Production of new functional coconut milk kefir with blueberry extract and microalgae: the comparison of the prebiotic potentials on lactic acid bacteria of kefir grain and biochemical characteristics.

Probiotic foods are recognized for their importance on human health. Kefir is a versatile probiotic food that can be made from non-dairy sources for vegan diet. This study evaluated the addition of microalga Haematococcus pluvialis (0.50% w/v) and blueberry Vaccinium myrtillus (0.50% w/v) extracts to compare their influence on the biochemical properties and the bacterial community of coconut milk kefir through Nanopore-based DNA sequencing. Results revealed that the V. myrtillus increased the microbial diversity in coconut milk kefir with more abundant Proteobacteria species such as Lacticaseibacillus paracasei (22%) and Lactococcus lactis (6.3%). Microalga demonstrated the opposite effect on C, making Firmicutes represent the whole of the microbiota. Biochemical analysis revealed increased fat content in the kefir samples, with the C1 registering 1.62% and the 1.07% in C2, in contrast to the control group's 0.87% fat content. The crude protein content exhibited a decrease in both samples compared to the control group (0.00% and 0.88% versus 1.07%). These findings suggest that fortifying vegan kefir with prebiotics has the potential to induce significant alterations in the kefir microbiota.

The prebiotic impacts of galactose side-chain of tamarind xyloglucan oligosaccharides on gut microbiota.

To explore the impacts of galactose side-chain on the prebiotic activity of xyloglucan oligosaccharides (XGOS), XGOS and de-galactosylated XGOS (DG-XGOS) were prepared from tamarind using an enzymatic method. The differences in structural features of XGOS and DG-XGOS were systematically analyzed. Their in vitro fermentation characteristics of human fecal microbiota were explored. These results indicated that both XGOS and DG-XGOS promoted short-chain fatty acids (SCFAs) production, decreased pH, and changed the microbiota composition of the fermentation broth. Comparatively, DG-XGOS was more effective than XGOS in producing SCFAs, inhibiting the phylum Proteobacteria prevalence, and promoting the phyla Bacteroidetes and Actinobacteria prevalence. In summary, the xyloglucan degradation products exert potential prebiotic activity. Removing the galactose side-chains further enhances oligosaccharide utilization by fecal microbiota, offering a valuable approach to improve the biological efficacy of oligosaccharides.

Optimization of Pectin Extraction from Melon Peel as a New Source of Pectin and Pectin Hydrolysate with Prebiotic Potential.

Food wastes have a large number of functional ingredients that have potential for valorization. Melon peels are increasingly produced as waste in food industries in Thailand. This study aimed to optimize pectin extraction conditions from melon peel for its prebiotic potential. Optimization was conducted using a response surface methodology and Box-Behnken experimental design. An analysis of variance indicated a significant interaction between the extraction conditions on extraction yield and degree of esterification (DE). These include pH and solvent-to-sample ratio. The conditions for the extraction of pectin with low DE (LDP), medium DE (MDP) and high DE (HDP) were optimized. Pectin hydrolysate from LDP, MDP and HDP was prepared by enzymatic hydrolysis into LPEH, MPEH and HPEH, respectively. LDP, MDP, HDP, LPEH, MPEH and HPEH were compared for their efficiency in terms of the growth of three probiotic strains, namely Lactobacillus plantarum TISTR 877, Lactobacillus casei TISTR 390 and Enterococcus faecium TISTR 1027. Among the samples tested, HPEH showed the highest ability as a carbon source to promote the growth and prebiotic activity score for these three probiotic strains. This study suggests that melon peel waste from agro-industry can be a novel source for prebiotic production.

Determination of prebiotic activity and probiotic encapsulation ability of inulin type fructans obtained from Inula helenium roots.

