IMR Press / FBE / Volume 16 / Issue 2 / DOI: 10.31083/j.fbe1602011
Open Access Original Research
Investigation on Fermented Milk Quality after the Addition of Flaxseed Mucilage and the Use of Lactobacillus delbrueckii subsp. bulgaricus and Lactiplantibacillus plantarum AG9
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1 Department of Meat and Milk Technology, Institute of Food Production and Biotechnology, Kazan National Research Technological University, 420015 Kazan, Russia
*Correspondence: ev-nikitina@inbox.ru (Elena Nikitina)
These authors contributed equally.
Front. Biosci. (Elite Ed) 2024, 16(2), 11; https://doi.org/10.31083/j.fbe1602011
Submitted: 23 September 2023 | Revised: 29 January 2024 | Accepted: 26 March 2024 | Published: 7 May 2024
Copyright: © 2024 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
Abstract

Background: Flaxseed mucilage (FSM) is one of the healthy components of flaxseed. FSM is an example of a material that can be used in the food, cosmetic, and pharmaceutical industries due to its rheological properties. FSM consists mainly of two polysaccharides, arabinoxylan, and rhamnogalacturonan I, and it also contains protein components and minerals. The prospect of using FSM in food is due to its gelling, water binding, emulsifying, and foaming properties. In addition, valuable natural sources of phenolic compounds such as lignans, phenolic acids, flavonoids, phenylpropanoids, and tannins are partially extracted from flaxseed in FSM. These antioxidant components have pharmacological properties, including anti-diabetic, anti-hypertensive, immunomodulatory, anti-inflammatory and neuroprotective properties. A combination of FSM and lactobacilli in dairy foods can improve their functional properties. This study aimed to develop dairy products by adding of FSM and using two lactic acid bacteria (LAB). FSM (0.2%) was used as an ingredient to improve both the texture and antioxidant properties of the product. Methods: Skim milk was fermented with 0.2% flaxseed mucilage using Lactobacillus delbrueckii subs. bulgaricus and the probiotic Lactiplantibacillus plantarum AG9. The finished fermented milk products were stored at 4 °C for 14 days. Quantitative chemical, textural, and antioxidant analyses were carried out. Results: Adding 0.2% FSM to the dairy product stimulated the synthesis of lactic acid. FSM increased the viscosity and water-holding capacity of L. bulgaricus or L. bulgaricus/L. plantarum AG9 fermented milk products. Combining these starter strains with FSM promoted the formation of a hard, elastic, resilient casein matrix in the product. When only L. plantarum AG9 was used for the fermentation, the dairy product had a high syneresis and a low viscosity and firmness; such a product is inferior in textural characteristics to the variant with commercial L. bulgaricus. The addition of FSM improved the textural properties of this variant. The use of L. plantarum AG9 and FSM makes it possible to obtain a fermented milk product with the highest content of polyphenolic compounds, which have the highest antioxidant properties and stimulate lipase and α-glucosidase inhibitor synthesis. Combining of L. bulgaricus and L. plantarum AG9 in the starter (20% of the total mass of the starter) and adding of 0.2% FSM is the optimal combination for obtaining a dairy product with high textural and antioxidant properties. Conclusions: The physicochemical properties (viscosity, syneresis, water holding capacity, texture) and antioxidant properties of fermented milk were improved. In the future, as part of the work to investigate the functional properties of dairy products with FSM, studies will be conducted using in in vivo models.

Keywords
flaxseed mucilage
lactobacilli
fermented milk
texture
physical–chemistry
antioxidants
microstructure
1. Introduction

Recently, the range of dairy and fermented milk products has increased. This is due to the increasing demand for healthy foods with good consumer properties [1, 2]. Various plant additives are used to increase the variety of products, which positively affect their consumer properties. Biopolymers of polysaccharide nature from plants, contained in the cladodes or seed shells of plants such as chia or flax, have recently attracted much attention as an alternative biopolymer for food applications [3, 4].

Flaxseed (Linum usitatissimum) and its constituents contain a range of fatty acids, polysaccharides, plant polyphenols (flavonoids, lignans), fiber, vitamins, and minerals [5]. Flaxseed influences glycemic control and insulin resistance in pre-diabetes and type 2 diabetes [6]. The addition of flaxseed to the diet has been shown to reduce glycated hemoglobin (HbA1c) in people with type 2 diabetes [7].

