Study of Probiotics as Biostimulants and Biofortifiers in Seed Germination

Abstract

Germination is a bioprocessing technique used by the food industry to improve the nutrient content of seeds. Imbibition is considered a crucial stage for optimal plant development. In this research, four different groups of seeds were studied to evaluate the effect of using probiotics during the imbibition phase on the germination process, as well as on the microbiological quality, as specified by Mexican regulations. The efficiency of probiotic production was also determined. Regarding the results, most seeds achieved a germination rate between 90–100%, with seeds inoculated with probiotics reaching a higher rate. This increase was related to the concentration of auxins. Importantly, the lowest levels of CFU/g counts for coliforms and fungi were found in the treated sprouts. Additionally, the sprouts exhibited values greater than 1 × 10⁶ CFU/g of probiotics. The imbibition of certain types of seeds with probiotics with Lactiplantibacillus plantarum and Saccharomyces boulardii increases the germination speed, as well as the biomass obtained, which suggests that it may be an alternative to treatments in the agricultural industry for greater production.

1. Introduction

Germination is a bioprocessing technique used by the food industry to enhance the nutrient content of seeds and grains such as cereals, oilseeds, legumes, and vegetable seeds, in addition to being considered an economical tool accessible to everyone [1,2]. The improved nutrient utilization in sprouts is due to the action of enzymes that convert macronutrients into smaller molecules [3,4,5]. Nowadays, the consumption of sprouts has increased due to consumer concerns about improving their health, with sprouts being considered a functional food. The sprout market has begun to promote the consumption of these sprouts worldwide, and it is estimated that by 2025, the market could reach a value of 1.9 billion dollars.

Within the germination cycle, several factors determine seedling growth, nutrient content, and active compounds. One of the crucial stages in the germination process is imbibition, as humidity, the presence of light, and the substance in which the seed or grain is soaked ensure the efficiency and quality of this process [6].

Additionally, biostimulants are substances that enhance plant growth and development, increase tolerance to stress conditions, reduce pathogens, and improve nutrient availability in the soil. The use of biostimulants in agriculture is becoming increasingly common, reducing the reliance on chemical substances [7,8].

Each mechanism of action of biostimulants depends on their components, which is why they have not yet been identified. One hypothesis suggesting the benefit of using biostimulants is that they stimulate the plant’s metabolism, thereby increasing its stress response and improving conditions for development [9]. Research has documented that using microorganisms can be an effective strategy to enhance crop growth without relying on chemical substances that can harm the soil and consumer health [10]. During the germination process, probiotics take advantage of the nutrients contained in the seed, making minerals from the seed shell such as Zn, Mg, Ca, and Na more available, which allows a better use for the development of the plant, creating a symbiotic relationship. Currently, efforts are being made to incorporate them into various food matrices and other products of interest in the pharmaceutical and agricultural industries [11]. According to the literature, soaking seeds and grains in probiotic-inoculated solutions improves their properties, ensuring better microbiological quality for the consumer in addition to health benefits [12]. During the imbibition stage, probiotic microorganisms are absorbed by the seeds, gaining greater access to the nutrients necessary for their development. Factors such as germination temperature, inoculation method, and seed type directly influence the production efficiency of probiotic-rich sprouts [11,13]. Studies conducted by Świeca in 2019 showed that sprouts are an excellent source of probiotics and can be used in the formulation of functional foods. In addition to enhancing the phenolic content of the sprouts, probiotics increase their microbiological quality by replacing the natural microbiome with lactic acid bacteria [14].

The objective of this study was to investigate the effect of probiotics during the imbibition stage as a biostimulant and their probiotic impact on sprouts for incorporation into functional foods.

2. Materials and Methods

2.1. Effect of Biostimulation with Probiotics on the Microbiological Quality and Growth of Sprouts

2.1.1. Germination Conditions

Nine different seeds were germinated: legumes (brown lentils and mung beans), oilseeds (sesame and linseed), vegetables (beet and radish), and cereals (millet, triticale, and rye grains). Each seed type was subjected to three different treatments during the soaking stage. The seeds were treated with a 2% (m/v) sodium hypochlorite solution for 10 min and then washed with distilled water until reaching a pH of 7. Subsequently, the seeds were soaked for 5 h with the following treatments: distilled water (control), a suspension of L. plantarum 14917 (treatment 1), a suspension of S. boulardii CNCM I-745 (treatment 2), and a mixture of both (treatment 3). The suspension was inoculated at a concentration of 1 × 10⁸ CFU per gram of seed at a temperature of 25–28 °C. In different bibliographic sources, we find that the adequate concentration of probiotics in an inoculated solution is 1 × 10⁸ CFU/g, in order to guarantee the survival of the microorganism in the sprout [12,15] (

Table 1. Design of the germination process in its different stages.