Inulin, a prebiotic utilized in the food and pharmaceutical industries, promotes the growth of beneficial bacteria in the colon, thereby enhancing human health. Although inulin is commercially produced from chicory and artichoke, Inula helenium roots offer a high potential for inulin production. The aim of this study is to investigate the prebiotic activity of inulin (inulin-P) from I. helenium roots on Lactobacillus rhamnosus, as well as its ability to produce synbiotic microcapsules and the effects on probiotic viability during freeze-drying, in vitro gastrointestinal (GI) digestion, and storage. First, the effect of inulin-P on L. rhamnosus viability and short-chain fatty acid (SCFA) production was compared to other commonly utilized prebiotics. The findings revealed that inulin-P remarkably promoted the growth and SCFA yield of L. rhamnosus for 48 h of fermentation and 28 days of storage. Then, L. rhamnosus was encapsulated with inulin-P and commercial inulin to compare its survival throughout storage and the GI tract. Inulin-P microcapsules outperformed in terms of viability during storage (7.98 log CFU/g after 30 days at 4°C). Furthermore, inulin-P microcapsules were heat-resistant and protected L. rhamnosus from GI conditions, resulting in a high survival rate (89.52%) following large intestine simulation, which is ideal for increasing customer benefits. Additionally, inulin-P microcapsules exhibited similar physical characteristics to commercial inulin. Consequently, this study revealed that inulin-P, which is easy to produce, low-cost, and has industrial application potential, could be used as a good carrier for the synbiotic encapsulation of L. rhamnosus. PRACTICAL APPLICATION: Inulin is a prebiotic that promotes the activity and growth of beneficial bacteria in the human gut. Although commercial inulin is currently produced from chicory root and artichoke, Inula helenium root is a potential raw material for inulin production. In this study, inulin was produced from I. helenium roots with a low-cost and easy production method, and it was determined that this inulin was an effective carrier in the synbiotic encapsulation of L. rhamnosus. This inulin exhibits superior prebiotic activity and encapsulation efficiency compared to commercial inulins like Orafti® GR and HPX and can be easily integrated into industrial production.

Exploring prebiotic properties and its probiotic potential of new formulations of soy milk-derived beverages.

Introduction: The food and beverage industry has shown a growing interest in plant-based beverages as alternatives to traditional milk consumption. Soy milk is derived from soy beans and contains proteins, isoflavones, soy bean oligosaccharides, and saponins, among other ingredients. Because of its high nutritive value and versatility, soy milk has gained a lot of attention as a functional food. Methods: The present work aims to explore the prebiotic properties and gastrointestinal tolerance potential of new formulations of soy milk-derived drinks to be fermented with riboflavin-producing probiotic Lactiplantibacillus plantarum MTCC (Microbial Type Culture Collection and Gene Bank) 25432, Lactiplantibacillus plantarum MTCC 25433, and Lactobacillus acidophilus NCIM (National Collection of Industrial Microorganisms) 2902 strains. Results and discussion: The soy milk co-fermented beverage showed highest PAS (1.24 ± 0.02) followed by soy milk beverages fermented with L. plantarum MTCC 25433 (0.753 ± 0.0) when compared to the commercial prebiotic raffinose (1.29 ± 0.01). The findings of this study suggested that the soy milk beverages exhibited potent prebiotic activity, having the ability to support the growth of probiotics, and the potential to raise the content of several bioactive substances. The higher prebiotics activity score showed that the higher the growth rate of probiotics microorganism, the lower the growth of pathogen. For acidic tolerance, all fermented soy milk managed to meet the minimal requirement of 106 viable probiotic cells per milliliter at pH 2 (8.13, 8.26, 8.30, and 8.45 logs CFU/mL, respectively) and pH 3.5 (8.11, 8.07, 8.39, and 9.01 log CFU/mL, respectively). The survival rate of soy milk LAB isolates on bile for 3 h ranged from 84.64 to 89.60%. The study concluded that lactobacilli could thrive in gastrointestinal tract. The sensory evaluation scores for body and texture, color, flavor, and overall acceptability showed a significant difference (p < 0.05) between the fermented probiotic soy milk and control samples. Soy milk fermented with a combination of L. plantarum MTCC 25432 & MTCC 25433 demonstrated the highest acceptability with the least amount of beany flavor. The findings of the study suggest soy milk's potential in plant-based beverage market.

Encapsulated Mn-Saturated Lactoferrin as a Safe Source of Manganese Ions for Restoring Probiotic Lactobacillus plantarum.

The growth of Lactobacillus plantarum, a member of the Lactobacillus genus, which plays a crucial role in the bacterial microbiome of the gut, is significantly influenced by manganese ions. They can be safely delivered to the intestines by exploiting the chelating abilities of lactoferrin. The aim of this work was to encapsulate lactoferrin saturated with manganese ions (MnLf) in a system based on the Eudragit® RS polymer to protect protein from degradation and manganese release in the gastric environment. The entrapment efficiency was satisfactory, reaching about 95%, and most importantly, manganese ions were not released during microparticles (MPs) formation. The release profile of the protein from the freshly prepared MPs was sustained, with less than 15% of the protein released within the first hour. To achieve similar protein release efficiency, freeze-drying was carried out in the presence of 10% (w/v) mannitol as a cryoprotectant for MPs frozen at -20 °C. MPs with encapsulated MnLf exhibited prebiotic activity towards Lactobacillus plantarum. More importantly, the presence of equivalent levels of manganese ions in free form in the medium, as well as chelating by lactoferrin encapsulated in MPs, had a similar impact on stimulating bacterial growth. This indicates that the bioavailability of manganese ions in our prepared system is very good.