One of the healthy components of flaxseed is mucilage [8], which is composed of polysaccharides. Flax seed mucilage (FSM) can be a functional component in fermented dairy products [3, 9, 10]. The qualitative composition of the polysaccharide monose is xylose, glucose, galactose, rhamnose, fucose, and galacturonic acid [11]. The chemical composition of linseed gum and the molecular properties of its polysaccharides are influenced by the origin and genotype of the linseed itself [12, 13]; it has been found that brown varieties of flaxseed contain more rhamnose and galacturonic acid than yellow varieties, while arabinoxylan contains less. This difference in chemical composition affects the properties of the extractable mucilage from the seeds; the mucilage obtained from brown flaxseed has more pronounced gel-forming properties, and the mucilage from yellow flaxseed has a more active liquefying ability [14]. Flaxseed mucilage has recently been shown to have several beneficial properties. FSM has antioxidant, antiviral and anticarcinogenic properties [15], improves digestion and gastrointestinal peristalsis [16, 17], has a beneficial effect on reactive hypoglycemia and reduces glucose diffusion [18, 19, 20], helps control body fat and weight [19], and is involved in the regulation and establishment of healthy gut microbiota [21, 22]. It is important to note that the use of FSM as a functional food additive (in dairy products fermented with probiotic cultures) has a positive effect on viscosity indicators and improves the texture properties of the product, which increases the consumer properties of the product [23, 24, 25]. Flaxseed gums at 0.6% benefit the viscosity, texture, and syneresis of yogurt [26]. However, despite its beneficial effect on the textural properties of stirred yogurt, a concentration of 0.6% or more reduces the sensory evaluation (taste and overall acceptability) due to a granular and slightly bitter aftertaste. Several studies have shown that FSM polysaccharides are potential prebiotic food ingredients that benefit human health through the gut microbiota [27].

Lactic acid bacteria (LAB) have a significant positive impact on human health. Thus, regular consumption of fermented dairy products improves digestion, enriches the gut microbiome, and is available for people with lactose intolerance [28]. Lactobacilli have probiotic and anti-diabetic properties; however, further research into their benefits for human health is needed [29]. The different botanical origins of the mucilage have prebiotic properties that help preserve the probiotics in dairy products [30, 31]. Combining FSM and lactobacilli enhances their properties. Adding mucilage to fermented dairy products can increase the viability of lactic acid microorganisms and improve the structural, physicochemical, textural, and sensory properties of yogurt [32, 33]. Flax gum also helps to encapsulate microorganisms such as Lactobacillus plantarum and Bifidobacterium infantis [34]. Lactobacilli, in turn, reduce the concentration of cyanogenic glycosides during fermentation, which negatively affects nutrient absorption and can lead to deterioration in human health [35].

We have previously shown a positive effect of FSM on the probiotic properties of Lactiplantibacillus plantarum AG9 and Lactobacillus delbrueckii subs. bulgaricus. Depending on the strain, the increase in their resistance to gastric juice varied with FSM application [36]. In addition, the antioxidant capacity of these strains and the products of their metabolism varied differently in the presence of FSM. Previous studies showed that FSM at a concentration of 0.4% in a nutrient medium reduces the probiotic properties of LABs, particularly hydrophobicity and auto-aggregation. Considering our studies and the possibility of taste defects when high concentrations of FSM are applied, a low dose of 0.2% FSM was used in this work. The issue of FSM application is relevant from the point of view of its use in functional products [36]. This work studied the effect of FSM addition to fermented products on their physicochemical, antioxidant, microstructural, and textural properties during storage. The relationship between FSM and L. plantarum AG9 or L. bulgaricus strains in this process was analyzed.

2. Materials and Methods
2.1 Strains, Flax Seed Mucilage and Milk Fermentation

Ultra-high-temperature skim milk (0.05% m.f., Viola, Russia) was used for the fermentation. The final milk values were 3.18% protein and 4.7% lactose. The flaxseed (Linum usitatissimum L.) mucilage was obtained from brown flax seeds and lyophilically dried; the procedure has been described previously [36]. FSM was added to milk at a concentration of 0.2% and pasteurized in a boiling water bath for 30 min with constant stirring for uniform mucilage distribution. A control milk sample was pasteurized using the same procedure.

Strains Lactobacillus delbrueckii subsp. bulgaricus (Lactosintez LLC, Russia), and Lactiplantibacillus plantarum AG9 (previously Lactobacillus plantarum AG9) were used for milk fermentation; they were described earlier [37]. L. plantarum AG9 was isolated from silage; the antibiotic resistance has been described previously [37]. L. plantarum AG9 strain grown on blood agar (Himedia, India) showed no zones of hemolysis (γ-hemolysis) around the colonies. LABs were stored in de Man, Rogosa, and Sharpe (MRS) broth (Himedia, Mumbai, India) with 50% glycerol at –80 °C. For culture activation, a 100 µL aliquot of each culture was individually transferred into MRS broth and incubated at 37 °C for 24 h. This culture was used for injection into skimmed milk for pre-cultures. The LAB pre-cultures were prepared by incubating in skimmed milk at 40 ºC for 16 h. After that, the starter culture was added to milk mixtures at a rate of 5% to the mass of raw milk. The milk was incubated for the fermented product at 40 °C for 8 h, then the finished product was cooled to stabilize at 4 °C for 16 h. After stabilization, each sample was analyzed and stored for 14 days at 4 °C.