Seed		Soaking Treatment				Germination
Cereals	Rye Triticale Millet	Water	LP	SB	LP-SB	Time: 5 days Temperature: 25 °C RH: 90% Sprinkled: Daily Spray type: Distilled water
Legume	Pardina lentil Mung bean
Oil seeds	Flax seed Sesame
Vegetable seeds	Beetroot Radish

LP: Lactiplantibacillus plantarum 14917; LP-SB: mixture of Lactiplantibacillus plantarum 14917 and Saccharomyces boulardii CNXM I-745; and SB: Saccharomyces boulardii CNCM I-745.

). The volume of liquid inoculated with probiotics used for each treatment was the same. After the soaking stage, the seeds were drained and germinated in trays under controlled conditions inside a bioclimatic chamber at 25 °C with 90% relative humidity for 5 days. The seeds were kept hydrated throughout the process and sprayed once daily with 10 mL of distilled water, without light. The strains used grow at temperatures of 28 °C and are considered facultative anaerobes. The origin of the strains is from their isolation from a sample of goat milk for L. plantarum. While S. boulardii is a commercial strain.

2.1.2. Determination of Seed Germination

The parameters of plant height and stem diameter, root/stem index, germination percentage, and biomass produced were determined. The seed was considered germinated when it reached a length of 0.2 cm [16]. The parameters were measured with a vernier caliper, taking the lower edge of the seed and the downward growth from day 2–3 of germination (radicle) as a base point for the measurement of the root, while for the stem, the upper edge of the seed and the hypocotyl were taken as a base point.

2.2. Microbiological Quality of Sprouts

The concentration of total coliforms [17], aerobic mesophiles [18], and fungi and yeasts [19,20] was determined. An amount of 10 g of fresh sample was weighed in 99 mL of PBS, making serial solutions. The media used were MRS (Man, Rogosa, Sharpe) for L. plantarum and LW (Lowenstain Jensen) for S. boulardii, with an incubation time of 5 h at a temperature of 25 °C.

2.3. Determination of Auxins

2.3.1. Extraction

For extraction, 100 mg of germinates was pulverized with liquid nitrogen and weighted into 2 mL tubes, and 2 mL of a methanol/water/formic acid (15/4/1 vol) solution was used as an extractant. The mixture was vortexed for 20 s and sonicated for 10 min. The samples were then centrifugated at 12,000 rpm for 10 min at 4 °C. The supernatant obtained was filtered with membranes of 0.45 µm.

Standards for auxin determination (3 [5(n)-3 H] indolyl acetic acid, Amersham, UK) were used [21].

2.3.2. Quantification

Auxins were determined using an Agilent 1120 compact LC (Agilent Technologies, Santa Clara, CA, USA) under the following conditions: column Luna C18 (2), 150 mm × 4.6 mm, 3 µm, Phenomenex, Torrance, CA, USA), injection volume 20 µL, mobile phase (a) 40 mM HCOOH pH 3 and (b) acetonitrile/methanol 1:1, and a flow rate of 0.60 mL/min [22].

2.4. Evaluation of the Efficiency of Probiotic Production

2.4.1. Lactiplantibacillus Plantarum Count

The viability of L. plantarum was determined by plate counting (PN-ISO 15214:2002 [23]), using dilutions (1 × 10⁻¹ to 1 × 10⁻⁶) subsequently seeded on plates with MRS agar [24]. One gram of fresh sprouts was homogenized with 9 mL of PBS. Serial decimal dilutions of the sprout samples (1 × 10⁻¹ to 1 × 10⁻⁶) were then prepared, and 0.1 mL aliquots were placed on MRS agar in triplicate. The plates were incubated at 37 °C for 48 h. Colonies were counted and expressed as CFU/g of fresh weight of sprouts [11]. The efficiency factor was calculated as follows:

Efficiency factor = (Seed germination percentage/Mass of 10 sprouts) × Amount of probiotics.

2.4.2. Saccharomyces Boulardii Count

The number of yeasts and molds was determined using the plate technique on glucose yeast extract agar, according to PN-ISO 21527-1:2009 [25]. Colonies were counted and expressed as CFU/g of fresh weight [12].

2.5. Statistical Analysis

The experiments were established under a completely randomized experimental design. The results obtained were analyzed by applying an ANOVA analysis, and if there was a significant difference, the Tukey test was performed to compare means. Statistical analyses were performed using InfoStat software (version 2018).

3. Results

3.1. Seed Germination Parameters

Regarding the parameters evaluated to determine the efficiency of the germination process, the germination percentage in most of the seeds reached values of 90–100% by day 5, except for beet seeds (

Table 2. Percentage of germination in the different seed treatments.

