paracasei ssp. paracacasei and Bifidobacterium animalis subsp. lactis were also carried out by the 14 volunteers (consisting of students, 7 males and 7 females) familiarized with the sensory descriptors and the attribute intensities. Sensory evaluation was carried out after 24 h storage at 48 °C. The drink samples were evaluated, in a sensory laboratory under white light, for attributes of color, flavor, taste, texture, and overall acceptability using a nine-point structured hedonic scale: nine being considered excellent, five acceptable, and one extremely poor. The drink samples were served at 8 ± 2 °C in plastic cups labeled with three-digit codes, and the panelists used water to rinse their mouths between samples.
Statistical analysis: The data were in triple and subjected to statistical analysis using Statgraphics plus v.5.1 package (Manugistics Inc., Rockville, MA, USA). The data was analyzed by multifactor analysis of variance, and differences between the treatment means were separated using Duncan’s multiple range tests, with a significance level of 0.05.

3. Results

3.1. Microbial Content and Physicochemical Properties Analysis during Fermentation

The population of Bb-12® and L. casei® 431 in fermented soymilk drink with the sea buckthorn syrup throughout fermentation period at 30 and 37 °C is shown in Figure 1.
The influence of sea buckthorn syrup concentration on the growth Bb-12® and L. casei® 431 could be observed after 2 h fermentation at both temperatures: 30° and 37 °C. As the fermentation progressed, the number of CFU of Bb-12® and L. casei® 431 increased throughout the entire fermentation period to both temperatures of fermentation. Growth of the strains was highly similar, and only occasional differences in growth with statistical significance were observed (p < 0.05). At the end of the fermentation period, the number of viable cells of Bb-12® for the sample with 20% syrup was 1.42 × 109 CFU·mL−1 and 1.45 × 109 CFU·mL−1, respectively, for L. casei® 431 at 30 °C. For the same samples at 37 °C, the number of viable cells of Bb-12® was 1.62 × 1010 CFU·mL−1, and 1.63 × 1010 CFU·mL−1 for L. casei® 431, respectively.
A primary effect of sugar fermentation is the lowering of the pH value of the medium and the increase of the titratable acidity (TA) as shown in Figure 2, a rapid pH value drop in all drinks was observed, reaching around 4.7 at 12 h after of fermentation, for both temperatures: 30 and 37 °C. All drinks exhibited a pH decrease (p < 0.05) throughout the fermentation period.
TA were initially of 0.21 and 0.37 g lactic acid 100 mL−1 for 5% sea buckthorn syrup and 20%, respectively, for samples fermented at 30 °C and 0.24 and 0.40 g lactic acid 100 mL−1 for the same drinks fermented at 37 °C. However, at the end of the fermentation, the acidity increased for the same samples to 0.78 and 0.95 g lactic acid 100 mL−1, respectively, in the case of fermentation at 30 °C and 0.82 and 0.94 g lactic acid 100 mL−1 for the samples fermented at 37 °C (Figure 3). The TA changed significantly (p < 0.05) from all drinks.

3.2. Microbial Content and Physicochemical Properties Analysis during Storage Period

The results for the effect of the storage time on microbial content have been depicted in Figure 4. This evaluation began after the ending of the 12 h of fermentation. The total viable cells of Bb-12® (p < 0.05) were decreased moderately to 1.06 × 109 CFU·mL−1 for the drink with 20% sea buckthorn syrup, fermented at 30 °C and 1.08 × 1010 CFU·mL−1, respectively, for drink fermented at 37 °C.
For L. casei® 431, the total viable cells (p < 0.05) were also moderately decreased to 1.12 × 109 CFU·mL−1 and 1.12 × 1010 CFU·mL−1 for the beverage with 20% sea buckthorn syrup, respectively, fermented at 30 and 37 °C at the storage period end.
The changes in the pH values of the drink samples during storage are shown in Figure 5. The pH values of the drinks from different treatments slightly significantly decreased (p < 0.05) during the storage period. In general, the pH values of all drinks were around 4.4. By contrast, the TA values increased (p < 0.05) in all drinks after 14 days of storage period. In Figure 6, the values for all drinks after the storage time are shown. The TA values range between 0.78 and 0.95 g lactic acid 100 mL−1 for the sample with 5% and 20% sea buckthorn syrup, respectively, for fermentation at 30 °C. For fermentation at 37 °C, the values range between 0.82 and 0.94 g lactic acid 100 mL−1 for the drink with 5% and 20% sea buckthorn syrup, respectively.
The effects of different concentrations of sea buckthorn syrup on the WHC are shown in Figure 7. During the period of storage, WHC was higher for all beverages. As can be seen from Figure 7, the WHC of all drinks varied between 74–77% at the end of the refrigeration period, and the values of the drinks from different treatments slightly significantly decreased (p < 0.05).