2.2 Analysis of Fermented Skimmed Milk Properties

Analysis of protein and carbohydrates was performed on an InfraLUM® FT-12 device (RF) with appropriate software “SpectraLUM/Pro®” and calibration data for the product “yogurt”, protein in whey salt was tested in the serum from sour milk product after centrifugation at 3000 g for 15 min using milk analyzer “Klever-2M” (Biomer, Moscow, Russia). Glucose was measured in the whey using an Accu-Chek active GC glucometer (Roche, Mannheim, Germany). The pH was measured using a digital pH meter (HI 2216, Hanna Instruments, Vöhringen, Germany). The methods for analyzing of titratable acidity, water holding capacity (WHC), and syneresis have been described previously [38]. The viscosity of the samples (100 mL) was measured at 8 °C using a Brookfield rotary viscometer NDJ-9T (Changsha Lonroy Technology, Changsha, China) equipped with rotor no. 3, stirring at 30 rpm, measured for 10 s. Measurements were carried out in triplicates for each treatment, and results were recorded in mPas.

The texture profile analysis test was conducted using an ST-2 texture analyzer (Quality Laboratory JSC, Moscow, Russia) with a 36 mm diameter and 35 mm height cylindrical probe, penetrating the undisturbed fermented milk samples. Two cycles were applied to a depth of 10 mm at the rate of 0.5 mm s-1, touch force 7 g. Fermented milk was tested in a chemical beaker with a diameter of 50 mm, and the height of the sample was 25 mm. As a result, a plot of force versus time was obtained for each sample using the software ST-2 for Windows (Quality Laboratory JSC).

Total phenolic compounds (TPCs) in water extract and cell-free supernatants were determined using Folin–Ciocalteu reagent [37]. The peptide concentration analysis using an O-phthaldialdehyde (OPA) assay was described previously [39].

2.3 Scanning Electron Microscopy

The microstructure properties of fermented milk samples were evaluated by Scanning Electron Microscopy (SEM) [38]. Briefly, these samples were fixed with 2.5% glutaraldehyde for 4–5 h, subsequently washed three times with 0.2 M Na–K phosphate buffer (pH 7.0), then dehydrated using 30%, 40%, 50%, 60%, 70%, and 80% (twice for each concentration) at 15 min, and dehydrated in 95% ethanol three times at 30 min. The samples were mounted on metal stubs and coated with gold-palladium alloy (~10 nm thickness) using the Quorum Q150T ES coating machine. Samples were then observed using a self-emission scanning electron microscope Merlin (Carl Zeiss, Jena, Germany) at an acceleration voltage of 5 kV, secondary electrons detector. Magnifications are indicated at the bottom of each figure.

2.4 Preparation of Water Extract and Protein Free Extract

Fermented milk (10 g) was mixed with 2.5 mL distilled water, and the fermented milk pH was adjusted to 4 using 1 M HCl. The fermented milk was then incubated at 45 °C for 10 min, and the precipitated proteins were removed by centrifugation (10,000 rpm, 10 min). The supernatant was separated, and the pH was adjusted to 7.0 using NaOH (0.5 M). Then, the supernatant was centrifuged again (10,000 rpm, 10 min) to remove residual salts and proteins. The supernatant (water extract, WE) was stored in the refrigerator and used within 24 hours for analysis.

Then, 5 mL of a 1% trichloroacetic acid solution was mixed with 5 g of fermented milk [39]. After 5 min of incubation at room temperature, the precipitate was removed by centrifugation for 15 min at 10,000 g. The supernatant was used as a protein-free extract (PFE).

2.5 Methods of Antioxidant Activities, in Vitro Lipase and α-Glucosidase Inhibition Assay

The ferric-reducing antioxidant power (FRAP) assay was carried out following the procedure described by Nikitina et al., 2022 [38]. The water extracts were 2-fold pre-diluted for analysis; PFE was used in the initial form.

Evaluation of radical-scavenging ability (RSA) by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) assay was described early in Nikitina et al., 2022 [38]. The water extracts were 10-fold pre-diluted; PFEs were 5-fold pre-diluted for analysis. OH-free radical scavenging ability (OH-FRSA) was analyzed following the procedure described by [37]. The water extracts were 10-fold pre-diluted; PFEs were 5-fold pre-diluted for analysis. Evaluation of lipase inhibition and α-glucosidase inhibition assays were described early [36].

2.6 Statistical Analysis

All experiments were carried out in three to five replicates. Significance was established at p < 0.05. The results were analyzed for statistical significance with two-way ANOVA by GraphPad Prism software 5 (San Diego, CA, USA) at a significance level of p < 0.05. The graphical representation of the principal component analysis (PCA) allows the data to be analyzed on a two-dimensional P1/P2 map (MDSA) by Statistica12 (Statsoft) and to identify the trends between variables at a significance level p < 0.05.

3. Results and Discussion
3.1 Chemical Composition of Fermented Milks

The total titratable acidity (TTA) of fermented milk during storage is shown in Fig. 1. Lactic acid bacterial activity during low-temperature storage, and residual lactic acid production resulted in a significant increase in TTA for all samples during storage. Similar observations were reported in previous studies [40, 41]. In addition, the TTA values of fermented milk increased significantly (p > 0.05) in the presence of FSM. The highest values were found in samples where both strains (LB/AG9 and LB/AG9_FSM) were used together in the presence and absence of FSM. Previously, flaxseed powder showed a different trend: Adding linseed powder inhibits lactic acid synthesis in yogurt [31]. The authors associate this with increased protein levels due to linseed powder and increased buffer capacity. In contrast, other studies have shown a stimulation of lactic acid accumulation in the presence of FSM [26]. When FSM is used, no protein components could affect lactic acid synthesis.

