**Soluble Extracts from Chia Seed (***Salvia hispanica* **L.) A**ff**ect Brush Border Membrane Functionality, Morphology and Intestinal Bacterial Populations In Vivo (***Gallus gallus***)**

### **Bárbara Pereira da Silva 1, Nikolai Kolba 2, Hércia Stampini Duarte Martino 1, Jonathan Hart <sup>2</sup> and Elad Tako 2,\***


Received: 6 August 2019; Accepted: 20 September 2019; Published: 14 October 2019

**Abstract:** This study assessed and compared the effects of the intra-amniotic administration of various concentrations of soluble extracts from chia seed (*Salvia hispanica* L.) on the Fe and Zn status, brush border membrane functionality, intestinal morphology, and intestinal bacterial populations, in vivo. The hypothesis was that chia seed soluble extracts will affect the intestinal morphology, functionality and intestinal bacterial populations. By using the *Gallus gallus* model and the intra-amniotic administration approach, seven treatment groups (non-injected, 18 Ω H2O, 40 mg/mL inulin, non-injected, 5 mg/mL, 10 mg/mL, 25 mg/mL and 50 mg/mL of chia seed soluble extracts) were utilized. At hatch, the cecum, duodenum, liver, pectoral muscle and blood samples were collected for assessment of the relative abundance of the gut microflora, relative expression of Fe- and Zn-related genes and brush border membrane functionality and morphology, relative expression of lipids-related genes, glycogen, and hemoglobin levels, respectively. This study demonstrated that the intra-amniotic administration of chia seed soluble extracts increased (*p* < 0.05) the villus surface area, villus length, villus width and the number of goblet cells. Further, we observed an increase (*p* < 0.05) in zinc transporter 1 (ZnT1) and duodenal cytochrome b (Dcytb) proteins gene expression. Our results suggest that the dietary consumption of chia seeds may improve intestinal health and functionality and may indirectly improve iron and zinc intestinal absorption.

**Keywords:** intra amniotic (in ovo) administration; zinc gene expression; iron gene expression; brush border membrane functional genes; intestinal bacterial populations; villus surface area

#### **1. Introduction**

Micronutrient deficiency affects approximately two billion people worldwide. Iron (Fe) and zinc (Zn) deficiencies are the most prevalent, affecting approximately 45% and 17%, respectively, of the world population [1–3]. Both mineral deficiencies are more prevalent in Africa, South East Asia and Latin America [4,5]. Among the dietary factors that contribute to Fe and Zn deficiencies is their low bioavailability due to dietary potential inhibitors, such as phytic acid and phenolic compounds [2,6,7]. Dietary Fe and Zn deficiencies affect normal cell division and differentiation, as well as growth and development, impair physical and cognitive development, and increase the risk of infection [4,7,8].

We have previously established the *Gallus gallus* as a model to assess dietary Fe and Zn bioavailability [9–15]. In addition, this experimental model presents a complex gut microbiota [16], as the phylum level was shown to be similar to humans [17,18]. Further, the intra amniotic administration

method has been widely used and demonstrates the potential prebiotic effects of soluble fibers from beans, chickpeas, lentil and wheat, with demonstrated effects on the intestinal functionality, morphology, and microbial populations [10,13,15].

Prebiotics are dietary substrates that selectively promote the proliferation and/or activity of health-promoting bacterial populations in the colon [19,20]. The soluble extracts are obtained by the isolation process of the prebiotics of the food matrix and are composed for the most part of soluble fiber. The most commonly used prebiotics, as inulin, raffinose and stachyose, are dietary fibers with a well-investigated and proven ability to promote the abundance of intestinal bacterial populations, which may provide additional health benefit to the host [21]. It is known that soluble extracts are responsible for improving gastrointestinal motility [22,23], intestinal functionality and intestinal morphology [10,13,24,25], and improving mineral absorption [10,26]. Recent Studies have shown that the consumption of plant seed origin soluble extracts can up regulate the gene expression of brush border membrane (BBM) proteins that contribute to the digestion and absorption of nutrients, such as sucrase-isomaltase, aminopeptidase and sodium glucose cotransporter-1 [10,11,13]. Further, soluble extracts can positively affect intestinal health by increasing mucus production, goblet cell number, goblet cell diameter, villus surface area, villus height, villus width, and crypt depth [10,13,15,27,28]. These functional and morphological effects appears to occur due to the increased motility of the digestive tract by the soluble extracts, leading to hyperplasia and/or hypertrophy of muscle cells [29]. In addition, plant origin soluble extract (with high fiber content and, therefore, potential prebiotic properties) administration may act, directly or indirectly, as a factor that increases iron and zinc bioavailability [30–32]. This event occurs due the lower intestine (colon) fiber fermentation process and the bacterial production of short-chain fatty acids (SCFAs) that reduce the intestinal pH, inhibiting the growth of potentially pathogenic bacterial populations and increasing the solubility and, therefore, the absorption of minerals [10,26]. The SCFAs can increase the proliferation of epithelial cells, which, in return, increases the absorptive surface area, which contributes to the absorption of dietary minerals [33]. Also, it was previously shown that the consumption of soluble extracts has a synergistic effect, as it promotes the metabolic interactions within the gastrointestinal microbial community via the production of organic acids, which provide an acidic environment in the colon, indirectly suppressing the growth of pathogens [34].

The use of iron- and zinc-rich foods may be a good strategy aimed to reduce the prevalence of iron and zinc deficiencies, respectively. Chia (*Salvia hispanica* L.) is an herbaceous plant with good nutritional and functional value with high concentrations of bioactive compounds such as dietary fiber and minerals, including iron and zinc [35]. Although iron and zinc are present in high concentrations, it is important to take into account the bioavailability of these minerals [36]. In the present study, chia was chosen as the soluble extract source, since the consumption of chia bacame extensively common worldwide, and specifically consumed with increasing amounts in Mexico, Argentina, Chile, New Zealand, Japan, USA, Canada and Australia [37], as in some of these geographical regions (e.g., South America), dietary Fe and Zn deficiencies are a major health concern [4,5]. Thus, the primary objective of this study was to assess the effects of the intra-amniotic administration of chia soluble extracts with a putative prebiotic effect on Fe and Zn status and brush border membrane functionality, in vivo. A secondary objective was to evaluate the effects of the tested extracts on intestinal bacterial populations. The third objective was to evaluate the effects of the chia soluble extracts on intestinal morphology. We hypothesized that the chia soluble extracts will affect the intestinal morphology, functionality and bacterial populations.

#### **2. Material and Methods**

#### *2.1. Sample Preparation*

Chia seeds (*Salvia hispanica* L.) grown in the state of Mato Grosso (Brazil) were used for this study. To obtain the flour, the seeds were ground up in three replicates, using a knife mill (Marconi Equipment,

Algodoal, Brazil), to a particle size of 850 μm. Subsequently, chia flour was packed in polyethylene aluminum bags and stored in a freezer (−20 ◦C) until analysis.

#### *2.2. Polyphenols Analysis*

#### 2.2.1. Chia Sample Preparation

A volume of 5 mL of methanol:water (50:50 *v*/*v*) was added to 0.5 *g* of chia flour. The resulting slurry was vortexed for 1 min before incubation in a 24 ◦C sonication water bath for 20 min at room temperature. Samples were again vortexed and placed on a rocker at room temperature for 60 min before centrifuging at 4000× *g* for 15 min. Supernatants were filtered with a 0.2 μm PTFE syringe filter and stored at −20 ◦C for later use.

#### 2.2.2. Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis

Extracts and standards were analyzed by an Agilent 1220 Infinity Liquid Chromatograph (LC; Agilent Technologies, Inc., Santa Clara, CA, USA) coupled to an Advion expressionL® compact mass spectrometer (CMS; Advion Inc., Ithaca, NY, USA). Ten-microliter samples were injected and passed through an XBridge Shield RP18 3.5 μm 2.1 × 100 mm column (Waters, Milford, MA, USA) at 0.6 mL/min. The column was temperature-controlled at 40 ◦C. The mobile phase consisted of ultra-pure water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Polyphenols were eluted using linear gradients of 94.0 to 84.4% A in 1.50 min, 84.4 to 81.5% A in 2.25 min, 81.5 to 77.0% A in 6.25 min, 77.0 to 55.0% in 1.25 min, 55.0 to 46.0% in 2.25 min, 46.0 to 94.0% in 2.25 min and hold at 94.0% A for 2.25 min for a total run time of 18 min. From the column, the flow was directed into a variable wavelength Ultraviolet (UV) detector set at 280 nm. The flow was then directed into the source of an Advion expressionL® CMS, and Electrospray ionization (ESI) mass spectrometry was performed in the negative ionization mode using selected ion monitoring with a scan time of 200 ms. The capillary temperature and voltages were 250 ◦C and 180 volts, respectively. The ESI source voltage and gas temperature were 2.5 kilovolts and 250 ◦C, respectively. The desolvation gas flow was 240 L/h. Advion Mass Express™ software (Advidon, Ithaca, USA) was used to control the LC and compact mass spectrometers (CMS) instrumentation and data acquisition. Individual polyphenols were identified and confirmed by comparison of *m*/*z* and LC retention times with authentic standards. The analysis of MS and UV data was performed using Advion Data Express™ software (Advidon, Ithaca, USA).