3.3. Antioxidant Properties in Fermented Drinks

Figure 8 represent the changes in DPPH radical scavenging activity in fermented drinks, and their combination using different concentration of sea buckthorn syrup after 14 days of storage. All treated drinks showed a lower percentage of scavenging activity than their respective controls. The drink with 20% of sea buckthorn syrup showed a significant (p < 0.05) increase in scavenging activity at the end of storage (68%). The drinks with 5% and 10% sea buckthorn syrup had lower scavenging activity around 57% than drinks with 15% sea buckthorn syrup which showed 60% and 64% scavenging activity, respectively, for both temperatures of fermentation.
Figure 9 represents the changes in the total phenolic compounds in fermented drink and their combination using different concentrations of sea buckthorn syrup after 14 days of storage. All drinks displayed greater total phenolic compounds (p < 0.05) than their respective controls after 14 days of storage. The total phenolic compounds were the lowest 0.36 and 0.39 mg gallic acid/mL for the drink with 5% and 20% sea buckthorn syrup fermented at 30 °C (p < 0.05), respectively, compared with drinks fermented at 37 °C. However, the drink with 20% sea buckthorn syrup fermented at 37 °C showed maximum activity of total phenolic compounds (0.68 mg gallic acid/mL) compared with the rest of the drinks fermented at 30 and 37 °C, respectively.

3.4. Gastrointestinal Simulation

The influence of in vitro gastrointestinal digestion on viability of the drinks is shown in Figure 10 and Figure 11. During the digestion process, the bacterial count of Bb-12® and L. casei® 431 in all the samples was reduced significantly (p < 0.05).
There were lower reductions after the gastric simulation compared with the intestinal simulation for all samples. The positive effects of the concentration of sea buckthorn syrup, in the gastric simulation can be observed in all drinks. For the drinks with 20% sea buckthorn syrup, the cell viability was significantly higher: 77% for the sample fermented at 30 °C and 79% for the sample fermented at 37 °C, respectively, for Bb-12® (p < 0.05). The decrease was significant for the same samples in the case of L. casei® 431- 43% for the sample fermented at 30 °C and 77% for the sample fermented at 37 °C, respectively (p < 0.05). The same positive effects of concentration of sea buckthorn syrup, in the intestinal simulation for Bb-12® drinks can be observed: 65% for drink fermented at 30 °C and 80% for drink fermented at 37 °C, respectively. For the same drinks in the case of L. casei® 431 results show a decrease of up to 47% for the sample fermented at 30 °C and 78% for the sample fermented at 37 °C, respectively (p < 0.05).

3.5. Sensory Evaluation

Descriptive sensory characteristics of fermented drinks with Bb-12® and L. casei® 431 were assessed on a 9-point hedonic scale, by a sensory panel, in terms of color, flavor, taste, texture, and overall acceptability. The scores are summarized in Table 1.
There were observed significant differences between all beverages (p < 0.05). The drink with 20% sea buckthorn syrup fermented at 37 °C, had a more intense color, and was therefore better scored by the panelists. Regardless of the attributes of flavor and taste, there are no significant differences (p > 0.05) within drinks (fermented at both temperatures) supplemented with sea buckthorn syrup. Formulation 20% sea buckthorn syrup, fermented at 37 °C) (Table 1) had the highest values for flavor, taste, and overall acceptability attributes, differing significantly from the other (p < 0.05).

