Selecting a preculture strategy for improving biomass and astaxanthin productivity of Chromochloris zofingiensis.

Chromochloris zofingiensis is a potential source of natural astaxanthin; however, its rapid growth and astaxanthin enrichment cannot be achieved simultaneously. This study established autotrophic, mixotrophic, and heterotrophic preculture patterns to assess their ameliorative effect on the C. zofingiensis heterotrophic growth state. In comparison, mixotrophic preculture (MP) exhibited the best improving effect on heterotrophic biomass concentration of C. zofingiensis (up to 121.5 g L-1) in a 20 L fermenter, reaching the global leading level. The astaxanthin productivity achieved 111 mg L-1 day-1, 7.4-fold higher than the best record. The transcriptome and 13C tracer-based metabolic flux analysis were used for mechanism inquiry. The results revealed that MP promoted carotenoid and lipid synthesis, and supported synthesis preference of low unsaturated fatty acids represented by C18:1 and C16:0. The MP group maintained the best astaxanthin productivity via mastering the balance between increasing glucose metabolism and inhibition of carotenoid synthesis. The MP strategy optimized the physiological state of C. zofingiensis and realized its heterotrophic high-density growth for an excellent astaxanthin yield on a pilot scale. This strategy exhibits great application potential in the microalgae-related industry. KEY POINTS: • Preculture strategies changed carbon flux and gene expression in C. zofingiensis • C. zofingiensis realized a high-density culture with MP and fed-batch culture (FBC) • Astaxanthin productivity achieved 0.111 g L-1 day-1 with MP and FBC.

Technological readiness of commercial microalgae species for foods.

Microalgae have great potential as a future source to meet the increasing global demand for foods. Several microalgae are permitted as safety sources in different countries and regions, and processed as commercial products. However, edible safety, economic feasibility, and acceptable taste are the main challenges for microalgal application in the food industry. Overcome such challenges by developing technology accelerates transition of microalgae into sustainable and nutritious diets. In this review, edible safety of Spirulina, Chlamydomonas reinhardtii, Chlorella, Haematococcus pluvialis, Dunaliella salina, Schizochytrium and Nannochloropsis is introduced, and health benefits of microalgae-derived carotenoids, amino acids, and fatty acids are discussed. Technologies of adaptive laboratory evolution, kinetic model, bioreactor design and genetic engineering are proposed to improve the organoleptic traits and economic feasibility of microalgae. Then, current technologies of decoloration and de-fishy are summarized to provide options for processing. Novel technologies of extrusion cooking, delivery systems, and 3D bioprinting are suggested to improve food quality. The production costs, biomass values, and markets of microalgal products are analyzed to reveal the economic feasibility of microalgal production. Finally, challenges and future perspectives are proposed. Social acceptance is the major limitation of microalgae-derived foods, and further efforts are required toward the improvement of processing technology.

Fucoxanthin from marine microalgae: A promising bioactive compound for industrial production and food application.

Fucoxanthin attracts increasing attentions due to its potential health benefits, which has been exploited in several food commodities. However, fucoxanthin available for industrial application is mainly derived from macroalgae, and is not yet sufficiently cost-effective compared with microalgae. This review focuses on the strategies to improve fucoxanthin productivity and approaches to reduce downstream costs in microalgal production. Here we comprehensively and critically discuss ways and methods to increase the cell growth rate and fucoxanthin content of marine microalgae, including strain screening, condition optimization, design of culture mode, metabolic and genetic engineering, and scale-up production of fucoxanthin. The approaches in downstream processes provide promising alternatives for fucoxanthin production from marine microalgae. Besides, this review summarizes fucoxanthin improvements in solubility and bioavailability by delivery system of emulsion, nanoparticle, and hydrogel, and discusses fucoxanthin metabolism with gut microbes. Fucoxanthin production from marine microalgae possesses numerous advantages in environmental sustainability and final profits to meet incremental global market demands of fucoxanthin. Strategies of adaptive evolution, multi-stage cultivation, and bioreactor improvements have tremendous potentials to improve economic viability of the production. Moreover, fucoxanthin is promising as the microbiota-targeted ingredient, and nanoparticles can protect fucoxanthin from external environmental factors for improving the solubility and bioavailability.

Butyrogenic effect of galactosyl and mannosyl carbohydrates and their regulation on piglet intestinal microbiota.