#### *2.3. Extraction of Soluble Extracts from Chia*

The extraction of prebiotics was performed according to Tako et al. [14], Hou et al. [13] and Pacific et al. [10]. Chia flour samples were dissolved in distilled water (50 g/L) (60 ◦C, 60 min) and centrifuged at 3000 rpm (4 ◦C) for 25 min, and then the supernatant was collected. The supernatant was then dialyzed (MWCO 12–14 kDa) (48 h) against distilled water. The dialysate was collected and lyophilized to yield a fine off-white powder [12].

#### *2.4. Phytate, Dietary Fiber, Iron and Zinc Analysis in Chia Seeds and Chia Extract*

Dietary phytic acid (phytate)/total phosphorous was measured as phosphorus released by phytase and alkaline phosphatase, according to manufacturer's instructions (*n* = 5) (K-PHYT 12/12. Megazyme International, Bray, Ireland). The determination of total fiber and soluble and insoluble fractions was performed by the enzymatic-gravimetric method according to AOAC [38], using enzymatic hydrolysis for a heat-resistant amylase, protease and amyloglucosidase (Total dietary fiber assay Kiyonaga, Sigma®, Kawasaki, Japan). For the determination of iron and zinc, chia seed and chia extract (0.5 g) were treated with 3.0 mL of a 60:40 HNO3 and HClO4 mixture in a Pyrex glass tube and left overnight to destroy organic matter. The analyses were performed using an inductively coupled

plasma atomic emission spectrometer (ICP-AES) (Thermo iCAP 6500 series, Thermo Jarrell Ash Corp., Franklin, MA, USA) [12,28].

#### *2.5. Animals and Design*

Cornish-cross fertile broiler eggs (*n* = 105) were obtained from a commercial hatchery (Moyer's chicks, Quakertown, PA, USA). The eggs were incubated under optimal conditions at the Cornell University Animal Science poultry farm incubator. All animal protocols were approved by the Cornell University Institutional Animal Care and Use committee (ethic approval code: 2007-0129).

#### Intra Amniotic Administration

Lyophilized soluble extracts were diluted in 18 Ω H2O and for sample osmolarity determination (≤320 OSM). At 17 days of embryonic incubation, eggs containing viable embryos were weighed and divided into 7 groups (*n* = 15). All treatment groups were assigned eggs of a similar weight frequency distribution. Each group was then injected with the specified solution (1 mL per egg), using a 21 gauge needle into the amniotic fluid, which was identified by candling. The 7 groups were assigned as follows: (1) non-injected; (2) 18 Ω H2O; (3) inulin (40 mg/mL); (4) chia seed extract 0.5% (5 mg/mL); (5) chia seed extract 1% (10 mg/mL); (6) chia seed extract 2.5%; (7) chia seed extract 5% (50 mg/mL). After the injections, the holes were sealed with cellophane tape and the eggs were placed in hatching baskets. Immediately after hatch (21 days), the chicks were euthanized by CO2 exposure and their small intestine, blood, pectoral muscle, cecum and liver were collected.

#### *2.6. Iron and Zinc Content in Serum and Liver*

Liver (0.5 g) and serum (50 μL) were treated with 3.0 mL of a 60:40 HNO3 and HClO4 mixture in a Pyrex glass tube and were incubated overnight. The mixture was then heated to 120 ◦C for two hours and 0.25 mL of 40 μg/g Yttrium was added as an internal standard. Next, the temperature of the heating block was raised to 145 ◦C for 2 h. Then, for 10 min, the temperature of the heating block was raised to 190 ◦C. The cooled samples were then diluted to 20 mL, vortexed and transferred into autosampler tubes to be analyzed via inductively coupled plasma atomic emission spectrometer (ICP-AES). (Thermo Jarrell Ash Corp., Franklin, MA, USA) [12,28].

#### *2.7. Isolation of Total RNA from Duodenum and Liver*

Total RNA was extracted from 30 mg of the proximal duodenal tissue or liver tissue (*n* = 10) using Qiagen RNeasy Mini Kit (RNeasy Mini Kit, Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. Total RNA was eluted in 50 μL of RNase-free water. All steps were carried out under RNase-free conditions. RNA was quantified by absorbance at A 260/280 and the integrity of the 18S ribosomal RNAs was verified by 1.5% agarose gel electrophoresis followed by ethidium bromide staining. RNA was stored at −80 ◦C.

#### *2.8. Real Time Polymerase Chain Reaction (RT-PCR)*

To create the cDNA, a 20 μL reverse transcriptase (RT) reaction was completed in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). The concentration of cDNA obtained was determined by measuring the absorbance at 260 and 280 nm using an extinction coefficient of 33 (for single stranded DNA). Genomic DNA contamination was assessed by a real-time RT-PCR assay for the reference gene samples [12].

#### *2.9. Primer Design*

The primers used in the real-time qPCR were designed based on 13 gene sequences from the Genbank database, using Real-Time Primer Design Tool software (IDT DNA, Coralvilla, IA, USA). The sequences and the description of the primers used in this work are summarized in Table 1. The specificity of the primers was tested by performing a BLAST search against the genomic National Center for Biotechnology Information (NCBI) database. The *Gallus gallus* primer 18S rRNA was designed as a reference gene. Results obtained from the qPCR system were used to normalize those obtained from the specific systems as described below.


**Table 1.** The sequences of the primers used in this study.

DMT1, Divalent metal transporter 1; Dcytb, Duodenal cytochrome b; Znt 1, Zinc transporter 1; SI, Sucrose isomaltase; AP, Amino peptidase; SGLT1, Sodium-Glucose transport protein 1; LPL, Lipoprotein lipase; CEL, Carboxyl ester lipase; 18S rRNA, 18S Ribosomal subunit. \* liver analyses.

#### *2.10. Real-Time qPCR Design*

All procedures were conducted as previously described [10–13]. The specific primers that were used are shown in Table 1.

#### *2.11. Collection of Microbial Samples and Intestinal Content DNA Isolation*

The cecum contents were removed under sterile conditions, placed into a sterile tube containing 9 mL of Phosphate buffered saline (PBS) and homogenized by vortexing with glass beads for 3 min [27,39]. All procedures were conducted as previously described [10–14].

#### *2.12. Primer Design and PCR Amplification of Bacterial 16S rDNA*

Primers for *Lactobacillus*, *Bifidobacterium*, *Clostridium* and *Escherichia coli* were used [16,39]. The universal primers were designed with the invariant region in the 16S rRNA of bacteria and were used as internal standards. The proportions of each bacterial group are presented. The PCR products were loaded on 2% agarose gel stained with ethidium bromide and quantified by Quantity One 1-D analysis software (Bio-Rad, Hercules, CA, USA) [12]. The evaluation of the relative abundance of each examined bacterium was conducted as previously described [10–14].

#### *2.13. Glycogen Analysis*

At hatch, the pectoral muscle (20 mg) was collected for glycogen analysis. The tissue samples were homogenized in 8% perchloric acid, and glycogen concentration was determined as previously described [40]. After homogenization, the samples were centrifuged at 12,000 rpm at 4 ◦C for 15 min. The supernatant was removed, and 1.0 mL of petroleum ether was added. After mixing, the petroleum ether fraction was removed, and samples from the bottom layer were transferred to a new tube containing 300 μL of color reagent. All samples were read at a wavelength of 450 nm in an ELISA reader and the amount of glycogen was calculated according to a standard curve. The amount of glycogen present in pectoral sample was determined by multiplying the weight of the tissue by the amount of glycogen per 1 g of wet tissue.

#### *2.14. Morphological Examination*

As previously described [10,41], liver and intestine samples were collected at the conclusion of this study. Samples were fixed in 4% (*v*/*v*) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Serial sections of 5 μm were obtained and were deparaffinized in xylene, rehydrated in a different concentration of alcohol, stained with hematoxylin/eosin or Alcian Blue/Periodic acid-Schiff, and examined by light microscopy. The following variables were measured in the intestine: villus height, villus width, depth of crypts, goblet cell number and goblet cell diameter in each segment, performed with a light microscope using EPIX XCAP software (Standard version, Olympus, Waltham, MA, USA). Four segments for each biological sample and five biological samples per treatment group were used. Villi height was measured using the lamina propria as the base; villi width, depth of the crypt and the number of goblet cell were measured per side of a longitudinal section through the villus; goblet cell size was measured as the diameter of the goblet cells (μm2). Villi surface area was calculated from the villus height and width at half height as according to Uni et al. [42]. For the Alcian Blue and Periodic acid-Schiff stain, the segments were only counted for the types of goblet cells in the villi epithelium, goblet cells within the crypts and the mucus layer thickness. Goblet cells were enumerated on 10 villi/sample, and the means were utilized for statistical analysis. The liver was stained with hematoxylin-esoin (H&E) for standard microscopy and visualized using the same light microscope. Mean adipocyte diameter was determined by random, utilizing the EPIX XCAP software (standard version, Olympus, Waltham, MA, USA), by enumerating 10 adipocytes/segment/sample, and the means were utilized for statistical analysis.

#### *2.15. Statistical Analysis*

All values are expressed as the means and standard deviations. Experimental treatments for the in ovo assay were arranged in a completely randomized design. The results were analyzed by ANOVA. For significant "*p*-value", post hoc Duncan test was used to compare test groups. Statistical analysis was carried out using SPSS version 20.0 software (IBM, Armonk, USA). The level of significance was established at *p* < 0.05.

#### **3. Results**

#### *3.1. Concentration of Iron, Zinc, Phytic Acid and Dietary Fiber and the Phytate:Iron Ratio in Chia Flour and in Chia Extract*

The iron and zinc concentrations, insoluble fiber content, phytic acid and the phytate:iron ratio were higher (*p* < 0.05) in the chia seed compared to the chia extract (Table 2). However, the content of soluble fiber was significantly greater (*p* < 0.05) in the chia extract relative to chia seed.