Fig. 1.

Changes in titratable acidity during storage of fermented milk. TTA, total titratable acidity; FSM, flaxseed mucilage.

Regarding chemical composition, adding 0.2% FSM did not affect the total amount of protein in the fermented milk. However, it caused a redistribution of the protein components. Thus, the percentage of proteins in the whey became higher in the variants with FSM (Table 1). This may be due to FSM’s emulsifying properties and mucilage’s surfactant effect on the milk proteins [42].

Table 1.Effect of FSM and different strains of lactic acid bacteria (LAB) on the chemical parameters of fermented milk (data are expressed as mean ± standard deviation (SD)).
Sample Storage time, days Protein, % Protein in whey, % Carbohydrates, % Glucose, mmol/L Salts, %
L. bulgaricus 1 4.11 ± 0.09 2.96 ± 0.06 4.37 ± 0.22 2.1 ± 0.2 0.69 ± 0.01
7 4.01 ± 0.10 2.92 ± 0.03 4.31 ± 0.09 1.7 ± 0.3 0.68 ± 0.01
14 3.85 ± 0.07 2.78 ± 0.12 4.11 ± 0.11 1.8 ± 0.3 0.65 ± 0
L. bulgaricus + FSM 1 4.05 ± 0.02 3.20 ± 0.09 4.52 ± 0.13 2.1 ± 0.1 0.75 ± 0.03*
7 4.10 ± 0.11 3.18 ± 0.10 4.50 ± 0.12 1.9 ± 0.2 0.74 ± 0.02*
14 3.87 ± 0.06 3.12 ± 0.06 4.41 ± 0.08 1.9 ± 0.1 0.73 ± 0.01
L. plantarum AG9 1 4.08 ± 0.09 2.93 ± 0.04 4.33 ± 0.24 3.8 ± 1.0* 0.68 ± 0.01
7 4.08 ± 0.03 2.81 ± 0.09 4.15 ± 0.22 2.9 ± 0.9 0.66 ± 0.02
14 3.91 ± 0.04 2.76 ± 0.03 4.07 ± 0.11 1.9 ± 02 0.64 ± 0.1
L. plantarum AG9 + FSM 1 4.06 ± 0.04 3.04 ± 0.04 4.49 ± 0.19 1.9 ± 0.4 0.71 ± 0.02
7 4.03 ± 0.06 2.95 ± 0.01 4.35 ± 0.14 2.2 ± 0.2 0.69 ± 0.01
14 3.71 ± 0.09 2.90 ± 0.05 4.28 ± 0.12 2.1 ± 0.1 0.68 ± 0.01
L. bulgaricus + L. plantarum AG9 1 4.01 ± 0.02 3.09 ± 0.10 4.37 ± 0.11 1.3 ± 0.9 0.72 ± 0.02
7 4.03 ± 0.08 2.91 ± 0.06 4.20 ± 0.09 0.9 ± 0.3 0.68 ± 0.02
14 3.65 ± 0.09 2.84 ± 0.08 4.10 ± 0.08 0.9 ± 0.2 0.66 ± 0.02
L. bulgaricus + L. plantarum AG9+FSM 1 4.06 ± 0.08 3.18 ± 0.11 4.50 ± 0.12 1.4 ± 0.3 0.74 ± 0.01*
7 4.07 ± 0.10 3.11 ± 0.03 4.39 ± 0.09 1.2 ± 0.3 0.72 ± 0.02
14 3.84 ± 0.09 3.08 ± 0.04 4.35 ± 0.13 1.2 ± 0.4 0.72 ± 0.01

*Asterisks indicate statistically significant differences, p < 0.05. The differences between the FSM and non-FSM variants are shown for each strain at similar storage times.

The amount of exopolysaccharide (EPS) in fermented milk is highest in the AG9 strain (Fig. 2). We have previously shown a greater ability by AG9 strains to synthesize EPS compared to L. bulgaricus on MRS medium [39]. The level of EPS accumulation during storage was lower when AG9 and FSM were used together. The amount of EPS synthesized was lower in the L. bulgaricus monoculture or L. bulgaricus + AG9 co-culture, but FSM application significantly stimulated EPS synthesis during storage. The effect of FSM on EPS synthesis by different LAB species can be explained by differences in the interaction mechanisms between the polysaccharide and these cells, which we have previously identified [36], due to metabolic specificity.

Fig. 2.

Milk exopolysaccharide (EPS) fermented by different LAB strains with/without FSM (mean values ± SD, n = 3).