4. Discussion

The success of the new probiotic drink does not only rely on the ability to provide enough probiotic culture that may survive the human gastrointestinal tract, but it is also very important and an appropriate selection of substrate composition. Also, strains are necessary for the efficiency of control over the distribution of metabolic end products [34]. The potential roles of probiotics in diet have still been under exploration and the contradictory results provided by different studies make it difficult to develop a general recommendation for obtaining a new innovative drink. For this reason, [35] reported that the presence of numerous probiotics with different characteristics can explain the requirement for further studies that explore the best combination with the highest efficiency. This study examined the possibility of producing an innovative drink based on soy milk–sea buckthorn syrup in different concentrations and fermented with a co-culture by Bb-12® and L. casei® 431 strains.
To the best of our knowledge, no studies are available reporting on the production of innovative drinks, based on soymilk with different concentrations of sea buckthorn syrup and fermented with a co-culture of probiotics; in this regard, it is much more difficult to compare our results with those reported for other products due to differences such as experimental conditions, food matrix, and the studied microorganisms.
The cell viability of Bb-12® and L. casei® 431 strains was not affected by either the time, concentration, or temperature. However, at the 6th h of fermentation, its number increased, and the results indicated that there was a competition for the nutrients. The increase in cell numbers was higher at 37 °C for the drink with 20% sea buckthorn syrup, compared to 30 °C. As shown in Figure 1 supplementation with different amounts of sea buckthorn syrup in drink affects the growth of Bb-12® and L. casei® 431; the cell numbers as the samples increased with the amount of sea buckthorn syrup.
During storage, the cell numbers of Bb-12® and L. casei® 431 highly increased for the drink fermented at 30 °C and 37 °C, respectively, (Figure 4). Generally, during the storage period, cell numbers of Bb-12® and L. casei® 431 increased slightly for all beverages at both temperatures of fermentation. Analysis of variance for the probiotic counts showed that supplementation with different concentrations of sea buckthorn syrup in the drink affects the growth of Bb-12® and L. casei® 431; the viable cell numbers as the drinks increased with the amount of concentration of sea buckthorn syrup both during fermentation and the storage period. At the time of food consumption, after 14 days of storage at 4 °C, the recommended level of viable probiotic cells is 107–108 CFU·mL−1 [36]. Our viable cells of Bb-12® and L. casei® 431 remained at the level mentioned. Also, [37] mentions that not less than a million viable cells/mL of probiotic product is needed to reach the minimum amount of health benefits, for consumers. Thus, in this study, Bb-12® and L. casei® 431 strains were selected to be co-cultured to obtain short fermentation periods and larger populations of the probiotic bacteria. Data from this study showed that high levels of the L. casei® 431 probiotic could be reached when this strain is inoculated with Bb-12®. When these two selected strains were co-incubated, the probiotic bacteria had their growth favored reaching the population of 109 L. casei® 431 CFU·mL−1 and 109 Bb-12® CFU·mL−1 within 12 h of fermentation at 30 °C, respectively, 1010 L. casei® 431 CFU·mL−1 and 1010 Bb-12® CFU·mL−1 for drinks fermented at 37 °C.
Our results demonstrate that Bb-12® and L. casei® 431 co-culture strains can be used as a probiotic for obtaining innovative drinks, based on soymilk, and sea buckthorn syrup. The results obtained in this study confirm that soymilk and sea buckthorn syrup substrates have nutrient content high enough to support the growth and metabolism of lactic acid bacteria, as earlier reported by other researchers for another substrate [38,39,40,41,42,43]. The results of this study indicated that the nutrient content of the drinks was just enough to maintain a probiotic population of about 108 CFU·mL−1. Kandler and Weiss [44] declared that the requirements for nutrients are specific characteristics for each strain of Lactobacillus. Also, [45] declared that the growth capacity of different lactic acid bacteria, isolated from different products, has a different result based on the nutrient content of the medium in which it is being grown.
In conclusion, we can declare that previously, it’s very important to test the compatibility between probiotic and food matrix to provide a positive interaction capable of increasing the cell viability during the fermentation and storage period.
Figure 2 presents the evolution of the pH during the fermentation process at the temperatures of 30 and 37 °C. He et al. [46] reported that the pH of the drinks, as the fermentation proceeds, lowers because the fermenting organisms use carbohydrates as their main carbon source to produce acid (mostly lactic acid), but also short-chain fatty acids. Kailasapathy and Chin [47] declared that a pH of around 3.5–4.5 reported for drink formulations aids the pH increase of the gastrointestinal tract, and, because of this fact, enhances the stability benefits of probiotic strains consumed.