**Table 2.** Concentration of iron, zinc, dietary fiber and phytic acid in chia flour and in chia extract.

Values are the means ± SEM, *<sup>n</sup>* <sup>=</sup> 5. a,b Treatment groups not indicated by the same letter are significantly different (*p* < 0.05).

#### *3.2. Polyphenol Profile in Chia Flour*

The concentration of the five most prevalent polyphenolic compounds found in chia is presented in Table 3. Chia presented high concentrations of rosmarinic acid and rosmarinyl glucoside. In addition, we observed the presence of ferulic acid, caffeic acid and protocatechuic acid.


**Table 3.** Polyphenol profile present in chia flour.

Values are the means ± SEM, *n* = 10. mAU: milli absorbance unit; min: minutes.

#### *3.3. In Ovo Assay (Gallus Gallus Model)*

#### 3.3.1. Hb Concentration

The Hb values were significantly (*p* < 0.05) higher in the "2.5% chia" extract treatment group compared to the 18 Ω H2O and non-inject group. The other treatments did not differ from each other (Table 4).


**Table 4.** Blood hemoglobin (Hb) concentrations (g/dL).

Values are the means ± SEM, *n* = 10. a,b Treatment groups not indicated by the same letter are significantly different (*p* < 0.05).

#### 3.3.2. Iron and Zinc Concentration in Liver and Serum

As shown in Table 5, there were no significant (*p* > 0.05) differences in liver iron concentration and serum zinc concentration between treatment groups. However, "1% chia" extract treatment increased (*p* < 0.05) the zinc liver content compared to non-inject treatment. In addition, we observed that "5% chia" extract treatment showed a lower (*p* < 0.05) serum iron concentration when compared to the 18 Ω H2O and inulin groups. In general, different concentrations of chia extract did not affect iron and zinc concentrations in liver and serum.



Values are the means ± SEM, *<sup>n</sup>* <sup>=</sup> 10. a,b,c Treatment groups not indicated by the same letter are significantly different (*p* < 0.05).

#### 3.3.3. Gene Expression of Fe- and Zn-Related Genes

The gene expression of DMT1 was lower (*p* < 0.05) in the group treated with 2.5% chia soluble extract compared to the inulin and 18 Ω H2O groups (Figure 1). However, other various concentrations of chia soluble extract did not affect the expression of DMT1 (*p* > 0.05). The relative expression of DcytB and hepcidin was significantly up-regulated (*p* < 0.05) in the 1%, 2.5% and 5% chia extract. The groups treated with 1%, 2.5% and 5% chia extract showed lower (*p* > 0.05) ferroportin gene expression compared to the 18 Ω H2O injected group. However, no differences (*p* > 0.05) were observed between chia treatment groups. The relative expression of ZnT1 was significantly up-regulated (*p* < 0.05) in the 1%, 2.5% and 5% chia extract.

**Figure 1.** Effect of the intra-amniotic administration of experimental solutions on intestinal and liver gene expression. Values are the means <sup>±</sup> SEM, *n* = 10. a–c Per gene, treatments groups not indicated by the same letter are significantly different (*p* < 0.05). DMT1, Divalent metal transporter 1; Dcytb, Duodenal cytochrome b; ZnT1, Zinc transporter 1; AP, Amino peptidase; SGLT1, Sodium-Glucose transport protein 1; SI, Sucrose isomaltase; CEL, Carboxyl ester lipase; LpL, Lipoprotein lipase.

#### 3.3.4. Gene Expression of BBM Functional Proteins

The gene expression of aminopeptidase (AP), sodium-glucose transport protein 1 (SGLT1) and sucrase isomaltase (SI) are used as biomarkers of brush border membrane digestive and absorptive functions. AP and SGLT1 gene expression did not differ (*p* > 0.05) between groups treated with chia extract. However, the gene expression of SI was higher (*p* < 0.05) in "5% chia" extract treatment group compared to the "2.5% chia" extract treatment group (Figure 1).

#### 3.3.5. Gene Expression of Lipids Metabolism Protein

The gene expressions of carboxyl ester lipase (CEL) and lipoprotein lipase (LpL) are used as biomarkers of lipid metabolism. As shown in Figure 1, the "2.5% chia" extract treatment group presented higher (*p* > 0.05) CEL expression compared to the control groups. However, the gene expression of LpL did not differ between chia extract groups and control groups (*p* < 0.05).

#### 3.3.6. Cecum-to-Body-Weight Ratio

As shown in Figure 2, the chia soluble extract treatment groups showed a higher (*p* < 0.05) cecum weight (B) and cecum weight/body weight ratio (C) compared to control groups (*p* < 0.05). However, no significant difference (*p* > 0.05) was observed in body weight (A) among treatment groups and controls.

**Figure 2.** The effect of chia on the: (**A**) body weight; (**B**) cecum weight; and (**C**) cecum weight/body weight ratio (%). Values are the means <sup>±</sup> SEM *<sup>n</sup>* <sup>=</sup> 15. a,b Within a column, means without a common letter are significantly different (*p* < 0.05).

#### 3.3.7. Microbial Analysis

As shown in Figure 3, the relative abundance of both *Bifidobacterium* and *Lactobacillus* genera, increased (*p* < 0.05) in the "0.5% chia" treatment, relative to the 18 Ω H2O group and non-injected group. The "5% chia" treatment group showed a lower (*p* < 0.05) concentration of these bacterial populations compared to the other groups. The relative abundance of *E. coli* significantly decreased (*p* < 0.05) in the 1%, 2.5% and 5% chia extract treatment groups compared to the control groups. The relative abundance of *Clostridium* was significantly (*p* < 0.05) lower in the non-inject group, 18 Ω H2O group and "5% chia" treatment group. These results suggest that a lower concentration of chia extract may positively affect gut health.

**Figure 3.** Genera- and species-level bacterial populations (AU) from cecal contents measured on the day of hatch. Values are the means <sup>±</sup> SEM, *n* = 10. a–d Per bacterial category, treatment groups not indicated by the same letter are significantly different.

#### 3.3.8. Glycogen Analysis

No significant difference was observed in pectoral muscle glycogen content between groups (Table 6, *p* > 0.05).


**Table 6.** Concentration of glycogen in pectoral muscle.

Values are the means ± SEM, *<sup>n</sup>* <sup>=</sup> 10. <sup>a</sup> Treatment groups not indicated by the same letter are significantly different (*p* < 0.05).

#### 3.3.9. Morphometric Data for Villi, Depth of Crypts and Goblet Cell

The villus surface areas, villi length, width and the number of goblet cells were significantly (*p* < 0.05) higher in all chia extract treatment groups compare to controls (Tables 7 and 8), indicating that soluble extracts from chia had a positive effect on intestinal development, through the proliferation of enterocytes, and the increased number in mucus-producing cells. However, there were no significant (*p* > 0.05) differences in crypt depth and mucus layer width between treatment groups. Further, all chia extract treatments increased (*p* < 0.05) the diameter of goblet cells compared to controls. In relation to the types of goblet cells observed (acidic, neutral, mixed), we can note that the administration of 2.5% chia soluble extracts reduced (*p* < 0.05) the number of neutrals goblet cells compared to the control groups. In addition, the administration of 2.5% and 5% chia soluble extracts increased (*p* < 0.05) the number of acidic goblet cells, whereas the administration of 1% and 2.5% chia extract caused an increase (*p* < 0.05) in mixed goblet cells, compared to controls. In relation to the types of goblet cells in the crypt epithelium, the administration of 0.5% chia soluble extract increased (*p* < 0.05) the number of neutrals goblet cells compared to controls. In addition, the administration of 2.5% chia extracts increased (*p* < 0.05) the number of mixed goblet cells compared to controls. The number of acid goblet cells did not differ (*p* > 0.05) between groups (Figure 4). No significant differences between treatment groups were measured in fat cell diameter (*p* > 0.05, Figure 5).

**Table 7.** Effect of the intra-amniotic administration of experimental solutions on the duodenal small intestinal villus and crypt.


Values are the means ± SEM, *<sup>n</sup>* <sup>=</sup> 5. a–d Treatment groups not indicated by the same letter are significantly different (*p* < 0.05).

**Table 8.** Effect of the intra-amniotic administration of experimental solutions on the goblet cells.


Values are the means ± SEM, *<sup>n</sup>* <sup>=</sup> 5. a–c Treatment groups not indicated by the same letter are significantly different (*p* < 0.05).

**Figure 4.** Representations of the intestinal morphology of each treatment group are shown (Alcian Blue and Periodic acid-Schiff Stain). The yellow circles indicate crypts within the villi and the red circles indicate goblet cells on the villi. Bar = 50 μm.

**Figure 5.** Fat cell diameter. Values are the means <sup>±</sup> SEM, *n* = 5. <sup>a</sup> Treatment groups not indicated by the same letter are significantly different.

#### 3.3.10. Hepatic Morphometric Measurement

As shown in Figure 4, no significant differences were observed in hepatic fat cell diameter between all treatment groups (*p* > 0.05).