	Seeds	Treatments	% Germination	Germination Speed
Oilseeds	Flax a	Control	98.5 ± ${1.5}^{ a}$	91.96 ± ${0.73}^{ a}$
		LP	92.0 ± ${2.0}^{ a}$	82.06 ± ${2.31}^{ b}$
		SB	100 ± ${0.0}^{ a}$	95.83 ± ${0.31}^{ a}$
		LP-SB	99.5 ± ${0.5}^{ a}$	92.15± ${0.60}^{ a}$
	Sesame b	Control	98.0 ± ${1.0}^{ a}$	83.45 ± ${0.50}^{ c}$
		LP	95.0 ± ${1.0}^{ a}$	87.06 ± ${0.66}^{ b}$
		SB	95.5 ± ${1.5}^{ a}$	91.00 ± ${0.06}^{ a}$
		LP-SB	96.0 ± ${1.0}^{ a}$	88.99 ± ${0.45}^{ \mathit{bc}}$
Legumes	Lentil a	Control	99.5 ± ${0.5}^{ a}$	96.52 ± ${0.55}^{ a}$
		LP	99.0 ± ${1.0}^{ a}$	93.66 ± ${0.80}^{ a}$
		SB	97.5 ± ${0.5}^{ a}$	93.11 ± ${2.60}^{ a}$
		LP-SB	100 ± ${0.0}^{ a}$	93.89 ± 0.${14}^{ a}$
	Mung bean a	Control	100 ± ${0.0}^{ a}$	95.72 ± ${3.75}^{ a}$
		LP	100 ± ${0.0}^{ a}$	93.03 ± ${0.60}^{ a}$
		SB	99 ± ${0.0}^{ a}$	97.35 ± ${0.65}^{ a}$
		LP-SB	100 ± ${0.0}^{ a}$	97.45 ± ${0.12}^{ a}$
Vegetables	Radish a	Control	99.5 ± ${0.5}^{ a}$	93.15 ± ${0.50}^{ \mathit{ab}}$
		LP	99.0 ± ${1.0}^{ a}$	95.68 ± ${0.35}^{ a}$
		SB	99.5 ± ${0.5}^{ a}$	95.05 ± ${0.02}^{ \mathit{ab}}$
		LP-SB	99.5 ± ${0.5}^{ a}$	92.75 ± ${0.91}^{ b}$
	Beetroot b	Control	12 ± ${1.0}^{ a}$	33.17 ± ${0.21}^{ c}$
		LP	16 ± ${1.0}^{ a}$	60.50 ± ${0.06}^{ a}$
		SB	16 ± ${2.0}^{ a}$	48.75 ± ${0.20}^{ b}$
		LP-SB	16 ± ${1.0}^{ a}$	52.83 ± ${0.01}^{ \mathit{bc}}$
Cereals	Rye a	Control	99.5 ± ${0.5}^{ a}$	96.54 ± ${0.04}^{ a}$
		LP	100 ± ${1.0}^{ a}$	95.60 ± ${1.06}^{ a}$
		SB	100 ± ${0.0}^{ a}$	93.63 ± ${1.15}^{ a}$
		LP-SB	99.0 ± ${1.0}^{ a}$	94.46 ± ${1.15}^{ a}$
	Triticale a	Control	100 ± ${0.0}^{ a}$	94.46 ± ${0.50}^{ a}$
		LP	100 ± ${0.0}^{ a}$	95.16 ± ${0.16}^{ a}$
		SB	99.5 ± ${0.0}^{ a}$	93.65 ± ${0.62}^{ a}$
		LP-SB	99.5 ± ${0.5}^{ a}$	93.30 ± ${0.64}^{ a}$
	Millet a	Control	95.5 ± ${1.5}^{ a}$	62.59 ± ${1.65}^{ c}$
		LP	98.5 ± ${1.5}^{ a}$	85.33 ± ${1.06}^{ b}$
		SB	100 ± ${0.0}^{ a}$	90.84 ± ${0.02}^{ a}$
		LP-SB	98.0 ± ${1.0}^{ a}$	85.14 ± ${0.42}^{ b}$

LP: Lactiplantibacillus plantarum 14917; LP-SB: mixture of Lactiplantibacillus plantarum 14917 and Saccharomyces boulardii CNCM I-745; SB: Saccharomyces boulardii CNCM I-745. The analysis used was an ANOVA together with a Tukey test to determine significant differences. In the first column, different letters refer to a significant difference when comparing the different seed groups with each other. In the subsequent columns, different letters imply a significant difference between the different treatments specifically in that seed group.

). The beet sprouts showed percentages of 12–16%, which can be attributed to the prior handling the seeds undergo, such as exposure to high temperatures and chemicals, which compromise optimal plant development. Factors like good production practices can also influence the food safety of the seeds [26]. Additionally, factors such as handling by food workers, transportation, and storage are crucial for the development of pathogenic microorganisms that can affect germination [27]. Another reason for the inadequate development of the beet seeds is that the appropriate germination time for beets is around 7–12 days [28].

On the other hand, the legume seeds group achieved an ideal growth rate in a shorter period than the other seeds analyzed, with a 100% germination rate in 4 days, and are considered by several authors as seeds with a high germination rate. Similarly, cereals showed comparable behavior, likely due to their nutritional composition, which supports seedling development [29]. The vegetable seeds exhibited germination rates of 90% within 4 days, which aligns with the values found in this study [30]. In the case of oilseeds, germination rates ranged from 80% to 95%, depending on the treatment they were subjected to. These values are significant because the literature mentions that, due to the high lipid content of these seeds, the germination process can take longer, with values of 80% typically achieved after 10 days [31].