The pH had a slight variation in the various tests, remaining within an interval of 4.4 to 4,8 between 1 and 14 days of storage (Figure 5). Instead, the pH-lowering during storage was an expected behavior by the high activity of lactic acid probiotic cultures that are also found in higher numbers. The pH value reached was below 4.5 in all co-cultured fermentations, which demonstrated that 109–1010 CFU·mL−1 of inoculum was sufficient to decrease the pH. Our results are like those from Rinaldoni et al. [48], who reported that ultrafiltrated soymilk concentrate fermented by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus did not reach a pH lower than 4.9. Also, Bianchi et al. [49] declared a reduction in pH during 28 days of storage in the fermented beverage formulated with aqueous extracts of quinoa and soy. However, lower pH values have been reported in other grain-based beverages obtained from oats, wheat, and quinoa by lactic acid fermentation for 12–24 h [38,50].
The titratable acidity increased with the decrease of pH for all drinks during fermentation for both temperatures, and the drinks with 20% sea buckthorn syrup have a higher value of titratable acidity compared with all the other drinks during the fermentation process and storage period (Figure 3, Figure 6, respectively). Generally, the results of the pH values followed an opposite trend to that observed for TA measurements, i.e., as the acidity increased, the pH decreased. Our results agree with the results declared by Isanga and Zhang [51]. The researchers developed fermented peanut milk with the mixed cultures of Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus. thermophilus and reported that fermentation with combined cultures had higher titratable acidity values (0.39%) than with the single cultures of these strains. The levels obtained for lactic agreed with values reported by other researchers for another substrate [38,42,50,52].
The marked lactic acid increase in co-culture fermentations with Bb-12® and L. casei® 431 could be due to interactions between the different species used. The results obtained in our study highlighted the importance of the selection of the substrate composition and inoculum in the development of the organoleptic properties of these fermented drinks.
The WHC values obtained on the first day of storage were higher than those found at the end of storage and significantly decreased with the increasing sea buckthorn syrup concentrations (Figure 7). The control of WHC is regarded as extremely important for the process due to the visual appearance of the final product. Due to this parameter, the final product can be often rejected by customers. Mondragón-Bernal et al. [53] declared that this defect is caused when the gel formed cannot retain the liquids of the product in the three-dimensional arrangement and are expelled, comparable to tears (the product is crying”). Our results agree with the results reported by [53,54]. Instead, Lin et al. [42] reported that during the storage period, for the drink based on L. chinense Miller juice, milk and soy milk which were fermented with mixed cultures (Bifidobacterium longum and Lactobacillus paracasei subsp. paracasei NTU101) no syneresis phenomenon was observed.
In conclusion, we can say that although the two probiotic strains from innovative drinks acted synergistically to produce a rapid fermentation, excessive fermentation resulted in a loose protein network structure during the same time, leading to a low WHC.
Figure 9 presents the values for total phenolic compounds after 14 days of storage at a temperature of 4 °C. Taşkın and Bağdatlıoğlu [55] reported that defenses against environmental stresses are provided by plant phenolic and flavonoid chemicals, which are beneficial to human health. In the present study, the addition of two probiotic strains Bb-12® and L. casei® 431 into soy milk during fermentation has been demonstrated to boost the phenolic content at varied concentrations of sea buckthorn syrup compared to the control after the storage period of 14 days. Accumulative scientific evidence from literature demonstrated that phenolic compounds counteract oxidative stress by sequestering oxidant nitrogen and oxygen species, transferring electrons to free radicals and activating antioxidant enzymes. Due to this fact, thusly, it contributes to the prevention of diabetes, cancer, cardiovascular diseases, and obesity [56]. However, phenolic compounds must be available in the target tissue to exert their biological activity. Also, it has been established the bioconversion of phenolic conjugated forms to the corresponding aglycones and the release of bound forms to the corresponding free forms during lactic acid fermentation [40,57]. Taking together all these findings suggests that fermentation of soymilk with different concentrations of sea buckthorn syrup and co-culture from Bb-12® and L. casei® 431 is a promising approach to manufacturing novel antioxidant drinks enriched in bioaccessible phenolic compounds. The results obtained in this study agree with the results reported by [58,59,60]. Alamed et al. [61] declared that probiotic bacteria possess certain enzymes (phenolic acid decarboxylases), which aid in reducing polyphenol concentrations in the end product.
Figure 8 represents the changes in DPPH radical scavenging activity, in fermented drinks, and their combination using a co-culture of Bb-12® and L. casei® 431 after 14 days of storage. All treated samples showed a higher percentage of scavenging activity. In the present research, depending on the probiotic starter cultures and sea buckthorn syrup concentration used during the fermentation, a slight variance in the DPPH values in the final products was noticed for both temperatures of fermentation. Wijeratne et al. [62] reported that the fermentation process decreases polyphenol content, and this may enhance protein and carbohydrate digestion and improve mineral absorption, therefore enhancing the antioxidant value of fermented drinks. Antioxidant activity inhibits the oxidation of molecules caused by free radicals and is important for the shelf life of foods and, to protect the human body against oxidative damage upon consumption. Bioactive compounds in foods play a crucial role in elevating the effect of reactive oxygen species such as hydroxyl, superoxide, and peroxyl radicals formed by cells under