#### **4. Discussion**

Chia is a good source of dietary fiber, which was demonstrated to have a beneficial effect on intestinal health [29]. However, until now, the potential effects of soluble extracts from chia seed on the intestinal microbiota, intestinal morphology and mineral bioavailability, such as iron and zinc, were not investigated. Further, it is important to highlight that the alterations in microbiota populations, due the consumption of dietary fiber, may be associated, directly or indirectly, to the increased dietary bioavailability of iron and zinc in vulnerable populations [13,15,18,27]. The present study indicates that the in ovo administration of soluble extracts from chia seed increased the intestinal villus surface area, villi length, villi width, goblet cell number and goblet cell size (diameter), as well as cecum weight (used as biomarker of microbial presence and activity). In addition, the administration of chia seed soluble extracts up-regulated the expression of proteins related to zinc metabolism. Further, the chia soluble extract (0.5%) increased the *Bifidobacterium* and *Lactobacillus* relative abundance in cecum content.

According to our results, the hemoglobin concentration results corroborate with our findings of serum iron. We did not observe a change in liver iron concentrations, due to the short time of exposure of the soluble extracts, which was not sufficient to cause a modification in hepatic iron storage. This was in agreement with previous observations that evaluated the effects of intra-amniotic raffinose and stachyose administration on Fe status, as the results showed no significant differences in hemoglobin values between treatment groups [10]. Further, another study that assessed the effect of the intra-amniotic administration of bean soluble extracts on iron status indicated that bean extracts did not affect serum or liver iron concentrations [12]. A similar result was observed post intra-amniotic administration of wheat extracts [14]. In addition, a BBM Fe metabolism-related gene expression analysis of DcytB, DMT, ferroportin and hepcidin was conducted. DcytB is the protein responsible for reducing Fe3<sup>+</sup> to Fe2<sup>+</sup> in the apical membrane of the enterocyte [10,43]. DMT1 plays a key role in Fe2<sup>+</sup> transport into the enterocyte, being considered the major Fe intestinal transporter [10,43], whereas ferroportin is the protein that transports Fe2<sup>+</sup> from the enterocyte into the bloodstream [10,43]. In the current study, the administration of 1%, 2.5% and 5% chia soluble extract solutions up-regulated the expression of DcytB, which in return may increase the transportation of Fe by DMT1 into the enterocyte, and as previously demonstrated, this effect can potentially increase iron absorption efficiency in a long-term feed trial [12]. Further, we investigated hepcidin gene expression as the key iron-regulatory hormone that controls systemic iron homeostasis, as hepcidin is able to down regulate the expression of ferroportin [44,45]. Further, the increase in hepcidin production is stimulated by iron loading and inflammation [46,47]. In the present study, hepcidin gene expression was lower (*p* < 0.05) in the 1%, 2.5% and 5% chia soluble extract groups compared to the inulin and water groups, which suggests that in a long-term feeding trial, the dietary inclusion of chia may have a positive effect on Fe-related proteins.

ZnT1 is the only transporter of the ZnT transporters family that is localized on the enterocyte's basolateral membrane and functions by exporting cytosolic zinc into the extracellular space [48], an up-regulation in ZnT1 mRNA gene expression may occur under increased cellular zinc levels [49]. In the current study, the groups treated with chia seed soluble extract (1%, 2.5% and 5%) shown a gene expression up-regulation (*p* < 0.05) of ZnT1 compared to the other groups, although the zinc serum concentrations did not differ between experimental groups.

Previous studies demonstrated the potential beneficial effects of soluble extract from various sources and plant origin compounds (such as raffinose, stachyose, diadzein, bean, and wheat) on BBM functionality and intestinal bacterial populations [10–13,27]. In the current study, the expression of BBM functional genes (AP, SI and SGLT1) was not affected by the chia seed soluble extract administration, due to the short exposure time. However, in relation to microbial populations, there was an increasing abundance of *Lactobacillus* (*p* < 0.05), and *Bifidobacterium* (*p* < 0.05) in the cecal contents of animals received 0.5% chia soluble extracts compared to the 18 Ω H2O and non-injected group. Further, we observed an increased abundance in *Lactobacillus* (*p* < 0.05), *Bifidobacterium* (*p* < 0.05), *E. Coli* (*p* < 0.05) and *Clostridium* (*p* < 0.05) in the cecal contents of the animals that received 0.5% chia seed soluble extracts compared to other groups treated with chia seed extract. It is important to highlight that the increase in *Lactobacillus* and *Bifidobacterium* abundance, due the consumption of dietary fiber, may further contribute, directly or indirectly, to the increased bioavailability of iron and zinc in vulnerable populations, as these bacterial genera produce short-chain fatty acids (SCFAs), which reduce the intestinal pH, and therefore, may increase mineral (as Fe and Zn) solubility and therefore absorption [50]. *Bifidobacterium* and *Lactobacillus* can break down non-digestible fiber (prebiotics), due to their 1,2-glycosidase activity, leading to greater SCFA production [16,27,39], culminating with the increase in the absorption of iron and zinc.

The morphological parameters described in the current study, including villi development parameters and the crypt depth, are used as indicators of intestinal health, functionality and development [51]. The administration of chia seed soluble extracts, regardless of the concentration used, increased all parameters related to intestinal villi. These values (villus surface area, villus length and width) were significantly higher (*p* < 0.05) in the 5.0% chia group and relative to all other groups. This can be explained by the potential increased proliferation of intestinal cells in the short term, due the presence of soluble fiber, leading to hyperplasia and/or hypertrophy of intestinal cells and potentially enhancing the absorptive and digestive capacity of the villi BBM [29]. Another explanation

is that the tested extracts had potentially increased butyrate production, which may lead to enterocyte proliferation [52]. Added to these factors, the soluble extract of chia seed contains a high concentration of phenolic compounds, among them are rosmarinic acid and rosmarinyl glucoside, which present the ability to affect intestinal morphology [53], increasing the villus height, crypt depth ratio, and muscularis thickness, as observed in the study that evaluated the administration of dietary polyphenol concentrate previously performed in *Gallus gallus* [54]. The morphological results agree with our cecum weight and cecum weight/body weight ratio observations. All experimental groups showed a higher (*p* > 0.05) cecal weight (Figure 2B) post intra-amniotic soluble extract administration, indicating, and as previously suggested, increased cecal bacterial populations activity [10,12,13]. As for crypt depth, no differences between the experimental groups were observed, since duodenal crypts require a longer time to allow cellular proliferation. However, the intestinal crypts are meager and are able to rise to the surface of the villus, increasing the number of enterocytes in intestinal villi [52]—a phenomenon that was observed in the current study. Additionally, we observed increased goblet cell number and goblet cell diameter, which suggests an increased production of mucus that coats the intestinal lumen. As previously suggested, this may increase the intestinal BBM digestive and absorptive capabilities, and may indirectly increase the bioavailability of dietary components as suggested by the effects observed on the morphometric parameters [55–57]. The increase in "acidic goblet cells", containing acidic mucin due to the administration of 2.5% and 5% chia soluble extracts, may contribute to the reduction of intestinal pH, which in the long term, may lead to increased solubilization and uptake of iron and zinc and affect intestinal microbial profile [14,39]. The increase in "acidic goblet cells" was previously observed in a study that evaluated the effects of the intra amniotic administration of carbohydrate solution (containing maltose, sucrose and dextrin) on mucin content, goblet cell development, and levels of mucin mRNA in the *Gallus gallus* small intestine [58].

In general, previous studies showed a positive effect of prebiotic administration on intestinal morphology [10,13,25,51,52], for example, the intra-amniotic administration of raffinose and stachyose increased villus surface area compared to the control [10]. Similar results were observed by Hou et al. [13], who evaluated the effect of chickpea and lentil prebiotics administration in ovo. In another study, the authors evaluated the development of morphological parameters in *Gallus gallus*, and the results showed that the administration of a synthetic prebiotic increased the villus width and crypt depth. The prebiotic had no impact on villus height, villus surface area, and muscular thickness compared to the animals that received saline solution administration [51]. Bogucka et al. [52] evaluated the effect of inulin administration on the development of the intestinal villi and the number of goblet cells in the small intestine on the 1st and the 4th day post hatch (*Gallus gallus*) and the study indicated that on day one, the villus height did not differ among experimental groups. However, the villus width, villus surface area and crypt depth were lower in the prebiotic group. On day four, the inulin group showed a lower villus width, villus surface area and crypt depth [52]. Another study that evaluated the effect of the intra aminiotic administration of wheat bran prebiotic extract indicated increased villus height, goblet cell diameter and number in all treatment groups [11]. Further, Mista et al. [25] evaluated the effect of intra amniotic administered prebiotics on the development of the small intestine (*Gallus gallus*) and found that prebiotics did not affect the villus length, but did increase the crypt depth.

The observations described in the current study suggest that dietary chia seed consumption may be an effective strategy to reduce dietary iron and zinc deficiency and to potentially improve intestinal health. Overall, the up-regulation of Zn gene expression and the DcytB-Fe metabolism protein, the increase in villus surface area, villus length, villus width, goblet cell number and goblet cell diameter as well as cecum weight suggest that chia is a promising food ingredient that may improve mineral bioavailability and intestinal morphology. Hence, long-term feeding trials assessing the dietary effects of chia are now warranted.

#### **5. Conclusions**

The intra-amniotic (in ovo) administration of chia seed soluble extracts with putative prebiotic effects improved the intestinal morphology and up-regulated Zn-related protein gene expression. Further, chia seed soluble extract administration affected the intestinal microbiota and iron-related gene expression. The current study is the first to investigate the effects of chia seed soluble extracts with a potential prebiotic effect in vivo; thus, future studies aimed to assess dietary chia seed in a long-term feeding trials should be conducted, since chia may be a viable dietary ingredient that may improve intestinal health and contribute to intestinal mineral absorption.