The total phenolic compound (TPC) contents in the aqueous extract of the samples without FSM addition increased during storage, except for the LB/AG9 sample (Fig. 3A). A similar trend was observed for the samples with FSM, except for in the AG9 sample, where the number of phenolic compounds decreased significantly by the last day of storage. It was found that when L. bulgaricus and FSM are present together, the accumulation of total TPC is more intense during storage. Certainly, FSM influences the metabolic processes of TPS synthesis and/or activates proteolysis, releasing free aromatic amino acids from whey proteins. The possibility of FSM metabolism with the release of phenolic compounds cannot be excluded. It may also result from redistributing a redistribution of protein fractions between insoluble casein milk and albumin whey proteins. These changes may be due to the surfactant and emulsifying properties of FSM, manifested by increased solubility and availability of some caseins [42, 43].

Fig. 3.

Chemical composition of water extracts and protein-free extract of milk fermented by different strains of LABs with/without FSM. (A) The concentration of total phenolic compounds (TPC) in water extract, (B) the concentration of TPC in protein-free extract (PFE) (by tyrosine), (C) PFE free peptides, reaction with O-phthaldialdehyde (OPA)-reagent, (mean values ± SD, n = 3).

The TPC in PFE did not change significantly during storage, whereas the TPC concentration was higher in variants with FSM than those without, regardless of the strains used (Fig. 3B). This result clearly shows that FSM is also the source of the phenolic compounds. The quantitative content of low molecular weight peptides in the PFE increased significantly during the two weeks of storage, particularly in samples with FSM (Fig. 3C). This indicates the activation of proteolytic processes in the presence of FSM. Furthermore, the proteolytic activity increased more with L. bulgaricus.

3.2 Textural Characteristics of Different Fermented Milks

Fig. 4A shows the change in viscosity during storage in the different treatments and the control. The sample fermented with AG9 had the lowest viscosity. Using FSM and AG9 resulted in the formation of a more viscous gel during storage. The viscosity of the LB and LB/AG9 samples was almost the same, but the addition of FSM to LB resulted in a decrease in viscosity, while the LB/AG9 variant showed practically no change in viscosity.

Fig. 4.

Viscosity (A), syneresis (B), and water-holding capacity (C) of skimmed milk fermented by different strains of LAB𝐛𝐈 with/without FSM. “a” indicate statistically significant differences, p < 0.05. The differences between the FSM and non-FSM variants are shown for each strain at similar storage times.

Syneresis of fresh fermented milk was lower in the samples with FSM addition (Fig. 4B), with the greatest decrease in the variant occurring with AG9. However, syneresis increased in all variants after 14 days of storage. Polysaccharides in flaxseed mucilage may inhibit excessive whey separation. Reducing syneresis positively affects the organoleptic and consumer properties of dairy products [44]. Neutral hydrocolloids (e.g., FSM) reduce syneresis by increasing the viscosity of the continuous phase [45].

The water-holding capacity of samples containing FSM increased during storage (Fig. 4C). This change improved the structural properties of the samples. Water holding capacity and syneresis are inversely correlated. The decrease in whey separation after the addition of FSM may be due to the absorption of water by this hydrocolloid, which may increase the strength of the hydrocolloid and potentially contribute to the strength of the interaggregate bonds in yogurt [46]. This limits the molecular mobility of caseins and prevents water loss [47]. In previous studies, a decrease in syneresis was observed when different hydrocolloids, such as guar gum [48], starch [49] including enzyme modified [50], and gelatin [51], were added to the yogurt structure. The emulsifying properties of FSM, which liquefies the consistency of the samples, may be responsible for this trend.

The textural properties of the fermented milk are shown in Table 2. L. bulgaricus fermented milk had higher adhesion firmness and elasticity values, but elasticity and stickiness values were lower than L. plantarum AG9 samples. This trend continued throughout storage. The pattern of textural properties changed with the addition of FSM. In the case of L. bulgaricus_FSM samples, the firmness, stringiness and adhesiveness decrease in freshly prepared samples; after 7 days of storage, these parameters increase and become the same as in the L. bulgaricus samples. On the contrary, the firmness, stringiness, and adhesiveness of the AG9_ FSM samples increased, although the elasticity and the stickiness decreased. In the variant with both L. bulgaricus and L. plantarum AG9 strains, the character of the change in the textural properties is similar to that of the variant with L. bulgaricus. The influence of the L. plantarum AG9 strain was observed in the FSM variant. The presence of L. plantarum AG9 in the starter stabilized the firmness, which decreased by only 0.4 g compared to the L. bulgaricus_L. plantarum AG9 sample. The firmness of the L. bulgaricus_FSM sample decreased by 2.5 g compared to the L. bulgaricus sample. Accordingly, we do not observe any significant changes in the texture of the L. bulgaricus_L. plantarum AG9_FSM variant in terms of stringiness, adhesion and elasticity. Similar data were obtained when linseed was used in yogurt at a concentration of 2.63% [52]. Yogurt firmness was increased by using cress seed mucilage at a concentration of 0.05% [53], and jujube mucilage at a concentration of 0.1–0.4% had a similar positive effect on the textural properties of yogurt [54]. All these differences in properties result from features formed in the microstructure of the acid–milk gel. Previously, we showed that fresh fermented milk had less hardness and chewiness when FSM was added, but elasticity increased in samples with FSM [55].