However, several authors have noted that germination efficiency is affected by factors such as soaking time, the type of solution used for soaking, humidity levels, light exposure, temperature, and water quality during germination, as well as the handling of seeds during shipping and storage [31,32]. These factors directly influenced our results. It should be noted that temperature is one of the most important conditions for germination (with optimal values between 30 and 34 °C), and the temperature range used in this study was favorable [26,33].

When analyzing the results obtained from the different groups of seeds according to the treatment they received, better results in stem height were observed in most seeds treated with the mixture of both probiotics. This suggests that probiotic microorganisms can be used as biostimulants in various crops [34]. Additionally, the seeds soaked with the combination of probiotics showed an increased germination speed, particularly in the oilseed and legume groups. These results can be attributed to the imbibition with the different inocula [7,35].

Phosphorus is also a critical nutrient in plant metabolism. The most relevant microorganisms involved in phosphorus fixation include Pseudomonas, Bacillus, Flavobacterium, Paenibacillus, Deftia, Azotobacter, and Klebsiella. Similarly, the synthesis of phytohormones is a key property of certain microbiomes due to the production of auxins, gibberellins, and cytokinins. Probiotic microorganisms have also been highlighted as biocontrol agents, as they have the ability to reduce the presence of pathogens in their environment through competition, forming a symbiotic relationship with the host [36,37].

Root/Stem Ratio, Stem Diameter, Germination Speed and Biomass

The root/stem ratio showed higher values in triticale, rye, and bean sprouts treated with S. boulardii, indicating greater biomass production due to the larger stem diameter. Treatment with L. plantarum increased shoot biomass production. Probiotic microorganisms promote plant growth through a symbiotic relationship, offering an alternative to chemical substances [37]. In the treatments where co-culture was used, optimal growth of both microorganisms was observed (regardless of the type of sprouts), indicating the effective mobilization of stored starch, as confirmed by high sprouting efficiency. S. boulardii can utilize starch as a substrate source; however, it prefers glucose and other monosaccharides for more efficient growth [38].

The use of a co-culture presents an innovative strategy to examine the interaction between the two strains and determine whether they could act synergistically or if metabolic competition occurs between them. Our results showed that co-culturing offers improved properties in terms of germination, microbiology, and nutrition.

Table 3. Germination parameters in the different seed treatments.

	Seeds	Treatment	Root/Shoot Ratio	Diameter (mm)
Oilseeds	Flax	Control	1.26 ± ${0.09}^{ a}$	1.17 ± ${0.04}^{ c}$
		LP	0.98 ± ${0.18}^{ a}$	0.82 ± ${0.04}^{ b}$
		SB	1.02 ± ${0.20}^{ a}$	0.65 ± ${0.03}^{ a}$
		LP-SB	0.97 ± ${0.11}^{ a}$	0.80 ± ${0.07}^{ b}$
	Sesame	Control	1.82 ± ${0.26}^{ b}$	0.80 ± ${0.02}^{ \mathit{ab}}$
		LP	0.74 ± ${0.07}^{ a}$	0.69 ± ${0.06}^{ \mathit{ab}}$
		SB	0.94 ± ${0.14}^{ a}$	0.83 ± ${0.09}^{ b}$
		LP-SB	0.98 ± ${0.03}^{ a}$	0.67 ± ${0.06}^{ a}$
Legumes	Lentil	Control	1.25 ± ${0.19}^{ a}$	2.75 ± ${0.18}^{ b}$
		LP	1.24 ± ${0.25}^{ a}$	2.19 ± ${0.19}^{ a}$
		SB	1.68 ± ${0.17}^{ b}$	1.98 ± ${0.14}^{ a}$
		LP-SB	1.60 ± ${0.13}^{ \mathit{ab}}$	1.88 ± ${0.22}^{ a}$
	Mung bean	Control	0.50 ± ${0.04}^{ a}$	0.79 ± ${0.03}^{ a}$
		LP	0.96 ± ${0.16}^{ b}$	1.23 ± ${0.09}^{ c}$
		SB	0.71 ± ${0.13}^{ \mathit{ab}}$	1.07 ± ${0.08}^{ b}$
		LP-SB	0.78 ± ${0.14}^{ \mathit{ab}}$	0.98 ± ${0.10}^{ b}$
Vegetables	Radish	Control	1.93 ± ${0.24}^{ b}$	1.06 ± ${0.03}^{ \mathit{ab}}$
		LP	2.29 ± ${0.28}^{ b}$	0.87 ± ${0.05}^{ a}$
		SB	0.39 ± ${0.11}^{ a}$	1.27 ± ${0.33}^{ b}$
		LP-SB	0.74 ± ${0.05}^{ a}$	0.99 ± ${0.08}^{ \mathit{ab}}$
	Beetroot	Control	0.50 ± ${0.14}^{ a}$	0.41 ± ${0.33}^{ a}$
		LP	0.43 ± ${0.03}^{ a}$	0.57 ± ${0.28}^{ a}$
		SB	0.28 ± ${0.01}^{ a}$	0.28 ± ${0.35}^{ a}$
		LP-SB	0.85 ± ${0.07}^{ a}$	0.49 ± ${0.25}^{ a}$
Cereals	Rye	Control	1.70 ± ${0.22}^{ a}$	1.18 ± ${0.06}^{ a}$
		LP	2.20 ± ${0.21}^{ a}$	0.82 ± ${0.07}^{ a}$
		SB	1.96 ± ${0.38}^{ a}$	0.89 ± ${0.01}^{ a}$
		LP-SB	3.34 ± ${0.47}^{ b}$	0.81 ± ${0.04}^{ b}$
	Triticale	Control	2.22 ± ${0.27}^{ a}$	1.12 ± ${0.08}^{ b}$
		LP	2.23 ± ${0.14}^{ a}$	0.94 ± ${0.14}^{ \mathit{ab}}$
		SB	3.27 ± ${0.82}^{ a}$	0.95 ± ${0.06}^{ \mathit{ab}}$
		LP-SB	2.58 ± ${0.51}^{ a}$	0.84 ± ${0.03}^{ a}$
	Millet	Control	1.89 ± ${0.28}^{ b}$	0.81 ± ${0.02}^{ d}$
		LP	0.95 ± ${0.04}^{ a}$	0.64 ± ${0.05}^{ c}$
		SB	1.03 ± ${0.23}^{ a}$	0.56 ± ${0.03}^{ b}$
		LP-SB	1.08 ± ${0.12}^{ a}$	0.39 ± ${0.01}^{ a}$