**Author Contributions:** Data curation, B.P.d.S., and E.T.; Formal analysis, B.P.d.S., N.K., and E.T.; Investigation, B.P.d.S. and E.T.; Methodology, N.K., J.H., and E.T.; Resources, H.S.D.M. and E.T.; Supervision, E.T.; Writing—original draft, B.P.d.S. and E.T.; Writing—review and editing, E.T.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

### **Non-Dairy Fermented Beverages as Potential Carriers to Ensure Probiotics, Prebiotics, and Bioactive Compounds Arrival to the Gut and Their Health Benefits**

### **Estefanía Valero-Cases 1, Débora Cerdá-Bernad 1, Joaquín-Julián Pastor <sup>2</sup> and María-José Frutos 1,\***


Received: 30 April 2020; Accepted: 28 May 2020; Published: 3 June 2020

**Abstract:** In alignment with Hippocrates' aphorisms "Let food be your medicine and medicine be your food" and "All diseases begin in the gut", recent studies have suggested that healthy diets should include fermented foods to temporally enhance live microorganisms in our gut. As a result, consumers are now demanding this type of food and fermented food has gained popularity. However, certain sectors of population, such as those allergic to milk proteins, lactose intolerant and strict vegetarians, cannot consume dairy products. Therefore, a need has arisen in order to offer consumers an alternative to fermented dairy products by exploring new non-dairy matrices as probiotics carriers. Accordingly, this review aims to explore the benefits of different fermented non-dairy beverages (legume, cereal, pseudocereal, fruit and vegetable), as potential carriers of bioactive compounds (generated during the fermentation process), prebiotics and different probiotic bacteria, providing protection to ensure that their viability is in the range of 106–107 CFU/mL at the consumption time, in order that they reach the intestine in high amounts and improve human health through modulation of the gut microbiome.

**Keywords:** intestinal microbiota; vegetable drink; fermentation; beneficial microorganisms; lactic acid bacteria; cereal; legume; pseudocereal; fruit; synbiotic

#### **1. Introduction**

Following Hippocrates' aphorism "Let food be your medicine and medicine be your food". In recent years, consumers have become more aware of the relationship between health and diet, and are now demanding healthier products with better nutritional characteristics and specific components to prevent health problems and improve their quality of life and life expectancy. This trend offers new opportunities for products with health benefits beyond basic nutrition, which meet consumer expectations and at the same time, drives the growth of the functional food market [1–3]. Nowadays, functional foods have been developed that incorporate different components with health benefits, such as bioactive compounds isolated from plants, polyunsaturated fatty acids, probiotics, prebiotics, minerals and vitamins, among others [4–7].

Twenty-five centuries ago, Hippocrates also stated that "All diseases begin in the gut" when referring to the relevance of the gastrointestinal system for human health. Currently, scientists studying the human microbiome suggest that healthy diets should include fermented foods to temporally enhance live microorganisms in our gut. The intestinal microbiota is a huge and varied collection of microorganisms. The large intestine hosts around 1013–1014 microorganisms (almost 10 times the

number of cells that make up the human body) and most consist of the bacteria phyla Firmicutes and Bacteroidetes. They play an important role in the health of the host, having effects on the regulation of energy metabolism and maturation of the immune system [8]. The administration of live probiotics maintains the balance of gut microbiota and contributes to the overall intestinal health. As a result, fermented foods have gained popularity and are highly demanded by the population [9,10]. Products with probiotics which contribute to gut health represent one of the largest and fastest growing sectors. Probiotics are defined as "live microorganisms that when are administered in an adequate amount confer a beneficial effect on the host health" [11]. Probiotics can be consumed as part of fermented foods or as dietary supplements. The most common genera that have been employed as probiotics and are available on the food market are the lactic acid bacteria (LAB) *Lactobacillus* and *Bifidobacterium*. Their species have mostly been given a generally-recognized-as-safe (GRAS) status and qualified presumption of safety (QPS) status, as their consumption does not present risks for the host's health [11–13].

A probiotic must survive during gastrointestinal digestion and adhere to the intestinal epithelium to exert its beneficial effects. The adherence ability depends on the hydrophobicity and autoaggregation capacity of the probiotic microorganisms [14]. The period of survival and residence of probiotics in the colon can be influenced by their duration and dose of probiotic, as well as by the matrix used as a carrier of the probiotics (Figure 1) [15,16].

**Figure 1.** Summary of the beneficial effects of probiotics in different fermented non-dairy beverage matrices. Primary effects: changes in non-dairy matrices during fermentation. Secondary effects: changes in the intestinal epithelium. Tertiary effects: positive changes in health. SCFAs: short chain fatty acids; EPS: exopolysaccharides; AOC: antioxidant capacity [14–16].

Dairy foods have traditionally been used as carriers for probiotic microorganisms. Therefore, foods such as kefir, milk, yogurts, and cheese have been widely explored as dairy matrices for probiotic bacteria [17–20]. However, certain sectors of the population such as those allergic to milk proteins, those who are lactose intolerant, and those who are strictly vegetarian, cannot consume dairy products. Therefore, a need has arisen to offer consumers an alternative to fermented dairy products by exploring new non-dairy matrices as probiotics carriers [21,22]. However, the viability of probiotic microorganisms is more difficult to maintain in non-dairy matrices than in dairy matrices. The physicochemical parameters must be carefully controlled to guarantee the probiotic viability and to achieve adequate organoleptic properties (mainly aroma and flavor) that can be modified by fermentation [23]. Nevertheless, to improve the probiotic viability in non-dairy beverages, prebiotics could be used as a supplement. Prebiotics can be defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one of a limited number of bacteria in the colon" [24]; thus, they can improve the gut microbiome by specific beneficial bacteria fermentation in the colon. Foods or beverages that contain probiotics and prebiotics are known as synbiotic foods. Therefore, the proper selection of food matrices as potential carriers of probiotics is an essential factor to consider in the development of probiotic foods. It is important to ensure the viability of probiotics during processing and storage, in order to maintain their concentrations at high levels (106–107 colony-forming units (CFU) per mL or g of food) at the time of consumption (Figure 2). It is also essential to ensure their survival during gastrointestinal digestion, and thus a high viability of the probiotics, so that a sufficient amount reach the large intestine to exert their beneficial effects. Accordingly, this review aims to explore the potential of different fermented non-dairy beverages (legume, cereal, pseudocereal, fruit, and vegetable), as carriers of bioactive compounds (also generated during the fermentation process), prebiotics and different probiotic bacteria. This review will also examine their effectiveness in providing protection to ensure high probiotic survival during processing, storage and gastrointestinal digestion, in order to make sure that they reach the intestine in sufficient amounts to improve human health.

**Figure 2.** Important factors to be considered when assessing afermented non-dairy beverage matrices as potential carriers for the viability of probiotic bacteria.

#### **2. Non-Dairy Fermented Beverages as Vehicles for Bioactive Compound, Probiotic, and Prebiotic Delivery to the Gut and their Health Benefits**

#### *2.1. Fermented Legume Beverages*

Several researchers have tried to produce non-dairy probiotic beverages based on legume, as a food matrix for the delivery of bioactive compounds, probiotics, or prebiotics, in order to enhance human intestinal health. Legumes are potential matrices as carriers of probiotics because they contain non-digestible oligosaccharides that can be metabolized by the microorganisms. Soybean legume (*Glycine max*, L.) is the most used since it has high quality proteins and minerals, and due to its isoflavones contents it has the potential to reduce the incidence of osteoporosis and menopausal symptoms [25]. However, due to soy allergies which affect about 0.5% of the general population, other legumes, such as chickpeas (*Cicer arietinum*, L.), are also used [26]. Chickpeas contain a high amount of resistant starch and amylose and some studies have proved that they can reduce the risks of high blood pressure and type-2 diabetes [27].

The production of soy beverages is one of the traditional unfermented food uses of soybeans. Recently, some studies have developed fermented soy drinks with probiotics, in order to improve their beneficial health properties and their flavor and texture [28]. Bedani et al. [29] prepared a soy beverage fermented with probiotic cultures and studied the viability and resistance of probiotics to simulated gastrointestinal digestion, as well as the effect of adding ingredients such as inulin as prebiotic and okara flour, which is a by-product of the soy milk industry. This study concluded that soy milk is a potential food matrix for the delivery of probiotics and prebiotics which could protect them against gastrointestinal juices. The probiotics used, *Bifidobacterium animalis* Bb-12 and *Lactobacillus acidophilus* La-5, survived at high concentrations maintaining their viability at above 8 Log CFU/mL during 28 days of storage at 4 ◦C with an acidic pH between 4.30 and 4.70. However, the survival was not affected by inulin and/or okara flour. In addition, during the in vitro gastrointestinal digestion, *B. animalis* Bb-12 maintained populations above 7 Log CFU/mL, but *L. acidophilus* La-5 was very sensitive and its viability was reduced, the final concentration being below 5 Log CFU/mL (the initial concentrations of both probiotics were above 8 Log CFU/mL). Therefore, soybeans could be a potential vehicle for probiotics, being able to maintain a high viability to exert their beneficial effects on human gut microbiota, although further studies are required.