Valorization of rice straw and vascular aquatic weeds for sustainable prebiotic hemicellulosic autohydrolysate production: Extraction, characterization and fermentability.

This study describes the extraction and characterization of the hemicellulosic autohydrolysates (HAHs) derived from rice straw (RS) and vascular aquatic weeds like Typha angustifolia (TA) and Ceretophyllum demersum (CD). It further explores their capacity to sustain the proliferation of selected lactic acid bacteria (i.e., prebiotic activity) isolated from milk samples. To fractionate HAH from RS, TA and CD hot water extraction (HWE) method was used and RS, TA, and CD biomasses yielded 6.8, 4.99 and 2.98% of HAH corresponding to the hemicellulose extraction efficiencies of 26.15 ± 0.8%, 23.76 ± 0.6%, and 18.62 ± 0.4% respectively. The chemical characterization of HAH concentrates through HPLC showed that they comprised galactose, arabinose, xylose and glucose. The total phenol content of the RS, TA and CD-derived HAH concentrates were 37.53, 56.78 and 48.08 mg GAE/g. The five lactic acid bacteria (LAB) isolates Q1B, Q2A, Q3B, G1C and G2B selected for prebiotic activity assays generated mixed responses with the highest growth in RS-HAH for Q2A and the least in TA-HAH for Q3B. Further, the isolates Q2A, Q3B, G1C, and G2B, which showed the highest growth performance, were identified through MALDI-TOF and 16S rRNA sequencing as Lactobacillus brevis. All the tested LAB isolates showed diauxic growth in crude HAH preparations to maximize the utilization of carbon resources for their proliferation. This suggests that the selected LAB isolates are efficient degraders of hemicellulosic sugars. This paves the way for the valorization of lignocellulosic biomass to produce prebiotic hemicellulosic autohydrolysate and consequently enhances environmental sustainability by improving resource efficiency.

Paotianxiong polysaccharides potential prebiotics: Structural analysis and prebiotic properties.

Paotianxiong (PTX) is a processing product of Aconitum carmichaelii Debx., often used as a tonic food daily. However, the structure and activity of the polysaccharide component that plays a major role still need to be determined. In our work, two new polysaccharides were purified from PTX and named PTXP-1 and PTXP-2. Structural analysis showed that PTXP-1 is a glucan with a molecular weight of 915 Da and a structure of 4)-α-D-Glcp-(1 â†’ as the main chain. PTXP-2 is a glucose arabinoglycan with 4)-α-D-Glcp-(1 â†’ as the main chain, containing 8 glycosidic bonds attached, and a molecular weight of 57.9KDa. In vitro probiotic experiments demonstrated that PTXP-1 could significantly promote probiotic growth and acid production. In vivo experiments demonstrated that both PTXP-1 and PTXP-2 exhibited significant effectiveness in promoting the growth of intestinal probiotics. These findings help expand the application of polysaccharide components extracted from tonic herbs as functional food ingredients.

Curdlan-polyphenol complexes prepared by pH-driven effectively enhanced their physicochemical stability, antioxidant and prebiotic activities.

In this study, the curdlan-polyphenol complexes were constructed by a pH-driven method. The interaction between curdlan and various hydrophobic polyphenols (curcumin, quercetin, and chlorogenic acid) was investigated. Curdlan could self-assemble into particles for loading polyphenols through hydrogen bonding and hydrophobic interactions. The three polyphenols were embedded in curdlan in an amorphous state. The curdlan-curcumin complex showed the lowest viscoelasticity but exhibited the highest curcumin loading ability (34.04 ± 1.73 mg/g). However, the curdlan-chlorogenic acid complex emerged the opposite trend, indicating that the loading capacity was associated with the hydrophobicity of polyphenols. The antioxidant activity of curdlan significantly increased after combining with polyphenols, which could be maintained during in vitro simulated gastrointestinal digestion. In particular, the curdlan-quercetin complex exhibited the highest antioxidant activity and short-chain fatty acid concentration, which could influence gut microbiota composition by promoting the proliferation of Prevotella and inhibiting the growth of Escherichia_Shigella. In conclusion, the curdlan-polyphenol complexes prepared by an alcohol-free pH-driven method could effectively enhance the gastrointestinal stability of polyphenols as well as increase the antioxidant and prebiotic activities of curdlan, which could be applied as a functional ingredient to improve gut health.