Diarrhea is a global problem that causes economic losses in the pig industry. There is a growing attention on finding new alternatives to antibiotics to solve this problem. Hence, this study aimed to compare the prebiotic activity of low-molecular-weight hydrolyzed guar gum (GMPS) with commercial manno-oligosaccharide (MOS) and galacto-oligosaccharide (GOS). We further identified their combined effects along with probiotic Clostridium butyricum on regulating the intestinal microbiota of diarrheal piglet by in vitro fermentation. All the tested non-digestible carbohydrates (NDCs) showed favorable short-chain fatty acid-producing activity, and GOS and GMPS showed the highest production of lactate and butyrate, respectively. After 48 h of fermentation, the greatest enhancement in the abundance of Clostridium sensu stricto 1 was observed with the combination of GMPS and C. butyricum. Notably, all the selected NDCs significantly decreased the abundances of pathogenic bacteria genera Escherichia-Shigella and Fusobacterium and reduced the production of potentially toxic metabolites, including ammonia nitrogen, indole, and skatole. These findings demonstrated that by associating with the chemical structure, GMPS exhibited butyrogenic effects in stimulating the proliferation of C. butyricum. Thus, our results provided a theoretical foundation for further application of galactosyl and mannosyl NDCs in the livestock industry. KEY POINTS: • Galactosyl and mannosyl NDCs showed selective prebiotic effects. • GMPS, GOS, and MOS reduced pathogenic bacteria and toxic metabolites production. • GMPS specifically enhanced the Clostridium sensu stricto 1 and butyrate production.

Microalgae-Derived Pigments for the Food Industry.

In the food industry, manufacturers and customers have paid more attention to natural pigments instead of the synthetic counterparts for their excellent coloring ability and healthy properties. Microalgae are proven as one of the major photosynthesizers of naturally derived commercial pigments, gaining higher value in the global food pigment market. Microalgae-derived pigments, especially chlorophylls, carotenoids and phycobiliproteins, have unique colors and molecular structures, respectively, and show different physiological activities and health effects in the human body. This review provides recent updates on characteristics, application fields, stability in production and extraction processes of chlorophylls, carotenoids and phycobiliproteins to standardize and analyze their commercial production from microalgae. Potential food commodities for the pigment as eco-friendly colorants, nutraceuticals, and antioxidants are summarized for the target products. Then, recent cultivation strategies, metabolic and genomic designs are presented for high pigment productivity. Technical bottlenecks of downstream processing are discussed for improved stability and bioaccessibility during production. The production strategies of microalgal pigments have been exploited to varying degrees, with some already being applied at scale while others remain at the laboratory level. Finally, some factors affecting their global market value and future prospects are proposed. The microalgae-derived pigments have great potential in the food industry due to their high nutritional value and competitive production cost.

Clostridium butyricum Prevents Dysbiosis and the Rise in Blood Pressure in Spontaneously Hypertensive Rats.

Hypertension is accompanied by dysbiosis and a decrease in the relative abundance of short-chain fatty acid (SCFA)-producing bacteria. However, there is no report to examine the role of C. butyricum in blood pressure regulation. We hypothesized that a decrease in the relative abundance of SCFA-producing bacteria in the gut was the cause of spontaneously hypertensive rats (SHR)-induced hypertension. C. butyricum and captopril were used to treat adult SHR for six weeks. C. butyricum modulated SHR-induced dysbiosis and significantly reduced systolic blood pressure (SBP) in SHR (p < 0.01). A 16S rRNA analysis determined changes in the relative abundance of the mainly SCFA-producing bacteria Akkermansia muciniphila, Lactobacillus amylovorus, and Agthobacter rectalis, which increased significantly. Total SCFAs, and particularly butyrate concentrations, in the SHR cecum and plasma were reduced (p < 0.05), while C. butyricum prevented this effect. Likewise, we supplemented SHR with butyrate for six weeks. We analyzed the flora composition, cecum SCFA concentration, and inflammatory response. The results showed that butyrate prevented SHR-induced hypertension and inflammation, and the decline of cecum SCFA concentrations (p < 0.05). This research revealed that increasing cecum butyrate concentrations by probiotics, or direct butyrate supplementation, prevented the adverse effects of SHR on intestinal flora, vascular, and blood pressure.

Genome analysis and 2'-fucosyllactose utilization characteristics of a new Akkermansia muciniphila strain isolated from mice feces.