Other studies have combined soy milk with cereals (sprouted wheat, barley, and pearl millet) and legumes (green gram), or with peanut milk, producing beverages with probiotic characteristics and high cell concentrations after fermentation [30,31]. Mridula and Sharma [30] analyzed fermented beverages based on soy milk combined with different cereals or legumes, such as green gram (*Vigna radiata*, L.). In the beverage based on green gram, after the fermentation at 37 ◦C for 8 h by *L. acidophilus* NCDC14, the probiotic count ranged from 10.36 to 11.32 Log CFU/mL with an acidity between 0.50% and 0.80% and a pH of 4.2–4.4. Besides, a high sensory acceptability score was obtained. Therefore, this fermented legume beverage might be a potential vehicle for probiotics. Santos et al. [31] developed a fermented beverage of soy and peanut milk, using these two substrates to improve the nutrient availability for probiotics. For the fermentation, six different LAB were used, including probiotic strains (*Pediococcus acidilactici* UFLA BFFCX 27.1, *Lactococcus lactis* CCT 0360, *Lactobacillus rhamnosus* LR 32, and *Lactobacillus acidophilus* LACA 4) and yeasts, in a binary culture or in co-culture. *L. acidophilus* LACA 4 and *P. acidilactici* UFLA BFFCX 27.1 reached counts above 8 Log CFU/mL after fermentation for 24 h at 37 ◦C and another 24 h at ±4 ◦C.Higher lactic acid contents were also obtained by co-culture with *Saccharomyces cerevisiae* yeast which might serve as a source of vitamin B and proteins. Therefore, a beverage based on peanuts may be a good carrier of probiotics, since it allows larger populations of the probiotic bacteria to be grown and maintained, but further studies are necessary.

There are few documented studies that have determined the effect of fermented legume beverages on modulation of the gut microbiome in either animal or humaninvestigations. Cabello-Olmo et al. [32] evaluated the impact of long-term supplementation with a non-dairy fermented beverage in the development of type-2 diabetes in rats, which is related to the host's intestinal microbiota since diabetic individuals present a characteristic intestinal microbial community. The plant-based beverage was composed of legumes and cereals such as alfalfa meal, soya flour, and barley sprouts with other minor components. It was fermented at 37 ◦C after the incorporation of LAB and debittered brewer's yeast, and the *Lactobacillus* genus was the most predominant. This research analyzed the fecal microbiota of rat feces collected at six months of study. The specimens had ad libitum access to food supplemented with the fermented beverages whose composition included a high level of fermentable carbohydrates. The study revealed that, at genus level, supplementation with the fermented beverage enriched the abundance of *Sutterella* which includes commensal species found in healthy humans and animals, and *Proteus*, for which there is no evidence for its function in diabetes. However some studies have suggested its role in several pathological conditions. Some *Barnesiella* species and the *Anaerococcus* genus were more numerous in the group treated with the beverage. The abundance of *Barnesiella* is related to a better glucose tolerance and important metabolic improvements, and the relative abundance of *Anaerococcus* includes many bacterial species which produce butyrate in experimental conditions. Butyrate, as well as acetate and propionate, are short-chain fatty acids (SCFAs) which produce beneficial effects on the gastrointestinal tract. SCFAs are produced by the probiotic fermentation of non-digestible carbohydrates and improve the intestinal barrier, inhibiting the development of pathogens and the production of toxic elements, and they are used by intestinal cells as colonocytes to grow [33]. Therefore, the administration of this plant-based beverage fermented with probiotics leads to an enrichment of the gut microbiota population, improving glucose metabolism and protecting against type-2 diabetes development in rats. These novel fermented beverages could be potential vehicles of probiotics, prebiotics, and/or bioactive compounds to protect against metabolic alterations of the diabetic pathology. However, further studies on microbial metabolites which are also important and responsible for gut health are required.

Another study on the effect of the intake of a soy milk beverage fermented with *Lactobacillus casei* Shirota on gut microbiota in sixty healthy premenopausal women twice a day for 8 weeks, reported that there was an increase of *Lactobacillaceae* and *Bifidobacteriaceae* levels and a decrease of *Enterobacteriaceae* and *Porphyromonadaceae* levels during the intake period. The results suggested that a daily intake of fermented soy milk beverage beneficially contributes to modulation of the gut microbiota in premenopausal healthy women [34].

Other studies have focused on the use of different legumes, such as chickpeas (*Cicer arietinum*, L.), as alternatives to soy in fermented plant-based beverages, which could be a promising carrier of probiotics. The results showed probiotic counts of about 6 Log CFU/mL after fermentation for 16 h at 42 ◦C, but more optimization studies are required to minimize syneresis and improve the sensory acceptability [35]. One study developed a non-dairy beverage fermented by *Lactobacilli acidophilus* probiotics using germinated and ungerminated seeds of moth bean (*Vigna aconitifolia*, L.) and cereals, obtaining promising results since the levels of microorganisms after fermentation for 6 h at 37 ◦C were above 8 Log CFU/mL and a good sensory acceptability was obtained [36]. The main studies that have been conducted to date on fermented legume beverages and their results are summarized in Table 1.


**Table 1.** Summary of fermented legume beverages as potential carriers for bioactive compound, probiotic, and prebiotic delivery to the gut.

#### *2.2. Fermented Cereal Beverages*

Cereals are consumed all over the world and are considered one of the most important sources of carbohydrates, proteins, dietary fiber, minerals, and vitamins in our diet. Therefore, they are a good option among non-dairy raw materials for producing fermented beverages [37]. Oat (*Avena sativa*, L.) is a potential functional ingredient, due to its proteins, soluble fiber, and antioxidant properties, with β-glucan being the most important carbohydrate fraction because of its prebiotic properties in the gut [38]. Kedia et al. [39] investigated the prebiotic potential of oat through in vitro fermentation for

24 h with human fecal cultures. Their results showed an increase in SCFAs and beneficial intestinal bacteria such as *Enterobacteria*, and a reduction in harmful bacteria such as anaerobes and clostridia. Oat beverages fermented with different *Lactobacillus* strains have also been reported to display a high antioxidant capacity and an increase in polyphenol content with respect to non-fermented beverages. However, only fermented oat with *L. plantarum* LP09 showed an in vitro decrease in the hydrolysis index of starch (used as a measure of the glycemic index in healthy subjects). The fermentation also increased the β-glucan content. However, the soluble and insoluble fiber ratio decreased after fermentation. At the same time, the aroma and flavor were better than in the unfermented control samples [40]. Other studies have also shown that fermented oat beverages may be potential probiotic carriers. They resulted in optimization of the fermentation process and the beverage formulations, reaching high microbial levels (>107 CFU/mL) during production and storage, and were able to maintain the β-glucan content at the end of storage [41–45]. The viability and in vitro probiotic potential were recently investigated by Funck et al. [46] in oat beverages fermented with *L. curvatus* P99. The results showed a high probiotic viability (above 7 log CFU/mL) during 35 days of being refrigerated at 4 ◦C. The acceptability of the fermented beverages was good, since most consumers gave high scores for all sensory attributes evaluated. Regarding the probiotic properties, *L. curvatus* P99 showed high gastrointestinal survival and antimicrobial activity against Gram-negative and Gram-positive bacteria. Additionally, it has the capacity of auto-aggregation and of blocking the adhesion of pathogenic bacteria to gut epithelial cells. Johansson et al. [47] carried out an in vivo controlled randomized double-blind study with a rose-hip beverage supplemented with oats fermented with *L. plantarum* DSM9843 in 48 healthy adults. The group receiving the administration of 400 mL/day of this beverage for three weeks showed an increase of total fecal carboxylic acid (lactic, acetic, and propionic acid) and the probiotic bacteria were found in feces at a high concentration, indicating their survival during gastrointestinal digestion. Decreased flatulence and a softer stool consistency were also reported.

Among other cereals, rice (*Oryza sativa*, L.) is also used in the production of fermented beverages that are very popular in Asian-Pacific countries. Ghosh et al. [48] demonstrated a probiotic role of *L. fermentum* KKL1 in a fermented rice beverage. The beverages fermented at 37 ◦C for 4 days consecutively in anaerobic conditions showed a strong antioxidant activity; the production of glucoamylase, α-amylase and phytase (therefore, an increase of phytate bioavailability) and a mineral increase (Ca, Fe, Mg, Mn and Na). Besides, the hydrosoluble vitamin content in fermented samples was higher than in unfermented control samples such as folic acid, thiamine, ascorbic acid and pyridoxine. However, riboflavin decreased due to bacterial metabolism during fermentation. At the same time, rice was a good carrier for maintaining the *L. fermentum* KKL1 at a high concentration after gastrointestinal digestion and showed sensitivity to the antibiotics tested, except for polymyxin. Nevertheless, this strain showed a moderate cell surface hydrophobicity. Another study reported similar results in fermented rice beverages, but with different species of *Lactobacillus* bacteria, such as *L. plantarum* L7. This strain showed good in vitro characteristics, such as high survival to gastrointestinal digestion, antimicrobial activity, autoaggregation to the intestinal cell surface, and susceptibility to some antibiotics, the latter is a desirable characteristic because probiotics should not carry transmissible antibiotic-resistance genes, in order to avoid the development of new antibiotic-resistant pathogens The fermented beverages increased the lactic, succinic and acetic acids during fermentation for 6 days under anaerobic conditions at 35 ◦C. At the same time, increases in phytase activity and minerals (Na, Mg, Mn, Fe and Ca) were also observed. Furthermore, the fermented rice beverage presented a high antioxidant capacity [49]. Therefore, rice is a good matrix for *L. fermentum* KKL1 and *L. plantarum* L7 growth and survival in adequate concentrations. Furthermore, these bacteria played an important role in improving the functional properties of rice beverages. However, in vivo investigations are required to explore and verify their probiotic properties.