oxidative stress. These bioactive compounds can donate electrons to neutralize free radicals and the presence of several amino acid residues in the peptide chains can enhance antioxidant properties [63]. Similar values were also mentioned by [64] for a fermented plant-based milk drink made from individual soy and almond milk and [65], which displayed an antioxidant activity of about 85.36% during the storage period of fermented soymilk and [66] reported that ferment soymilk with L. plantarum B1-6 had a DPPH activity ranging from 27.79 to 94.92%. Since oxidative stress plays a crucial role in the pathogenesis of several chronic diseases, daily consumption of our innovative drink based on soymilk, sea buckthorn syrup fermented with a co-culture of Bb-12® and L. casei® 431 could have a beneficial impact on health. However, the explanation for the contrast detected between the decreased concentration of total phenolic compounds and the increased antioxidant activity for our drinks could be that: some lactic acid bacteria can degrade some phenolic compounds, and this determines producing compounds that increased antioxidant activity. Another important point is that once the co-culture strains are subjected to the acidic conditions of the drinks, an adaptation to the environment can occur. From this point of view, this thing favors the survival of the bacteria to acid stress during the passage through the human gastrointestinal tract, as assessed in the next section.
The survival of the co-culture Bb-12® and L. casei® 431 in a soy milk and sea buckthorn syrup matrix under SGJ and SIJ conditions was analyzed (Figure 10 and Figure 11). Plate counts showed enhanced protection of the Bb-12® strain compared to L. casei® 431 for both temperatures of fermentation. With our matrix, a significant reduction in the viability of L. casei® 431 was observed at the end of the incubation under SGJ conditions, corresponding to 43% and 77% survival (as compared to the starting point) for the drink with 20% sea buckthorn syrup fermented at 30 and 37 °C. Under SIJ conditions, for the same sample, the viability slightly increased (47% and 78% for L. casei® 431) yet remained significantly lower compared to the Bb-12® strain. Regarding the concentration of sea buckthorn syrup and fermentation temperature, the drink with a concentration of 20% was more effective than the drink with a concentration of 5%. The results obtained in this study agree with previous studies [67,68,69].
Survival under conditions simulating those of the gastrointestinal tract, particularly resistance to bile and low pH, is a crucial requirement of microorganism (probiotic) strains. The results obtained in our study show there to be great strain-dependent variability with respect to these traits, as reported conclusion by other authors [70,71]. Another important feature of potential probiotic strains is their ability to bind to the intestinal mucosa which may lead to the competitive exclusion of pathogenic bacteria [72]. Great heterogeneity in adhesion among different strains has been reported in previous surveys because adherence of bifidobacteria to enterocytes has been found to be related to their surface properties: hydrophobicity and auto-aggregation ability [72,73]. Refs. [67,74] declared that probiotics are degraded not only by the acidity in the stomach but also by salts and enzymes (pepsin and lysozyme). Thus, we were able to observe in this study, the impact of digestive enzymes and stomach hydrochloric acid on bacterial stability, highlighting the lower viability of the L. casei® 431 strain and the higher viability of the Bb-12® strain in acidic media.
To exert beneficial effects, the probiotic strains used in the food matrix should be delivered into the intestine in sufficient concentration. Therefore, an optimum carrier for oral probiotic delivery should be (1) suitable for human consumption, (2) deliver the probiotics to the colon, (3) protect during passage through the upper intestine, and (4) support the strains’ viability during the manufacturing and commercial life of the product.
The sensory attributes of taste, flavor, texture, color, and overall acceptability to sea buckthorn syrup and soymilk fermented with a co-culture were evaluated. The results were exhibited in Table 1. The scores for taste, flavor, texture, and overall acceptability of the soymilk fermented at 37 °C were all > 5, which presented neither like nor dislike. However, all the scores of five evaluated attributes of the soymilk with sea buckthorn syrup fermented with Bb-12® and L. casei® 431 at 30 °C were between 5 (neither like nor dislike) and 8 (like very much). These results indicated that soymilk with sea buckthorn syrup fermented with Bb-12® and L. casei® at 37 °C obtains a product with higher sensory quality than that fermented at 30 °C. From the results obtained, it can be concluded that the product had a good overall acceptability. Similar results were also mentioned by [75,76,77].
As can be seen, the probiotic strains Bb-12® and L. casei® used the concentration of sea buckthorn syrup, which was another important factor regarding the functionality of the drinks analyzed in this study. The concentration of the syrup influenced the viability of cells, pH, TA (during fermentation and storage period), WHC, antioxidant capacity, phenolic compounds, sensory analysis, and tests performed in SGJ and SIJ conditions for both temperatures. Also, the results obtained in this study suggest the potential of soymilk as a functional food. Fermented soymilk with sea buckthorn syrup containing probiotics such as Lactobacillus and Bifidobacterium may also be a candidate symbiotic.
In conclusion, incorporating sea buckthorn syrup into a fermented soy drink with a co-culture of Bb-12® and L. casei® 431 is an innovative approach that could provide nutritional and health benefits using a natural resource.