Akkermansia muciniphila is considered to be a next-generation probiotic, and closely related to host metabolism and immune response. Compared with other probiotics, little is known about its genomic analysis. Therefore, further researches about isolating more A. muciniphila strains and exploring functional genes are needed. In the present study, a new strain isolated from mice feces was identified as A. muciniphila (MucX). Whole-genome sequencing and annotation revealed that MucX possesses key genes necessary for human milk oligosaccharides (HMO) utilization, including α-L-fucosidases, ß-galactosidases, exo-α-sialidases, and ß-acetylhexosaminidases. The complete metabolic pathways for γ-aminobutyric acid and squalene and genes encoding functional proteins, such as the outer membrane protein Amuc_1100, were annotated in the MucX genome. Comparative genome analysis was used to identify functional genes unique to MucX compared to six other A. muciniphila strains. Results showed MucX genome possesses unique genes, including sugar transporters and transferases. Single-strain incubation revealed faster utilization of 2'-fucosyllactose (2'-FL), galacto-oligosaccharides, and lactose by MucX than by A. muciniphila DSM 22959. This study isolated and identified an A. muciniphila strain that can utilize 2'-FL, and expolored the genes related to HMO utilization and special metabolites, which provided a theoretical basis for the further excavation of A. muciniphila function and the compound application with fucosylated oligosaccharides.

Complete-genome sequence and in vitro probiotic characteristics analysis of Bifidobacterium pseudolongum YY-26.

AIMS: The aim was to isolate a neotype bifidobacteria strain and evaluate its in vitro probiotic potential. METHODS AND RESULTS: Bifidobacterium pseudolongum YY-26 (CGMCC 24310) was isolated from faeces of mice treated with low-molecular-weight hydrolyzed guar gum (GMPS) and identified based on 16S rRNA sequence and genome sequence. Whole-genome sequencing obtained using PacBio's single-molecular and Illumina's paired-end sequencing technology. A genome of 2.1 Mb in length, with 1877 predicted protein-coding sequences was obtained. Carbohydrate-Activity enZyme analysis revealed that YY-26 encodes 66 enzymes related to carbohydrate metabolism. Whole genome sequence analysis revealed the typical probiotic characteristics of YY-26, including safety in genetic level and ability to produce beneficial metabolites and extracellular polysaccharides. Ability of extensive carbon source utilization and short-chain fatty acid production was observed with single YY-26 cultivation. Considerable acetic acids and lactic acids were determined in GMPS utilization. YY-26 showed tolerance to simulated gastrointestinal tract and displayed appreciable antioxidant activity of free radical scavenging. CONCLUSIONS: B. pseudolongum YY-26 was identified with numerous probiotic-associated genes and its probiotic characteristics were verified in vitro. SIGNIFICANCE AND IMPACT OF STUDY: This study supplemented with limited publicly information regarding the genomes of B. pseudolongum strains and revealed the probiotic potential of YY-26.

Response mechanism of Vibrio parahaemolyticus at high pressure revealed by transcriptomic analysis.

Vibrio parahaemolyticus is a common pathogen in aquatic products, such as shellfishes. Laboratory-based simulated studies demonstrated that V. parahaemolyticus can tolerate high hydrostatic pressure (HHP) up to 20 MPa. However, the molecular mechanisms of high-pressure adaptation remain unclear. Herein, we analyzed the physiological changes and transcriptomic responses of V. parahaemolyticus ATCC 17,802 under HHP conditions to determine the possible survival mechanisms. Under HHP conditions, the morphology of V. parahaemolyticus was notably changed exhibiting the coccoid microbial cells. The transcriptome analysis revealed that there were 795 differentially expressed genes (DEGs) under the 20 MPa condition, including 406 upregulated DEGs and 389 downregulated DEGs. Most of the downregulated DEGs encoded proteins related to energy metabolism, such as citrate synthase (gltA), pyruvate kinase (pyk), and glyceraldehyde-3-phosphate dehydrogenase (gapA). Many of the upregulated DEGs encoded proteins related to adhesion and virulence factors, such as RNA polymerase σ factor (rpoE), L-threonine 3-dehydrogenase, and bacterial nucleotide signal c-di-GMP (WU75_RS02745 and WU75_RS07185). In our proposed mechanism model, V. parahaemolyticus responds to HHP stress through RNA polymerase σ factor RpoE. These findings indicate that V. parahaemolyticus cells may adopt a complex adaptation strategy to cope with HHP stress. KEY POINTS: •The transcriptomic response of Vibrio parahaemolyticus under HHP conditions was studied for the first time. •V. parahaemolyticus may adopt a complex adaptation strategy to cope with HHP stress. •ToxRS and RpoE played an important role in sensing and responding the HHP signal.

Regulation of Virulence Factors Expression During the Intestinal Colonization of Vibrio parahaemolyticus.