Maize (*Zea mays*, L.) is another highly consumed cereal that contains about 72% starch, 10% proteins, and 4% fiber, together with vitamin B and essential minerals [50]. Menezes et al. [51] used the *L. paracasei* LBC-81 with the yeast *S. cerevisiae* CCMA 0731 and *L. paracasei* LBC-81 with *S. cerevisiae* CCMA 0732 in combination, for the fermentation of maize beverages at 30 ◦C for 24 h. The results showed a high microorganism viability of above 7 Log CFU/mL during fermentation and during 28 days of refrigerated storage (4 ◦C). The beverages achieved a score of 5 out of 9 points for general acceptance, corresponding to the descriptor "neither dislike nor like". Lactic acid was the main organic acid produced during the fermentation time and low concentrations of acetic acid and ethanol were also detected. A total of 70 volatile compounds were identified. Although the physicochemical results presented in this study were interesting, more in vivo studies on the viability and health benefits of these microorganisms are needed.

The fermentation process can be used for the delivery of probiotic bacteria and for food detoxification. Probiotic growth in the fermented food medium reduces toxins in raw materials. Aflatoxins can suppress the activity of the human immune system, affect nutrient absorption, and induce liver cancer. This was evaluated by Wacoo et al. [52] in a modification of the traditional method of the production of the Kwete beverage (traditional African fermented maize). Kwete was produced by fermentation with *L. rhamnosus* yoba 2012 and *S. thermophilus* C106 at 30 ◦C for 24 h. The results showed that the beverage was stable for a month under refrigeration storage at 4 ◦C, with a mean pH of 3.9 and titratable acidity of 0.6%; the bacteria could also reduce the aflatoxins to undetectable levels during fermentation. The aflatoxins reduction is a novel approach to detoxification of this kind of beverage widely consumed in Africa.

The use of cereal mixtures for the development of fermented beverages has also been investigated. Rathore et al. [5] made comparisons of single and mixed cereal beverages fermented with different strains of LAB. The results of this study indicated that the organic acid production in mixed cereal substrates was lower than in the single cereal beverages. However, the microbial populations were similar for all substrates. These results are very interesting for future investigations on sensorial properties and consumer acceptance. Nevertheless, malt was the best substrate for microbial growth used as single or mixed beverages. Freire et al. [53] developed mixed beverages from rice and maize fermented with *L. acidophilus* LACA 4 and *L. pantarum* CCMA 0743 for 36 h at 37 ◦C and supplemented with frutooligosaccharides (FOS). The FOS were an important prebiotic for maintaining the microbial viability in high concentrations (>10<sup>7</sup> CFU/mL) during 28 days of refrigerated storage (4 ◦C). The main organic acids were lactic and acetic acid. The sensory acceptance of the beverages was good with high scores with respect to unfermented ones, indicating their high potential for the market.

Maize beverages have been shown to be a potential matrix for the growth and survival of the bacteria studied, with important changes in their composition. However, in the future, further studies focused on the identification, quantification and potential effect of bioactive compounds, as well as the modulation of intestinal microbiota, will be of great interest to elucidate the functionality of maize beverages in the human organism. A summary of the studies on fermented cereal beverages conducted to date and their results is presented in Table 2.


**Table 2.** Summary of fermented cereal beverages as potential carriers form bioactive compounds, probiotics, prebiotics delivery to de gut.

<sup>1</sup> SCFAs: short chain fatty acids.

#### *2.3. Fermented Pseudocereal Beverages*

Several studies have focused on the development of non-dairy probiotic beverages using pseudocereals as vehicles for the delivery of bioactive compounds, probiotics, and prebiotics. Pseudocereals are viable potential substrates, as they contain nutrients easily metabolized by probiotic microorganisms. They are a good source of high-quality proteins comparable to those of cereals, minerals (Ca, Cu, Fe, Mg, Mn and Zn) in higher amounts than in conventional cereals, carbohydrates, and fiber [54]. Quinoa (*Chenopodium quinoa*, L.) is the pseudocereal most widely used as a food matrix. It is the only plant-based food that has all of the essential amino acids (lysine, methionine, and threonine), trace elements and vitamins, and its protein quality to matches that of milk [55]. It can also decrease the risk of type-2 diabetes and cardiovascular diseases [56]. Furthermore, it is gluten-free, so its consumption is suitable for celiacs and people with gluten-allergy problems.

One study reported the use of two varieties of quinoa (Rosada de Huancayo and Pasankalla) as suitable food matrices for the development of fermented beverages [57]. The fermentation was carried out by the probiotics *Lactobacillus plantarum* Q823, *Lactobacillus casei* Q11, and *Lactococcus lactis* ARH74 for 6 h at 30 ◦C. After 28 days of storage (5–7 ◦C), *L. plantarum* Q823 and *L. casei* Q11 were detected at levels higher than 9 Log CFU/mL, which is a concentration above the recommended minimum for probiotic effects, with initial concentrations of 8 Log CFU/mL. Vera-Pingitore et al. [58] have previously reported that *L. plantarum* Q823 can survive during the passage through the human gastrointestinal tract. In this study, seven healthy female volunteers consumed 20 mL of quinoa-based beverage containing 9.19 Log CFU/mL on a daily basis or 7 days, and microbial counts were analyzed in feces. Levels between 5 and 7 Log CFU/mL were detected for at least 7 days after the end of the intake. Therefore, quinoa-based fermented beverages contain high amounts of protein, fiber, vitamins and minerals, with probiotics which could exert health benefits on the human gastrointestinal microbiota. However, more long-term human clinical trial studies are needed to demonstrate that these beverages have probiotic properties.

There are few documented studies that have attempted to determine the in vivo effect of fermented pseudocereal beverages on the modulation of the gut microbiome in either animal or human investigations. However, one study has evaluated the impact of several beverages based on aqueous extracts of soy and quinoa with prebiotics (FOS) and/or with probiotics (*Lactobacillus casei* Lc-01) on the human intestinal microbiota, using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) [59]. The SHIME is a five-stage sequential reactor system simulating the different parts of the gastrointestinal tract in vitro, representing the human gut microbiota [60]. The study of Bianchi et al. [59] reported that a synbiotic beverage fortified with both a probiotic and a prebiotic showed the best beneficial effect on the gut microbiota, as the oligosaccharides used were hydrocolloids, which protected the microorganism. The concentration used in the SHIME was a proportion equivalent to 8 Log CFU/mL in the beverage. *L. casei* Lc-01 of the synbiotic beverage survived the stomach and intestinal conditions and reached the colon, maintaining its functionality, and the concentration of SCFAs was stable during the in vitro gastrointestinal digestion. Furthermore, the growth of several species of *Lactobacillus* spp. and *Bifidobacterium* spp. in the colon were stimulated by the synbiotic beverage. At the same time, the growth of potential enteropathogenic bacteria such as *Clostridium* spp., enterobacteria and other pathogenic bacteria such as *Bacteroides* spp. and *Enterococcus* spp. was reduced. Another positive effect of the beverage on the gastrointestinal tract was the significant decrease in the ammonia ion production. Ammonia can stimulate the development of colon carcinogenesis, since it can affect intestinal cells, changing their morphology and intermediary metabolism by increasing DNA synthesis [61]. Therefore, this beverage based on quinoa and soybean with FOS and fermented by *L. casei* Lc-01 positively modulates the gut microbiota improving the diversity and richness of beneficial bacteria without affecting their functionality and reducing the growth of the pathogenic ones, and decreasing the production of toxic elements such as ammonia.

Some studies have investigated the use of other pseudocereals, such as chia (*Salvia hispanica*, L.), amaranth (*Amaranthus*, L.) or buckwheat (*Fagopyrum esculentum*, L.), as food carriers to develop pseudocereal probiotic beverages, obtaining interesting results. In a study on the elaboration of a beverage with the probiotic *Lactobacillus rhamnosus* GG (5–6 Log CFU/mL), using mashed buckwheat previously fermented with LAB, the results showed that the levels of the microorganism were higher than 6 Log CFU/mL after 14 days of cold storage at 6 ◦C [62]. Kocková and Valík [63] also produced beverages based on buckwheat or dark buckwheat fermented with *L. rhamnosus* GG ATCC 53,103 (6 Log CFU/mL) at 37 ◦C for 10 h. This probiotic was able to grow and metabolize buckwheat and dark buckwheat and to survive during 21 days of a refrigerated storage period at 5 ◦C, with the probiotic counts being above the minimum recommended (>7 Log CFU/mL). Kocková et al. [64] studied the use of different pseudocereals as substrates for the production of different beverages fermented with *L. rhamnosus* GG (5 Log CFU/mL) for 10 h at 37 ◦C, using amaranth flour, amaranth grain, buckwheat flour, or whole buckwheat flour. The results showed that the probiotic could grow and metabolize these pseudocereals and after 21 days of storage at 5 ◦C, the probiotic levels were higher than 6 Log CFU/mL, and thus over the limit required for probiotic food, except for the beverage with whole buckwheat flour. Another study reported that chia can be fermented by *L. plantarum* C8, and that after 24 h of fermentation, its overall characteristics were improved, such as the phenolic compound concentration and antioxidant activity [65]. Therefore, this pseudocereal could be a good matrix for the development of probiotic beverages. The main studies conducted to date on fermented pseudocereal beverages and their results are summarized in Table 3.