5. Conclusions

This research showed that the addition of 20% sea buckthorn syrup in a fermented soy-based product positively influences the growth and development of Bb-12® and L. casei® 431. The viability of these two strains increased significantly after 12 h of fermentation, and the pH decreased at values around 4.70. Also, this study proved that the concentration of the syrup influenced the viability of cells, pH, TA (during fermentation and storage period), WHC, antioxidant capacity, phenolic compounds, and sensory analysis. A significant enhancement of antioxidant capacities was observed after Bb-12® and L. casei® 431 fermentation, positively correlated to some lactic acid bacteria, which can degrade some phenolic compounds, and this determines the production of compounds that increased antioxidant activity. The sea buckthorn syrup in the fermented drink was found to have positively impacted the growth of probiotics. However, the soy drink with sea buckthorn syrup, because of the bacterial count, also qualifies as a fermented probiotic drink. Through SGJ and SIJ conditions, the low pH of the stomach and the antimicrobial action of pepsin are known to provide an effective barrier against the entry of bacteria into the intestinal tract. Regarding the viability of the probiotics after gastrointestinal simulation, the protective effect given by sea buckthorn syrup can be observed in the case of the co-culture of Bb-12® and L. casei® 431 drinks. Accordingly, according to the results obtained after digestion, the supportive impact of syrup depends on the bacteria used. Thus, enriching soy beverages with sea buckthorn syrup and fermenting with these two strains, Bb-12® and L. casei® 431, presents a functional improved product. Further investigation on probiotic encapsulation techniques and another concentration of sea buckthorn syrup is required to understand the components responsible for the effective distribution of Bb-12® and L. casei® 431 through the gastrointestinal tract.

Author Contributions

Conceptualization, N.-M.M., A.Y.R.-V. and A.-V.I.; methodology, N.-M.M., D.B.M. and M.B.; software, N.-M.M., A.Y.R.-V. and R.E.G.B.; validation, N.-M.M., A.Y.R.-V. and A.-V.I.; formal analysis, N.-M.M., R.E.G.B., D.B.M. and M.B.; investigation, N.-M.M., D.B.M., M.B. and A.-V.I.; resources, N.-M.M.; data curation, N.-M.M. and D.B.M.; writing—original draft preparation, N.-M.M.; writing—review and editing, N.-M.M., A.Y.R.-V. and A.-V.I.; visualization, N.-M.M., A.Y.R.-V. and R.E.G.B.; supervision, N.-M.M., A.Y.R.-V. and R.E.G.B.; project administration, N.-M.M.; funding acquisition, A.-V.I. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the “Dunarea de Jos” University of Galati, VAT 3127522 and The APC was paid by the “Dunarea de Jos” University of Galati, VAT 3127522.