LP: Lactiplantibacillus plantarum 14917; LP-SB: mixture of Lactiplantibacillus plantarum 14917 and Saccharomyces boulardii CNCM I-745; SB: Saccharomyces boulardii CNCM I-745. The analysis used was an ANOVA together with a Tukey test to determine significant differences. In the first column, different letters refer to a significant difference when comparing the different seed groups with each other. In the subsequent columns, different letters imply a significant difference between the different treatments specifically in that seed group.

analyzes the relationship between the root and stem of the seeds, stem diameter, germination speed, and biomass produced. Substances classified as biostimulants include humic substances, hydrolyzed proteins, nitrogenous compounds, algae and plant extracts, microorganisms, and inorganic compounds. This suggests that probiotics, based on the proposed definition, could be classified as biostimulants, as they enhance plant growth and development, leading to accelerated growth compared to the use of other substances [39].

3.2. Microbiological Quality of Sprouts

It is currently known that the consumption of sprouts can pose a health risk, as many outbreaks associated with the consumption of this type of food have been identified.

The microbiological quality of the sprouts improved in those treated with probiotic microorganisms. Figure 1 and Figure 2 show total coliforms present in sprouts; as can be seen, lentil sprouts showed the lowest amount of CFU/g of total coliforms and aerobic mesophiles (Figure 3), while triticale and rye sprouts had the highest content of mesophiles. Regarding fungi and yeast, triticale had lowest counts for fungi and yeasts (Figure 4). Abadias et al. (2018) [40] reported that in more than 40% of sprouted foods, mesophilic bacteria counts exceeded 8 log CFU/g. Sprouts enriched with yeast and probiotic bacteria were characterized by significantly higher yeast counts; however, the fungal population was significantly reduced. The total fungal count may have been influenced by water activity values. The concentration of aerobic mesophiles can be attributed to factors such as the origin of the seed, contaminated soils, irrigation water, cross-contamination with other batches, inadequate handling and storage, and the proliferation of microorganisms on the grain’s surface due to the conditions of the germination process, such as soaking (Figure 3 and Figure 5) [41,42]. It can be assumed that ecological competition plays a key role in reducing mold and coliform populations. Antagonistic effects of probiotic microorganisms against foodborne pathogens have been previously reported in sprouts [43], dairy products [44], and fruits and fresh vegetables [45]. It is important to note that good sanitation practices are essential for reducing pathogenic microorganisms, with the use of chlorinated water, disinfectants, radiation, heat, and high pressure being recommended [28].

3.3. Determination of Auxins

Table 4. Concentration of auxins present in the different seed treatments.