#### *2.4. Fermented Fruit and Vegetable Beverages*

Fruit and vegetable beverages are an excellent source of vitamins, antioxidants, minerals, and bioactives. At the same time they represent a good alternative to dairy matrices and a good choice for the entire human population, because they have hydration properties, are refreshing and have attractive flavors [21,66]. Therefore, different fruits and vegetable juices are used to develop fermented beverages in combination or alone as an alternative to fermented dairy products. The fermentation process can increase the shelf life of fruit and vegetable beverages, improving their nutritional and functional properties, with beneficial effects on health [16]. Recently, a wide variety of research has been focused on the production of fermented non-dairy synbiotic beverages, including different types of vegetables or fruits, such as blended carrot-orange juices and nectars with different inulin concentrations [67,68], pomegranate juices, and Cornelian cherry beverages using delignified wheat bran [69,70], clarified apple juice with oligofructose [71], orange juice with oligofructose [72], orange juices and hibiscus tea mixed beverage with oligofructose [73], and blended red fruit beverages (strawberry, blackberry and papaya) supplemented with three separate prebiotics: FOS, inulin and galactooligosaccharides [74]. Generally, the findings have indicated a good compatibility among prebiotic ingredients and vegetable beverage matrices. Furthermore, prebiotic supplementation can improve the viability of the different probiotic strains so that they are above the minimum concentration recommended during the beverages processing and storage and are also able to survive during gastrointestinal digestion in order to reach the colon, promoting the growth of beneficial bacteria. Furthermore, a recent randomized, controlled, triple-blinded, parallel trial study of polycystic ovarian syndrome (POCS) showed the effect of synbiotic pomegranate juice (containing inulin and three species of *Lactobacillus*) in terms of improving the testosterone level, body mass index, insulin, insulin resistance, weight, and waist circumference in POCS. However, neither group showed a significant change in the fasting blood sugar, luteinizing hormone, and follicle-stimulating hormone [75].

**Table 3.** Summary of fermented pseudocereal beverages as potential carriers for bioactive compound, probiotic, and prebiotic delivery to the gut.


Several LAB can biotransform polyphenols into phenolic compounds with an improved bioavailability and bioactivity during the fermentation time. Several studies have investigated phenolic compound biotransformation during fermentation and gastrointestinal digestion. The studies on fermented fruit or vegetable beverages have shown an improved antioxidant capacity and phenolic composition modification. Apple juice fermented with *L. plantarum* ATCC14917 at 37 ◦C improved the antioxidant capacity by increasing the quercetin, phloretin and 5-O-caffeoylquinic acid contents during 24 h of fermentation [76]. Yang et al. [77] reported an increase of the total flavonoids content as well as the antioxidant activity in fermented mixed beverages from apples, carrots, and pears during fermentation with two commercial *L. plantarum* 115 *L. plantarum* Vege Start 60. On the other hand, the biotransformation of phenolic compounds during fermentation and gastrointestinal digestion in fermented pomegranate juices ensured the survival of *L. plantarum* CECT220, *L. acidophilus* CECT903, *B. longum subsp. infantis* CECT4551, and *B. bifidum* CECT870, suggesting a prebiotic effect [78]. Furthermore, one study on a fermented tea infusion found an increase in the overall antioxidant capacity, a modification in the phenolic composition and an increase in their cellular uptake after in vitro digestion [79]. Oolong tea polyphenols have been reported to improve host health through the generation of SCFAs and modulation of the human gut microbiota, leading to potential applications for anti-obesity therapies [80]. Specifically, (-)-Epigallocatechin 3-O-(3-O-methyl) gallate showed a prebiotic effect with the modulation of gut microbiota and obesity prevention in high-fat diet-fed

mice [81,82]. Phenolic compounds are recognized as antioxidants, but some of them also exhibit antimicrobial activity. Cueva et al. [83] showed that phenolic compounds generated from probiotic metabolism (phenylpropionic, benzoic and phenylacetic) can inhibit the growth of intestinal pathogens and prevent intestinal dysbiosis.

The ability of probiotic microorganisms to metabolize phenolic compounds is known to depend on the species or strains. However, differences in the total polyphenol content and antioxidant capacity have been shown between different vegetable beverages for the same probiotic strains. These differences may be related to the variability in the phytochemical composition of the different vegetable and fruit matrices [84,85]. In addition, the matrix also has an influence on the exopolysaccharides (EPS) production during fermentation, improving the consistency and antioxidant capacity of fermented juices [86]. At the same time, the EPS also have a significant role as prebiotics and can enhance probiotic colonization in the gut. They have also been used as immunomodulatory, immunostimulatory, antidiabetic, and hypocholesterolemic agents [87,88].

Bearing in mind the nutritional importance of fruit and vegetable beverages, some of them, such as fresh prickly pear juices, present hazardous volatile components in negligible quantities. Therefore, the reduction of risky compounds by modification or decomposition during the fermentation time is an interesting strategy. Panda et al. [89] demonstrated prickly pear quality enhancement by fermentation with *Lactobacillus fermentum* ATCC 9338 for 48 h at 28 ◦C. The study demonstrates the decomposition of several risky organic compounds present in the fresh juice, such as 2-propenenitrile, 2-(acetyloxy); furfuryl alcohol; acetaldehyde; 2,2-diethyl-3-methyloxazolidine; 4h-Pyran-4-one; 3,5-dihydroxy-2-methyl; and furan.

Beyond the nutritional and physicochemical advantages, recent studies have shown that different fermented fruit and vegetable beverages also have some physiological functions. For example, some studies have shown a stable α-glucosidase inhibitory activity with an anti-hyperglycemic in vitro effect in a pumpkin beverage fermented by *L. mali* K8 [90]. Gamboa-Gómez et al. [91] also showed an anti-hyperglycemic effect with an infusion of oak leaves and fermented beverages from *Quercus convallata* and *Q. arizonica* in vitro and in vivo studies with female mice. On the other hand, blended fermented blueberry pomace by *L. rhamnosus* GG, *L. plantarum*-1, and *L. plantarum*-2 showed in in vitro hypocholesterolaemic effect. In addition, this fermented beverage exhibited an outstanding performance in terms of anti-fatigue in a mouse weight swimming experiment [92]. According to Harima-Mizusawa et al. [93], citrus beverage fermented with *L. plantarum* YIT0132 had a good effect in relieving the perennial allergic rhinitis symptoms in a double-blind, placebo-controlled trial. Other fermented beverages, such as those of tomato, feijoa, blueberry-blackberry, cactus pear, and prickly pear fruits exhibit a great in vitro anti-inflammatory capacity and help maintain the integrity of intestinal barrier [85,86,94]. However, the results are influenced by the different vegetable beverage matrices. For example, Valero-Cases et al. [94] showed the best improvement of the intestinal barrier with fermented tomato juices with respect to fermented feijoa ones. The vasorelaxant capacity was proposed for fermented jabuticaba berry beverages through an in vivo study of vascular reactivity in male Wistar rats. Theses beverages could act as an interesting cardiovascular protector [95]. Cheng et al. [96] reported an increase of SCFAs production and an improvement of the fecal microbiota community structure in vitro with blueberry pomace fermented by *L. casei* CICC20280. Wang et al. [97] showed that the consumption of fermented beverages of Changbai Mountain vegetables and fruits can reduce the Firmicutes/Bacteroidetes ratio and increase the Bacteroidales S24–7 group, Bacteroidaceae, the genus *Bacteroides*, and Prevotellaceae in a mouse model study. However, these are results from in vitro and in vivo studies with animal models, so future research in humans is required to evaluate the physiological effects on health improvement. A summary of studies conducted to date on fruit and vegetable fermented beverages and their results is shown in Table 4.


**4.**Summaryoffruitandvegetablefermentedbeveragespotentialcarriersformbioactivecompounds,probiotics,prebioticsdelivery

 lactic acid bacteria; SCFAs: short chain fatty acids; EPS: exopolysaccharides.

#### **3. Conclusions and Future Perspectives**

The studies carried out to date have provided useful information and a deeper understanding of the metabolic mechanisms of growth, probiotic viability, and the microbial biotransformation or production of bioactive compounds in fermented non-dairy beverages. Non-dairy matrices (legumes, cereals, pseudocereals, fruits, and vegetables) represent potential carriers of probiotics, prebiotics, and bioactive compounds. They are a good alternative to dairy matrices because it has been proven that the fermentation of these vegetable matrices can improve the shelf life and their safety due to the organic acids generated during the fermentation period, their nutritional and functional composition, and their digestibility. Moreover, in all the matrices reviewed, the probiotic concentrations are above the minimum recommended (>7 Log CFU/mL). Therefore, they are a good alternative to the dairy products on the market that can also be consumed by people intolerant or allergic to milk proteins, those who are hypercholesterolemic, or those who are vegetarian, among others. However, to corroborate the health benefits of fermented non-dairy beverage consumption, further in vivo research, including human clinical studies addressing matrix combinations and doses in different populations, is needed.

**Author Contributions:** Conceptualization: E.V.-C.; methodology: E.V.-C. and M.-J.F.; writing-original draft preparation: E.V.-C. and D.C.-B.; writing—review and editing: E.V.-C., D.C.-B., J.-J.P. and M.-J.F.; project administration: J.-J.P.; supervision: E.V.-C., D.C.-B. and M.-J.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** E.V.-C.: Thanks to Generalitat Valenciana and the European Social Found for the Vali+d Postdoctoral fellowship. D.C.-B.: Thanks to the Spanish-Ministry of Education, Culture and Sport for the FPU-PhD fellowship.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


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