Colonization and adhesion are the key steps for Vibrio parahaemolyticus to infect human body and cause seafood poisoning. However, at present, there is a lack of systematic review on the regulation of virulence factors expression during the intestinal colonization of V. parahaemolyticus. This review aims to describe the virulence factors associated with the colonization and adhesion of V. parahaemolyticus (multivalent adhesion molecule 7, enolase secretion, use of flagella, biofilm formation, and the action of secretion systems) and focuses on the aspects that affect these processes in V. parahaemolyticus, including secretion systems, quorum sensing (QS), and the human gastrointestinal tract. V. parahaemolyticus regulates the expression of virulence factors by forming a virulence regulation network through QS and the core regulator, ToxR, which contributes to the early colonization of the pathogen. In the virulence regulation network, the secretion systems, type III and type VI secretion systems, help V. parahaemolyticus adhere to the distal end of the small intestine by secreting effectors that induce the lysis of epithelial cells and change the shape of the intestinal lining, which provides nutrients and a suitable environment for its growth. This review summarizes the research progress in recent years on the virulence factors associated with the colonization and adhesion of V. parahaemolyticus, which provides valuable information for the safety control of marine food.

Characterization of Recombinant Antimicrobial Peptide BMGlv2 Heterologously Expressed in Trichoderma reesei.

Antimicrobial peptides (AMPs) serve as alternative candidates for antibiotics and have attracted the attention of a wide range of industries for various purposes, including the prevention and treatment of piglet diarrhea in the swine industry. Escherichia coli, Salmonella, and Clostridium perfringens are the most common pathogens causing piglet diarrhea. In this study, the antimicrobial peptide gloverin2 (BMGlv2), derived from Bombyx mandarina, was explored to determine the efficient prevention effect on bacterial piglet diarrhea. BMGlv2 was heterologously expressed in Trichoderma reesei Tu6, and its antimicrobial properties against the three bacteria were characterized. The results showed that the minimum inhibitory concentrations of the peptide against E. coli ATCC 25922, S. derby ATCC 13076, and C. perfringens CVCC 2032 were 43.75, 43.75, and 21.86 µg/mL, respectively. The antimicrobial activity of BMGlv2 was not severely affected by high temperature, salt ions, and digestive enzymes. It had low hemolytic activity against rabbit red blood cells, indicating its safety for use as a feed additive. Furthermore, the measurements of the leakage of bacterial cell contents and scanning electron microscopy of C. perfringens CVCC 2032 indicated that BMGlv2 exerted antimicrobial activity by destroying the cell membrane. Overall, this study showed the heterologous expression of the antimicrobial peptide BMGlv2 in T. reesei and verified its antimicrobial properties against three common pathogenic bacteria associated with piglet diarrhea, which can provide a reference for the applications of AMPs as an alternative product in industrial agriculture.

Genome sequence analysis of Cronobacter phage PF-CE2 and proposal of a new species in the genus Pseudotevenvirus.

The genome of a Cronobacter sakazakii M1 phage named PF-CE2 was characterized in this work, and a new species named "Cronobacter virus PF-CE2", in the genus Pseudotevenvirus of the subfamily Tevenvirinae of the family Myoviridae is proposed. The Gp190 gene of phage PF-CE2 is predicted to encode a bacteriophage-borne glycanase that is capable of degrading fucose-containing exopolysaccharides produced by C. sakazakii M1. Furthermore, we propose changing the taxonomic status of eight additional phages based on nucleotide sequence comparisons. This work provides a theoretical basis for subsequent heterologous expression of the phage PF-CE2 glycanase and provides an important reference for the preservation and sharing of these phages.

Application of Microalgal Stress Responses in Industrial Microalgal Production Systems.

Adaptive laboratory evolution (ALE) has been widely utilized as a tool for developing new biological and phenotypic functions to explore strain improvement for microalgal production. Specifically, ALE has been utilized to evolve strains to better adapt to defined conditions. It has become a new solution to improve the performance of strains in microalgae biotechnology. This review mainly summarizes the key results from recent microalgal ALE studies in industrial production. ALE designed for improving cell growth rate, product yield, environmental tolerance and wastewater treatment is discussed to exploit microalgae in various applications. Further development of ALE is proposed, to provide theoretical support for producing the high value-added products from microalgal production.

Potassium Alginate Oligosaccharides Alter Gut Microbiota, and Have Potential to Prevent the Development of Hypertension and Heart Failure in Spontaneously Hypertensive Rats.