	Seeds	Treatments	Auxins (mg/kg)
Oilseeds	Flax b	Control a	53.27 ± ${9.17}^{ a}$
		LP b	6.26 ± ${1.45}^{ b}$
		SB b	0.22 ± ${0.00}^{ b}$
		LP-SB b	0.38 ± ${0.07}^{ b}$
	Sesame b	Control a	0.19 ± ${0.18}^{ b}$
		LP	0.23 ± ${0.21}^{ b}$
		SB	3.28 ± ${0.09}^{ a}$
		LP-SB	3.11 ± ${0.25}^{ a}$
Legumes	Lentil b	Control	1.81 ± ${0.91}^{ b}$
		LP	0.16 ± ${0.02}^{ b}$
		SB	0.13 ± ${0.04}^{ b}$
		LP-SB	29.17 ± ${14.28}^{ a}$
Vegetables	Radish b	Control	2.47 ± ${0.59}^{ a}$
		LP	3.76 ± ${1.17}^{ a}$
		SB	0.84 ± ${0.84}^{ a}$
		LP-SB	1.37 ± ${0.45}^{ a}$
	Beetroot a	Control	1840.10 ± ${260.55}^{ a}$
		LP	1.51 ± ${1.40}^{ b}$
		SB	0.00 ± ${0.00}^{ b}$
		LP-SB	2.66 ± ${0.45}^{ b}$
Cereals	Rye b	Control	5.03 ± ${2.57}^{ a}$
		LP	0.00 ± ${0.00}^{ b}$
		SB	0.19 ± ${0.20}^{ b}$
		LP-SB	0.65 ± ${0.08}^{ b}$
	Triticale b	Control	52.96 ± ${71.76}^{ a}$
		LP	1.77 ± ${0.65}^{ a}$
		SB	0.18 ± ${0.08}^{ a}$
		LP-SB	0.00 ± ${0.00}^{ a}$

Where the letter in the seed’s column refers to the significant difference from a means test between the seeds. In the treatments column, the letter refers to the significant difference between the treatments in general. The analysis used was an ANOVA together with a Tukey test to determine significant differences. In the first column, different letters refer to a significant difference when comparing the different seed groups with each other. In the subsequent columns, different letters imply a significant difference between the different treatments specifically in that seed group.

shows the auxin concentrations found in different germinations (control and treatment) [46,47]. The results revealed significant differences between the control sample and various treatments. The treatment containing both probiotic microorganisms exhibited the highest auxin concentration, followed by the treatment with L. plantarum. Among the different seeds used, beetroot showed the highest auxin concentration, followed by linseed, lentil, and radish. These findings indicate that the number of auxins present in sprouts depends on the seed type and the treatment it undergoes.

The literature has identified that the development of storage roots, such as in beetroot, involves the action of auxins [48]. Auxins influence the biosynthesis of various compounds like starch, storage proteins, and an increase in cell growth and size for starch storage [49]. Furthermore, auxin content in developing underground storage roots increases during the early stages of root growth [50,51]. In crops like carrot, radish, and sugar beet, high auxin concentrations have been observed in stolons or storage roots [52,53,54]. However, auxin concentration is affected during the later stages of development, suggesting that high auxin levels are crucial during the beginning of storage root formation, while low auxin concentration is necessary for an increase in the volume or size of storage organs [55].

Recent studies have shown that auxin concentrations in radish did not differ between shoots in the first 15 days of germination. However, a decrease was observed at 21 days of sowing, indicating that in the final stages of plant development, auxin levels decrease to promote an increase in the size of the hypocotyl. Reduced levels of auxins are found in shoots and leaves [52,56,57]. In our study, beetroot reported higher auxin concentrations compared to other seed groups, and the control treatment of beetroot had the highest auxin percentages.

By linking auxin concentration to the germination parameters, we can correlate the height of the stem and root, as well as germination percentage, to the auxin concentration in specific shoots of beetroot, flax, radish, and lentil. In the case of beetroot, the auxin concentration was characterized by an increase in root diameter and an increase in the volume of the hypocotyl and root system, primarily due to an increase in starch [5,58,59]. Conversely, flaxseed sprouts exhibited a behavior opposite to that of beetroot sprouts, as the auxin content allowed for an increase in stem and leaf length, as shown in Table 3. It is worth noting that within the germination speed, the time established in the literature for each seed differs. In this study, a base time of 5 days was established for all seeds. Therefore, beetroot, being a hard seed requiring more time to germinate, may have had its stem and root length affected by this time constraint [60].

Regarding seed imbibition with probiotics, we can determine that auxin concentration was mediated by the action of microorganisms. The IAA molecule affects the physiological processes of bacteria and fungi, facilitating plant growth and leading to a response to environmental stress by stimulating root growth and carbohydrate filtration. Some bacteria and fungi can also degrade IAA. We hypothesize that auxin synthesis in sprouts may change in response to the interaction with probiotic microorganisms, primarily bacteria [61,62,63].