Table 2.Textural profile of different fermented milks (mean values ± SD, n = 3).
Sample Storage time, days Firmness, g Elasticity Adhesive-ness Cohesive-ness Gumminess Stringiness, m Chewiness
L. bulgaricus 1 48.00 ± 0.14 0.40 ± 0.02 31.26 ± 1.42 0.92 ± 0.01 44.22 ± 0.55 8.04 ± 0.37 1429 ± 17
7 47.45 ± 1.2 0.46 ± 0.04 30.62 ± 5.73 0.93 ± 0.03 43.93 ± 0.24 7.96 ± 0.77 1439 ± 16
14 47.30 ± 0.33 0.42 ± 0.05 30.68 ± 2.06 0.91 ± 0.01 42.86 ± 0.54 7.82 ± 0.13 1391 ± 8
L. bulgaricus + FSM 1 45.40 ± 0* 0.51 ± 0.01 23.65 ± 0.53 0.93 ± 0 42.26 ± 0.05* 6.65 ± 0.06* 1399 ± 46
7 48.75 ± 0.49 0.42 ± 0.04 29.47 ± 3.41 0.92 ± 0.03 44.91 ± 1.00 7.75 ± 0.77 1464 ± 52
14 47.90 ± 0.43 0.45 ± 0.02 33.20 ± 2.76* 0.91 ± 0.01 42.46 ± 0.76* 8.41 ± 0.42* 1389 ± 30
L. plantarum AG9 1 46.00 ± 0.14* 0.61 ± 0.01 22.00 ± 0.04 0.95 ± 0.01 43.32 ± 0.09 6.46 ± 0.03* 1460 ± 3
7 45.50 ± 0.71* 0.62 ± 0.04 21.52 ± 0.83 0.94 ± 0.02 42.75 ± 0.37 6.51 ± 0.28* 1448 ± 28
14 45.45 ± 0.64* 0.59 ± 0.03 25.53 ± 0.89 0.93 ± 0.01 42.28 ± 0.22* 7.20 ± 0.01* 1416 ± 6
L. plantarum AG9 + FSM 1 47.55 ± 0.78 0.48 ± 0.03 27.63 ± 0.20 0.9 ± 0.01 42.97 ± 0.01* 7.46 ± 0.06* 1407 ± 8
7 47.45 ± 0.78 0.46 ± 0.04 32.03 ± 0.49 0.91 ± 0.03 43.28 ± 0.68 8.11 ± 0.01 1413 ± 32
14 46.65 ± 0.34 0.51 ± 0.05 28.17 ± 1.30 0.91 ± 0.03 42.5 ± 0.04* 7.31 ± 0.14 1396 ± 11
L. bulgaricus + L. plantarum AG9 1 47.35 ± 0.35 0.41 ± 0.02 31.78 ± 4.69 0.91 ± 0 43.25 ± 0.39 8.19 ± 0.83 1391 ± 3
7 48.10 ± 0.71 0.40 ± 0.05 32.80 ± 1.18 0.92 ± 0.04 44.10 ± 0.21 7.95 ± 0.37 1423 ± 62
14 47.85 ± 0.64 0.42 ± 0.06 37.87 ± 7.49 0.91 ± 0.04 43.47 ± 0.46 8.23 ± 0.47 1407 ± 76
L. bulgaricus + L. plantarum AG9 + FSM 1 46.95 ± 0.21* 0.45 ± 0.02 28.41 ± 3.93 0.92 ± 0.02 43.31 ± 0.92 7.54 ± 0.63 1416 ± 60
7 47.00 ± 0.57 0.45 ± 0.04 31.11 ± 3.91 0.91 ± 0.04 42.96 ± 0.47 7.89 ± 0.57 1404 ± 26
14 47.30 ± 0.57 0.49 ± 0.02 31.38 ± 1.73 0.9 ± 0.01 42.74 ± 0.10 7.71 ± 0.57 1393 ± 22

*Asterisks indicate statistically significant differences, p < 0.05. The differences between the FSM and non-FSM variants are shown for each strain at similar storage times.

3.3 Microstructure of Fermented Milk

A dense but sparsely cohesive protein network with many cavities was observed in the control variants of fermented milk (Fig. 5A–C). In the control variants, L. bulgaricus cells (Fig. 5A) were weakly immersed in the protein matrix; the cells were tightly attached in the form of chains. L. plantarum AG9 cells (Fig. 5B) were loaded into the protein matrix; they were smaller than L. bulgaricus cells and had typical extracellular formations, allowing the cells to form conglomerates with milk proteins. When L. bulgaricus and L. plantarum AG9 were used together (Fig. 5C), both cell types were observed in the image with the characteristic differences described above.

Fig. 5.

Microstructure of fermented milk in the absence (A–C) and presence of 0.2% FSM (D–F) after 7 days storage. (A) L. bulgaricus; (B) L. plantarum AG9; (C) L. bulgaricus_L. plantarum AG9, (D) L. bulgaricus + FSM, (E) L. plantarum AG9_FSM, (F) L. bulgaricus_L. plantarum AG9_FSM. Scale bar: 200 nm.