Potential prebiotic and antibacterial activities of fucoidan from Laminaria japonica.

Fucoidan from Laminaria japonica became sterilized with an autoclave and ultraviolet (UV) radiation. Potential prebiotic and antibacterial activities of sterilized fucoidans (SF) were the subject of investigation. Molecular weight, monosaccharide composition, FTIR, and NMR spectra of SF underwent evaluations to elucidate the relationship between the structure and activities of SF. The growth of Lactobacillus rhamnosus GG and L. acidophilus with autoclave sterilized fucoidan (ASF) and the growth of L. plantarum, L. gasseri, L. paracasei, and L. reuteri with UV sterilized fucoidan (USF) increased significantly. Also, fucoidan was vastly more effective than fructooligosaccharides in improving the growth of L. gasseri, L. reuteri, and L. paracasei. The growth of Escherichia coli and Bacillus cereus decreased at each SF concentration. ASF was more effective against E. coli, B. cereus, and Staphylococcus aureus than the USF efficiency. However, USF exhibited more inhibitory effects on the growth of Enterobacteriaceae compared to the ASF efficiency. When comparing the ASF and USF, autoclave caused a considerable decrease in molecular weight and uronic acid content, increased fucose and galactose, and made no significant changes in NMR spectra. Fucoidan effectively promoted probiotic bacterial growth and reduced pathogenic outbreaks in the medium. Therefore, it can occur as a new algal prebiotic and antibacterial agent.

Simultaneously efficient dissolution and structural modification of chrysanthemum pectin: Targeting at proliferation of Bacteroides.

Pectic polysaccharide is a bioactive ingredient in Chrysanthemum morifolium Ramat. 'Hangbaiju' (CMH), but the high proportion of HG domain limited its use as a prebiotic. In this study, hot water, cellulase-assisted, medium-temperature alkali, and deep eutectic solvent extraction strategies were firstly used to extract pectin from CMH (CMHP). CMHP obtained by cellulase-assisted extraction had high purity and strong ability to promote the proliferation of Bacteroides and mixed probiotics. However, 4 extraction strategies led to general high proportion of HG domain in CMHPs. To further enhance the dissolution and prebiotic potential of CMHP, pectinase was used alone and combined with cellulase. The key factor for the optimal extraction was enzymolysis by cellulase and pectinase in a mass ratio of 3:1 at 1 % (w/w) dosage. The optimal CMHP had high yield (15.15 %), high content of total sugar, and Bacteroides proliferative activity superior to inulin, which was probably due to the cooperation of complex enzyme on the destruction of cell wall and pectin structural modification for raised RG-I domain (80.30 %) with relatively high degree of branching and moderate HG domain. This study provided a green strategy for extraction of RG-I enriched prebiotic pectin from plants.

Banana Peel (Musa ABB cv. Nam Wa Mali-Ong) as a Source of Value-Adding Components and the Functional Properties of Its Bioactive Ingredients.

Banana peel (BP) is the primary by-product generated during banana processing which causes numerous environmental issues. This study examines the physical attributes, proximate analysis, glycoarray profiling, antioxidant abilities, and prebiotic activity of BP. The analysis demonstrated that carbohydrates constituted the primary components of BP and the glycoarray profiling indicated that BP contains multiple pectin and hemicellulose structures. BP also contained phenolic compounds, including (+)-catechin and gallic acid, flavonoid compounds, and antioxidant activities. BP demonstrated prebiotic effects by promoting the proliferation of advantageous gut bacteria while inhibiting the growth of harmful bacteria. The prebiotic index scores demonstrated that BP exhibited a greater capacity to promote the growth of beneficial bacteria in comparison to regular sugar. The study demonstrated the potential of the BP as a valuable source of dietary fibre, bioactive compounds, and prebiotics. These components have beneficial characteristics and can be utilised in the production of food, feed additives, and functional food.