Concerning the conditions under which the seeds were germinated, it has been documented that local auxin production is mediated by the presence or absence of light. Auxin levels are stimulated in response to shade, being transported to the lower part of the plant and affecting hypocotyl growth [64,65,66]. The metabolism of phytohormones present in the roots and hypocotyl is associated with the growth-promoting effect on the cotyledons. One of the metabolic pathways for IAA synthesis is through tryptophan, which explains why flaxseed germinations, being a significant source of tryptophan from plant sources, reached the reported auxin levels [67,68]. The lowest levels were found in cereal sprouts. Studies have shown that the amount and type of carbohydrates contained in the seed are determining factors during the auxin synthesis process in the early stages of cultivation, with higher auxin concentrations found in germinations where simple carbohydrates predominate. The greater or lesser amount of auxin depends greatly on the seed used, since the results are obtained from vegetable seeds imbibed with probiotics. The probiotic intervenes in the different biochemical processes for obtaining energy, specifically starch, therefore there is a greater release of minerals such as Ca, Na, K, Zn, and Mg, which allows the increase in the synthesis of auxins, depending on the composition of the seed used, mainly in seeds with a high tannin content. One of the mechanisms that establish a substance as a biostimulant is improved nutrient utilization, such as nitrogen fixation, better solubilization of phosphates, and hormone synthesis, as well as the increased response of plants to pathogens [7]. However, nitrogen, in its available form, cannot be used by biological organisms, which justifies the need for nitrogen fixation before it can be assimilated. Microbial metabolism contributes to an increase in nitrogen fixation. The genera of microorganisms capable of fixing nitrogen include Azospirillum, Azotobacter, Bacillus, and Nitrobacter [35].

3.4. Viability of Probiotics

Probiotic Production Efficiency Factor

Table 5. Probiotic production efficiency factor in the different seed treatments.

	Seeds	Treatments	Efficiency Factor Production of Probiotics
			Lactiplantibacillus plantarum	Saccharomyces boulardii
Oilseeds	Flax	Control	1.82 × 109 ± 4.56 × 108 a	1.03 × 1010 ± 4.79 × 109 a
		LP	1.12 × 1011 ± 6.22 × 109 c	7.69 × 1011 ± 3.83 × 1011 a
		SB	6.52 × 1010 ± 5.92 × 109 b	1.38 × 1011 1.17 × 1010 a
		LP-SB	6.02 × 1010 ± 3.84 × 109 b	2.15 × 1011 ± 4.35 × 109 a
	Sesame	Control	7.78 × 1010 ± 3.33 × 1010 a	1.01 × 1012 ± 5.56 × 1010 c
		LP	1.60 × 1010 ± 6.39 × 108 a	6.39 × 1011 ± 6.39 × 1010 b
		SB	1.42 × 1011 ± 4.75 × 1010 a	4.32 × 1011 ± 3.47 × 1010 ab
		LP-SB	3.07 × 1010 ± 1.02 × 1010 a	2.63 × 1011 ± 2.59 × 1010 a
		Control	6.98 × 1011 ± 2.33 × 1011 a	2.14 × 1012 ± 7.45 × 1010 a
	Lentil	LP	3.33 × 1010 ± 7.40 × 108 a	1.94 × 1011 ± 2.14 × 1010 a
		SB	4.55 × 1010 ± 4.14 × 109 a	7.94 × 109 ± 1.32 × 109 b
Legumes		LP-SB	2.32 × 1011 ± 4.83 × 1010 a	9.06 × 1011 ± 4.59 × 1010 c
		Control	2.57 × 109 ± 5.14 × 108 a	5.65 × 109 ± 5.14 × 107 a
	Mung Bean	LP	4.80 × 109 ± 4.00 × 108 b	1.21 × 1011 ± 9.01 × 109 ab
		SB	3.93 × 109 ± 9.14 × 107 ab	2.05 × 1011 ± 7.31 × 109 ab
		LP-SB	9.57 × 109 ± 0.00 × 100 c	3.09 × 1011 ± 7.18 × 1010 c
		Control	9.98 × 1011 ± 2.22 × 1011 b	4.17 × 1011 ±7.76 × 109 a
	Radish	LP	4.44 × 1010 ± 5.92 × 109 a	9.28 × 1011 ± 1.85 × 1010 a
		SB	3.19 × 1010 ± 5.24 × 108 a	3.01 × 1011 ± 7.85 × 109 a
Vegetables		LP-SB	9.00 × 1010 ± 1.42 × 1010 a	7.34 × 1011 ± 1.94 × 1011 a
		Control	1.88 × 1010 ± 1.05 × 109 a	1.64 × 1010 ± 1.05 × 108 a
	Beetroot	LP	5.41 × 1010 ± 5.05 × 109 a	5.22 × 1011 ± 1.23 × 1010 b
		SB	6.34 × 1010 ± 3.17 × 1010 a	1.84 × 1010 ± 1.59 × 108 a
		LP-SB	9.97 × 1010 ± 1.11 × 1010 a	1.35 × 1012 ± 8.46 × 1010 c
		Control	7.18 × 1011 ± 3.59 × 1011 a	2.09 × 1012 ± 4.56 × 1011 b
	Rye	LP	1.12 × 1011 ± 5.09 × 1010 a	1.04 × 1012 ± 1.54 × 1011 ab
Cereals		SB	2.56 × 109 ± 1.22 × 108 a	9.51 × 1011 ± 8.91 × 1010 ab
		LP-SB	7.68 × 109 ± 7.20 × 108 a	4.60 × 1010 ± 1.32 × 109 a
		Control	2.13 × 1011 ± 7.08 × 1010 b	1.52 × 1010 ± 7.08 × 107 a
	Triticale	LP	9.10 × 109 ± 0.00 × 100 a	6.54 × 1011 ± 6.52 × 1010 c
		SB	4.65 × 109 ± 1.65 × 109 a	2.20 × 1010 ± 1.65 × 109 a
		LP-SB	1.76 × 1011 ± 0.00 × 100 ab	3.77 × 1011 ± 1.76 × 1010 b
		Control	1.13 × 1012 ± 1.13 × 1012 a	3.58 × 1012 ± 5.20 × 1011 b
	Millet	LP	2.90 × 1011 ± 3.62 × 1010 a	3.26 × 1011 ± 5.25 × 1010 a
		SB	1.33 × 1011 ± 1.21 × 1010 a	2.69 × 1010 ± 5.24 × 108 a
		LP-SB	3.67 × 1010 ± 3.05 × 1010 a	3.81 × 1011 ± 7.52 × 1010 a