The microscopic picture when FSM is used differs from the variants without FSM (Fig. 5D–F). Micrographs of the LB+FSM variant (Fig. 5D) show fragments of mucilage in the form of strands and flat areas covering cells and milk aggregates. FSM is visualized as broken pieces and remnants; LAB enzymes might hydrolyze the polysaccharide. L. plantarum AG9 cells adhere to the protein matrix and are covered by an encapsulating formation with no clear boundaries (Fig. 5E) and no ruptures. When L. bulgaricus is used together with L. plantarum AG9, the cells are embedded in the protein matrix, firmly adhering to each other, forming a chain and covered by an enveloping formation, which was also observed in the previous image (Fig. 5F), in addition, there are no large bonds similar to the LB + FSM variant. Certainly, the incorporation of FSM into the structure of the milk gel affects its texture. Changes in the textural properties, viscosity, and syneresis depend on the strains used in the starter. The properties of the strains influence the textural properties of the fermented milk in terms of acid production and the ability to synthesize EPS.

3.4 Antioxidant Activity of Fermented Milk

Antioxidant activity was tested in fermented milk water extract and protein-free fermented milk extract systems. The antioxidant activity of the water extracts in the FRAP test is higher in the samples with added FSM compared to the control samples (Fig. 6A). FRAP decreases slightly towards the end of its shelf life but remains high. The water extractable (WE) components of fermented milk show high RSA (DPPH test). In samples with FSM, the radical scavenging activity is higher after 14 days of storage (Fig. 6B), which seems to be due to the release of antioxidant components from the linseed gums under the LAB effects. The WE components bound OH-free radicals more actively in the L. bulgaricus starter and FSM variants after 14 days of storage (Fig. 6C). The use of strain AG9 as a mono-starter led to a higher formation of OH-FRSA in the variant without FSM at the end of storage. The combination of the two strains and FSM resulted in a stabilization of OH-FRSA throughout the storage period.

Fig. 6.

Antioxidant properties of water extracts of milk fermented by different strains of LABs with/without FSM. (A) ferric reducing antioxidant power (FRAP); (B) radical-scavenging ability (DPPH); (C) OH-free radical scavenging ability (OH-scav). “a” indicates statistically significant differences, p < 0.05. The differences between the FSM and non-FSM variants are shown for each strain at similar storage times.

FRAP of PFEs in samples with FSM was higher than control samples (Fig. 7A). The amount of low molecular weight reducing compounds in PFEs, including glucose, decreased, resulting in a decrease in FRAP. In most cases, the highest RSA was detected after 7 days of storage (Fig. 7B). As with the aqueous extract RSA above when FSM is used, the highest value was detected in the AG9 + FSM sample. The OH-FRSA of PFE was highest after 14 days of storage, except for the LB sample (Fig. 7C). The low molecular weight complex with the highest OH-FRSA is formed in the L. bulgaricus/L. plantarum AG9+FSM sample.

Fig. 7.

Antioxidant properties of protein-free extracts (PFE) of milk fermented by different LAB strains with/without FSM. (A) ferric reducing antioxidant power (FRAP); (B) radical-scavenging ability (DPPH); (C) OH-free radical scavenging ability (OH-scav). “a” indicates statistically significant differences, p < 0.05. The differences between the FSM and non-FSM variants are shown for each strain at similar storage times.

Hadinezhad et al. [10] showed the high antioxidant properties of FSM in oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and beta-carotene tests. The highest antioxidant activity was found in brown flaxseed [56]. In addition, LAB can synthesize a wide range of bioactive substances in milk that benefit human health [29]. We have previously shown that combining FSMs with dairy whey increases antioxidant activity [36]. The present studies revealed a synergistic interaction between FSM and different LAB strains in forming dairy antioxidant properties. Inhibition of pancreatic lipase PFE is higher in samples with linseed gum (Fig. 8). All variants showed a decrease in lipase inhibition during storage. After 14 days of storage, the highest inhibitory activity was retained in variants AG9 and LB/AG9 with the addition of FSM. The demonstrated ability of metabolites to inhibit lipase may be the basis for using these products to correct metabolic diseases [7].

Fig. 8.

Inhibition of panceatic lipase (A) and glucosidase (B) by the protein-free extracts (PFE) of milk fermented by different strains of LAB with/without FSM. “a” indicates statistically significant differences, p < 0.05.

In the LB_FSM sample, the PFE components are more active in inhibiting glucosidase. This trend is maintained throughout the storage period. When the AG9 starter is used for milk fermentation, the percentage of glucosidase inhibition is low throughout the storage period. The exception is the LB/AG9 + FSM variant, where an increase in inhibitory activity is observed after 14 days of storage. This may be due to the metabolism of linseed gums, specifically by L. bulgaricus. It is likely that during storage, the inhibitory effect of AG9 is reduced and L. bulgaricus starts to degrade FSM more actively, releasing phenolic compounds and low molecular weight peptides. Phenolic compounds in linseed gums can inhibit glucosidase and pancreatic lipase, which have anti-diabetic effects [6].