In vitro human gut microbiota fermentation of litchi pulp polysaccharides as affected by Lactobacillus pre-treatment.

In this study, litchi polysaccharides were obtained from unfermented or fermented pulp by Lactobacillus fermentum (denoted as LP and LPF, respectively). The differences between LP and LPF in the colonic fermentation characteristics and modulatory of gut microbiota growth and metabolism were investigated with an in vitro fecal fermentation model. Results revealed that the strategies of gut bacteria metabolizing LP and LPF were different and LPF with lower molecular weight (Mw) was readily utilized by bacteria. The monosaccharide utilization sequence of each polysaccharide was Ara > Gla > GalA > GlcA ≈ Glu ≈ Man. Moreover, LPF promoted stronger proliferation of Bifidobacterium, Megamonas, Prevotella, and Bacteroides and higher SCFAs production (especially acetic and butyric acids) than LP. Correlation analysis further revealed that Mw could represent an essential structural feature of polysaccharides associated with its microbiota-regulating effect. Overall, Lactobacillus fermentation pre-treatment of litchi pulp promoted the fermentation characteristics and prebiotic activities of its polysaccharide.

The Regulatory Effect of Huangshui Polysaccharides on Intestinal Microbiota and Metabolites during In Vitro Fermentation.

Huangshui polysaccharides (HSPs) have attracted extensive attention recently for their biological activity and physicochemical property. This research investigated the extraction, structural characterization, and prebiotic activity of three different HSPs (HSP40-0, HSP60-0, and HSP80-0) in vitro to reveal the scientific support for the high-value utilization of Huangshui. HSPs were heteropolysaccharide with diverse structures and surface morphologies. Comprehensive analysis was conducted through 16S rRNA gene sequencing and metabolite profiling techniques, and results showed that HSPs had different potentials to regulate the gut microbiota due to their different structures; for instance, both HSP40-0 and HSP80-0 could notably increase the relative abundance of Bacteroidota, whereas HSP60-0 could increase the relative abundance of Phascolarctobacterium. In addition, HSPs upregulated beneficial differential metabolites, especially short-chain fatty acids (SCFAs). Fermentation products containing these metabolites exhibited anti-inflammatory effects on LPS-treated Caco-2 cells. This study will provide reference for exploring the relationship between the natural polysaccharide structure and the prebiotic activity and widen the application of Huangshui.

Characterization of Various Noncovalent Polyphenol-Starch Complexes and Their Prebiotic Activities during In Vitro Digestion and Fermentation.

This study explores the structural characterization of six noncovalent polyphenol-starch complexes and their prebiotic activities during in vitro digestion and fermentation. Ferulic acid, caffeic acid, gallic acid, isoquercetin, astragalin, and hyperin were complexed with sweet potato starch (SPS). The polyphenols exhibited high binding capacity (>70%) with SPS. A partial release of flavonoids from the complexes was observed via in vitro digestion, while the phenolic acids remained tightly bound. Molecular dynamics (MD) simulation revealed that polyphenols altered the spatial configuration of polysaccharides and intramolecular hydrogen bonds formed. Additionally, polyphenol-SPS complexes exerted inhibitory effects on starch digestion compared to gelatinized SPS, owing to the increase in resistant starch fraction. It revealed that the different complexes stimulated the growth of Lactobacillus rhamnosus and Bifidobacterium bifidum, while inhibiting the growth of Escherichia coli. Moreover, in vitro fermentation experiments revealed that complexes were utilized by the gut microbiota, resulting in the production of short-chain fatty acids and a decrease in pH. In addition, the polyphenol-SPS complexes altered the composition of gut microbiota by promoting the growth of beneficial bacteria and decreasing pathogenic bacteria. Polyphenol-SPS complexes exhibit great potential for use as a prebiotic and exert dual beneficial effects on gut microbiota.

Eat your beets: Conversion of polysaccharides into oligosaccharides for enhanced bioactivity.