Institutional Review Board Statement

The study did not require ethical approval.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors acknowledge the research staff of “Dunarea de Jos” University” Galati for the financial support of publishing our study.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00806 g001
Figure 1. Cell viability profile of the fermentation with Bb-12® and L. casei® 431. Values for Bb-12® and L. casei® 431viable cell growth is displayed as mean values, CFU·mL−1, n = 3.
Figure 1. Cell viability profile of the fermentation with Bb-12® and L. casei® 431. Values for Bb-12® and L. casei® 431viable cell growth is displayed as mean values, CFU·mL−1, n = 3.
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Figure 2. Changes in pH value during fermentation process.
Figure 2. Changes in pH value during fermentation process.
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Figure 3. Changes in titratable acidity of drinks during fermentation process.
Figure 3. Changes in titratable acidity of drinks during fermentation process.
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Figure 4. Survivability of Bb-12® and L. casei® 431 in drinks during storage at 4 °C.
Figure 4. Survivability of Bb-12® and L. casei® 431 in drinks during storage at 4 °C.
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Figure 5. Changes in pH during storage period.
Figure 5. Changes in pH during storage period.
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Figure 6. Changes in titratable acidity during storage period.
Figure 6. Changes in titratable acidity during storage period.
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Figure 7. Changes in water holding capacity during storage period.
Figure 7. Changes in water holding capacity during storage period.
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Figure 8. Antioxidant capacity in fermented drinks after storage period.
Figure 8. Antioxidant capacity in fermented drinks after storage period.
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Figure 9. Changes in total phenolic content after 14 days of storage.
Figure 9. Changes in total phenolic content after 14 days of storage.
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Figure 10. Survival rates of Bb-12® and L. casei® 431 incorporated into the drinks fermented at 30 °C after continuous gastrointestinal simulations.
Figure 10. Survival rates of Bb-12® and L. casei® 431 incorporated into the drinks fermented at 30 °C after continuous gastrointestinal simulations.
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Figure 11. Survival rates of Bb-12® and L. casei® 431 incorporated into the drinks fermented at 37 °C after continuous gastrointestinal simulations.
Figure 11. Survival rates of Bb-12® and L. casei® 431 incorporated into the drinks fermented at 37 °C after continuous gastrointestinal simulations.
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Table 1. Sensory evaluation parameters of fermented drinks.
Table 1. Sensory evaluation parameters of fermented drinks.
CharacteristicsBeverage Sample
Fermented at 30 °CFermented at 37 °C
5%10%15%20%5%10%15%20%
Color5.8 c7.3 a,b7.6 a,b7.9 a5.85 c7.3 a,b7.6 a,b8.9 a,b
Flavor6.9 b7.3 a,b7.3 a,b7.8 a7.1 b7.4 a,b7.6 a,b8.8 a
Taste6.9 b7.4 a7.3 a,b7.4 a6.8 b7.3 a,b7.3 a,b8.6 b
Texture6.7 b7.3 a,b7.4 a7.5 a6.8 b7.3 a,b7.4 a8.5 a
Overall acceptability6.9 b7.2 b7.9 a8.1 a7.1 b7.4 b76.9 a8.3 a
a,b,c Different superscript letters in the same rows indicate statistically significant difference (p < 0.05) according to Duncan multiple-range test. Values are expressed as means ± SD (n = 3).
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MDPI and ACS Style

Maftei, N.-M.; Bogdan, R.E.G.; Boev, M.; Marin, D.B.; Ramos-Villarroel, A.Y.; Iancu, A.-V. Innovative Fermented Soy Drink with the Sea Buckthorn Syrup and the Probiotics Co-Culture of Lactobacillus Paracasei ssp. Paracasei (L. Casei® 431) and Bifidobacterium Animalis ssp. Lactis (Bb-12®). Fermentation 2023, 9, 806. https://doi.org/10.3390/fermentation9090806

AMA Style

Maftei N-M, Bogdan REG, Boev M, Marin DB, Ramos-Villarroel AY, Iancu A-V. Innovative Fermented Soy Drink with the Sea Buckthorn Syrup and the Probiotics Co-Culture of Lactobacillus Paracasei ssp. Paracasei (L. Casei® 431) and Bifidobacterium Animalis ssp. Lactis (Bb-12®). Fermentation. 2023; 9(9):806. https://doi.org/10.3390/fermentation9090806

Chicago/Turabian Style

Maftei, Nicoleta-Maricica, Roxana Elena Goroftei Bogdan, Monica Boev, Denisa Batîr Marin, Ana Yndira Ramos-Villarroel, and Alina-Viorica Iancu. 2023. "Innovative Fermented Soy Drink with the Sea Buckthorn Syrup and the Probiotics Co-Culture of Lactobacillus Paracasei ssp. Paracasei (L. Casei® 431) and Bifidobacterium Animalis ssp. Lactis (Bb-12®)" Fermentation 9, no. 9: 806. https://doi.org/10.3390/fermentation9090806

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