3.5 Principal Component Analysis

PCA revealed a high degree of difference between the complex properties of fermented milk with/without FSM when the starter was varied (Fig. 9A). The character of the change in the properties of the fermented samples during storage has a similar pattern and direction (tending from the upper region to the lower region). It can be seen that the coordinates of samples of LB:LB/AG9 and LB + FSM:LB/AG9 + FSM pairs after 14 days of storage are close to each other, which indicates the leading role of commercial starter strain of L. bulgaricus in the formation of fermented milk product properties at late storage periods. As a mono-starter, the L. plantarum AG9 strain produces different fermented milk properties from other samples. The use of FSM with AG9 leads to changes in the properties of fermented milk. MDSA showed a high degree of correlation between the antioxidant properties of RSA/OH-FRSA and peptide levels (Fig. 9B). The amount of glucose is negatively correlated with most of the parameters characterising the textural and chemical properties of the product. In fact, the lower the glucose content, the more intensively it is metabolized with the release of lactic acid. The acidification of milk leads to the formation of casein gelation and a specific texture of the dairy product.

Fig. 9.

Principal component analysis (A) and multidimensional scaling analysis (B) of milk fermented properties.

The present work revealed differences in the effect of FSM on the complex properties in fermented milk depending on the strains used (starter or probiotic). We have previously shown that flaxseed mucilage affects the cell differently depending on the LAB species (L. bulgaricus, L. plantarum AG9) co-cultured on MRS medium [30].

Currently, researchers have identified the following applications for seed mucilage: bakery products, emulsified meat products, ice cream, egg and fat replacers, gluten-free pasta, and fermented dairy products [57]. Using flaxseed gums at low concentrations (0.1–0.2%) in the low-fat yogurt increased viscosity, consistency, and water retention, decreased syneresis, and increased a* and b* values [58]. However, the authors reported adequate sensory properties at 0.15% FSM concentration, comparable to our studies. An increase in the concentration of dry FSM in yoghurts has a negative effect on sensory attributes. Another important effect of mucilage is its related to their ability to stimulate lactic acid bacteria to grow and persist. For example, using Plantago ovata forsk seed mucilage promoted the survival of Lactobacillus acidophilus in symbiotic low-fat yogurt [59]. FSM was used to make functional fat-free cream cheese. The mucilage improved the texture, enhanced the survival of the probiotic bacteria, and improved the overall sensory characteristics of the cheese [60]. Our study shows stimulation of lactic acid accumulation in variants with FSM, which indicates the stimulation of lactic acid bacteria proliferation and metabolism in fermented milk. The effect of increasing the antioxidant properties of yogurt in the presence of FSM, as shown by us, was also noted by other authors. The present work results parallel Azarpazhooh et al. 2021 [61], who showed that flaxseed-mucilage powder increased the DPPH values in a coconut milk-based beverage. As in our studies, several studies show a similar change in the microstructure of milk products. For example, low-fat cream cheese containing flaxseed and Lepidium perfoliatum seed gums forms a porous matrix of cream cheese [62, 63]. Such changes are due to the breakage of casein bonds and the dispersion of gums deep into the milk matrix. Therefore, using flax seed mucilage at a concentration of 0.2% in the formulation of non-fat fermented milk improved physicochemical, textural, and antioxidant parameters and stimulated the metabolism of LAB.

4. Conclusions

The results of this work contribute to the study of the effect of a plant polysaccharide, flaxseed mucilage, on the chemical, textural, and antioxidant properties of dairy products by varying lactic acid bacteria. It should be noted that the use of L. plantarum AG9 and FSM makes it possible to obtain fermented milk characterized by the highest content of polyphenolic compounds, with the highest antioxidant properties and leading to the synthesis of lipase and α-glucosidase inhibitors. However, such a product is inferior to the commercial L. bulgaricus variant in terms of textural properties. A starter with a combination of L. bulgaricus and L. plantarum AG9 (20% of the total weight) can be used optimally to develop health properties combined with high textural qualities. Adding 0.2% linseed mucilage to fermented milk stimulated the accumulation of lactic acid. In addition, the physico-chemical properties (viscosity, syneresis, WHC, texture) and antioxidant properties of the fermented milk were improved. In the future, studies using in vivo models will be carried out to investigate the functional properties of dairy products with FSM.

Availability of Data and Materials

There is no supplementary data in the article, all data is described in the article

Author Contributions

EN – Conceptualization and design, Project administration, Writing – original draft, Writing – review & editing. TP – Formal Analysis, Software, Writing – original draft. AS – Resources, Visualization, Writing – review & editing. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Ethics Approval and Consent to Participate

Not applicable.

Acknowledgment

The team of authors would like to express gratitude to Mikshina P.V. (Kazan Institute of Biochemistry and Biophysics of the Kazan Scientific Center of the Russian Academy of Sciences, Kazan, Russian Federation) for the provided flaxseed mucilage for experimental studies.

Funding

The study was supported financially by the Russian Science Foundation and the Academy of Sciences of the Republic of Tatarstan under scientific project No. 22-26-20022 “Mechanisms of interaction between lactic acid bacteria and plant mucus enriched with polysaccharides as a basis for the creation of new functional foods”.

Conflict of Interest

The authors declare no conflict of interest.

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