Bioactive oligosaccharides with the potential to improve human health, especially in modulating gut microbiota via prebiotic activity, are available from few natural sources. This work uses polysaccharide oxidative cleavage to generate oligosaccharides from beet pulp, an agroindustry by-product. A scalable membrane filtration approach was applied to purify the oligosaccharides for subsequent in vitro functional testing. The combined use of nano-LC/Chip Q-TOF MS and UHPLC/QqQ MS allowed the evaluation of the oligosaccharide profile and their monosaccharide complexity. A final product containing roughly 40 g of oligosaccharide was obtained from 475 g of carbohydrates. Microbiological bioactivity assays indicated that the product obtained herein stimulated desirable commensal gut bacteria. This rapid, reproducible, and scalable method represents a breakthrough in the food industry for generating potential prebiotic ingredients from common plant by-products at scale. INDUSTRIAL RELEVANCE: This work proposes an innovative technology based on polysaccharide oxidative cleavage and multi-stage membrane purification to produce potential prebiotic oligosaccharides from renewable sources. It also provides critical information to evidence the prebiotic potential of the newly generated oligosaccharides on the growth promotion ability of representative probiotic strains of bifidobacteria and lactobacilli.

The Prebiotic Activity of a Novel Polysaccharide Extracted from Huangshui by Fecal Fermentation In Vitro.

A novel polysaccharide, HSP80-2, with an average molecular weight of 13.8 kDa, was successfully isolated by the gradient ethanol precipitation (GEP) method from Huangshui (HS), the by-product of Chinese Baijiu. It was mainly composed of arabinose, xylose, and glucose with a molar ratio of 4.0:3.1:2.4, which was completely different from the previous reported HS polysaccharides (HSPs). Morphological observations indicated that HSP80-2 exhibited a smooth but uneven fragmented structure. Moreover, HSP80-2 exerted prebiotic activity evaluated by in vitro fermentation. Specifically, HSP80-2 was utilized by gut microbiota, and significantly regulated the composition and abundance of beneficial microbiota such as Phascolarctobacterium, Parabacteroides, and Bacteroides. Notably, KEGG pathway enrichment analysis illustrated that HSP80-2 enriched the pathways of amino sugar and nucleotide sugar metabolism (Ko00520), galactose metabolism (ko00052), and the citrate cycle (TCA cycle) (ko00020). Meanwhile, the contents of short-chain fatty acids (SCFAs) mainly including acetic acid, propionic acid, and butyric acid in the HSP80-2 group were remarkably increased, which was closely associated with the growth of Lachnoclostridium and Parabacteroides. These results showed that HSP80-2 might be used as a potential functional factor to promote human gut health, which further extended the high value utilization of HS.

Evaluation of Prebiotic Activity of Stellariae Radix Polysaccharides and Its Effects on Gut Microbiota.

This study aims to evaluate the prebiotic potential of polysaccharides derived from Stellariae Radix (SRPs) and explore their influence on the gut microbiota composition in mice. Lactobacillus acidophilus and Bifidobacterium longum were cultivated in an MRS medium, while their growth kinetics, clumping behavior, sugar utilization, pH variation, growth density, and probiotic index were meticulously monitored. Additionally, the impact of crude Stellariae Radix polysaccharides (CSRP) on the richness and diversity of gut microbiota in mice was assessed via 16S rDNA sequencing. The results demonstrated the remarkable ability of CSRPs to stimulate the proliferation of Lactobacillus acidophilus and Bifidobacterium longum. Moreover, the oral administration of CSRPs to mice led to a noticeable increase in beneficial bacterial populations and a concurrent decrease in detrimental bacterial populations within the intestinal flora. These findings provided an initial validation of CSRPs as a promising agent in maintaining the equilibrium of gut microbiota in mice, thereby offering a substantial theoretical foundation for developing Stellariae Radix as a prebiotic ingredient in various applications, including food, healthcare products, and animal feed. Furthermore, this study presented novel insights for the exploration and utilization of Stellariae Radix resources.

Isolation and structural elucidation of prebiotic oligosaccharides from Ziziphi Spinosae Semen.

Six oligosaccharides were discovered and isolated for the first time from Ziziphi Spinosae Semen. On the basis of spectroscopic analysis, their structures were determined to be verbascose (1), verbascotetraose (2), stachyose (3), manninotriose (4), raffinose (5), and melibiose (6). The prebiotic effect of the oligosaccharide fraction was assayed by eight gut bacterial growth in vitro, revealing a significant increase in cell density, up to 4-fold, for Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus johnsonii. The impact of six oligosaccharides with different degrees of polymerization (DPs) and structures on the growth of Lactobacillus acidophilus was evaluated. As a result, stachyose and raffinose demonstrated superior support for bacterial growth compared to the other oligosaccharides. This study explored the structure-activity relationship of raffinose family oligosaccharides (RFOs) and showed that the more the monosaccharide type, the more supportive the gut bacteria growth when oligosaccharides have the same molecular weight.