LP: Lactiplantibacillus plantarum 14917; LP-SB: mixture of Lactiplantibacillus plantarum 14917 and Saccharomyces boulardii CNCM I-745; SB: Saccharomyces boulardii CNCM I-745. The analysis used was an ANOVA together with a Tukey test to determine significant differences. In the columns, different letters imply a significant difference between the different treatments specifically in that seed group.

shows the efficiency of probiotic production in different sprouts, where significant differences were observed among the seed groups. Beet sprouts demonstrated greater viability for S. boulardii (1.35 × 10¹² ± 8.46 × 10 ^c), while millet sprouts exhibited higher viability for L. plantarum (2.90 × 10¹¹ ± 3.62 × 10 ^a). During the imbibition stage, probiotics are absorbed by the seeds, gaining better access to available nutrients and thus achieving optimal development. Factors such as imbibition and germination temperature, the method of inoculating the imbibition medium, the seed type, and post-germination conditions directly influence the efficiency of probiotic-rich sprout production [11]. Additionally, the microorganism used to inoculate the seeds can change its metabolism depending on the seed occupied.

In oilseed sprouts, flaxseed inoculated with L. plantarum exhibited values considered suitable for a food to be classified as carrying probiotics (minimum amount of 1 × 10⁶). However, no significant differences were found within this seed group. For legume seeds, the highest values occurred in lentils inoculated with the combination of both probiotic microorganisms. The same was true for vegetables in beets inoculated with the mixture. In cereals, triticale with L. plantarum reached higher counts. Statistical analysis revealed significant differences among the seeds, which can be attributed to their varying nutritional compositions. A common factor among the treatments with better counts for efficiency and probiotic production was the presence of L. plantarum [69].

The literature mentions that using probiotics in biostimulant formulations involves microbial consortia that modulate the various mechanisms of probiotic–plant interaction. These modified effects include nutrient acquisition, the secretion of secondary metabolites in response to stress, and an increase in defense mechanisms against pathogens [70,71]. Therefore, incorporating probiotics in agriculture can provide stability to plants during their development and stimulate the production of secondary metabolites [72]. Moreover, including probiotics in a food matrix is an excellent way to enhance the benefits of consuming probiotics. The sprouts of different seeds are considered probiotic-carrying foods due to their high microorganism content (minimum amount of 1 × 10⁶). The treatment offering the best results is the one that presents the combination of both microorganisms [73].

Studies by Świeca in 2019 [12] demonstrated that sprouts inoculated with probiotics enhance the phenolic content of these foods, improve nutrient digestibility (reducing antinutrients in seeds), and increase microbiological quality, replacing the characteristic microbiome of the sprouts with probiotic microorganisms. It is important to note that probiotic efficiency and production are affected by the number of inoculated microorganisms and the conditions they must tolerate to reach the large intestine (pH, gastric acid, bile salts) [10,12,74].

4. Conclusions

Inoculating the imbibition substance with probiotics improves various germination parameters, such as speed, particularly in oilseeds and legumes on day 5 of growth. Treatment with L. plantarum increased biomass production in shoots to a greater extent. The concentration of auxins was higher in the control treatment of the shoots, indicating that germination time is crucial for enhancing the amount of auxins in probiotic-imbibed shoots.

The microbiological quality of the sprouts improved significantly in those subjected to imbibition with probiotic microorganisms. Probiotics function as a protective component against phytopathogens, reducing the incidence of pests or carriers of pathogenic microorganisms.

The sprouts of different seeds can be considered probiotic-containing foods due to their high microorganism content (minimum quantity of 1 × 10⁶).

The imbibition of certain types of seeds with probiotics increases the germination rate through auxin synthesis, as well as the biomass obtained, which suggests that it may be an alternative to treatments in the agricultural industry for greater production.

Within the future perspectives, the use of probiotics as a biostimulant is contemplated due to its increase in the synthesis of hormones during the germination process. It is worth mentioning that more studies are needed to determine other types of hormones that support the above.