Food-derived oligosaccharides show promising therapeutic potential in lowering blood pressure (BP), but the mechanism is poorly understood. Recently, the potential role of gut microbiota (GM) in hypertension has been investigated, but the specific GM signature that may participate in hypertension remains unclear. To test the potassium alginate oligosaccharides (PAO) mechanism in lowering BP and specific microbial signature changes in altering GM, we administered various dosages of PAO in 40 spontaneously hypertensive rats for a duration of six weeks. We analyzed BP, sequenced the 16S ribosomal DNA gene in the cecum content, and gathered RNA-seq data in cardiac tissues. We showed that the oral administration of PAO could significantly decrease systolic BP and mean arterial pressure. Transcriptome analyses demonstrated that the protective effects of developing heart failure were accompanied by down-regulating of the Natriuretic Peptide A gene expression and by decreasing the concentrations of angiotensin II and atrial natriuretic peptide in plasma. In comparison to the Vehicle control, PAO could increase the microbial diversity by altering the composition of GM. PAO could also decrease the ratio of Firmicutes to Bacteroidetes by decreasing the abundance of Prevotella and Phascolarctobacterium bacteria. The favorable effect of PAO may be added to the positive influence of the abundance of major metabolites produced by Gram-negative bacteria in GM. We suggest that PAO caused changes in GM, and thus, they played an important role in preventing the development of cardiovascular disease.

A thermostable glucose oxidase from Aspergillus heteromophus CBS 117.55 with broad pH stability and digestive enzyme resistance.

In this study, the heterologous expression of an engineered thermostablle glucose oxidase from Aspergillus heteromophus CBS 117.55 was achieved in P. pastoris. This recombinant GoxAh was thermostable, with an optimal temperature range 25 °C-65 °C, and it was capable of retaining greater than 90% of its initial activity following a 10-min incubation at 75 °C. This enzyme had an optimum pH of 6.0, and it could retain above 80% of its initial activity following a 2-h incubation at a broad pH range (2.0-8.0). Moreover, GoxAh displayed excellent pepsin and trypsin resistance, and highly resistant to a range of tested metal ions and chemical reagents. These good properties make GoxAh a promising candidate for feed additive. The Km and kcat/Km values of GoxAh were 187 mM and 1.09/mM/s, which limited its widespread application to some degree. However, due to its excellent characteristics, GoxAh is still of potential economic value for high value-added areas, as well as a good initial enzyme for developing applicable feed enzyme by protein engineering.

Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris.

Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a ß-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2-4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.

Nondigestible carbohydrates, butyrate, and butyrate-producing bacteria.

Nondigestible carbohydrates (NDCs) are fermentation substrates in the colon after escaping digestion in the upper gastrointestinal tract. Among NDCs, resistant starch is not hydrolyzed by pancreatic amylases but can be degraded by enzymes produced by large intestinal bacteria, including clostridia, bacteroides, and bifidobacteria. Nonstarch polysaccharides, such as pectin, guar gum, alginate, arabinoxylan, and inulin fructans, and nondigestible oligosaccharides and their derivatives, can also be fermented by beneficial bacteria in the large intestine. Butyrate is one of the most important metabolites produced through gastrointestinal microbial fermentation and functions as a major energy source for colonocytes by directly affecting the growth and differentiation of colonocytes. Moreover, butyrate has various physiological effects, including enhancement of intestinal barrier function and mucosal immunity. In this review, several representative NDCs are introduced, and their chemical components, structures, and physiological functions, including promotion of the proliferation of butyrate-producing bacteria and enhancement of butyrate production, are discussed. We also describe the strategies for achieving directional accumulation of colonic butyrate based on endogenous generation mechanisms.

Study on expression and action mode of recombinant alginate lyases based on conserved domains reconstruction.

Understanding the effect of conserved domains reconstruction of alginate lyases on action mode is essential for their application and in-depth study. We report the expression and action mode of recombinant alginate lyase (AlyM) and its conserved domain reconstruction forms (AlyMΔCBM, cAlyM, and AlyMΔ58C). The enzymatic activities of AlyM, AlyMΔCBM, cAlyM, and AlyMΔ58C were 61.77, 150.57, 388.97, and 308.21 U/mg, respectively. The transcription level of cAlyM was 49.89-fold of AlyM. cAlyM and AlyMΔ58C showed higher thermal stability than AlyM, indicating that the removal of F5_F8_type_C domain was beneficial for the increase of thermal stability. The enzymes were bifunctional alginate lyases and preferred polyG to polyM. The enzymes degraded alginate to produce unsaturated disaccharide, trisaccharide, and tetrasaccharide as the main end-products. Pentamannuronic acid and pentaguluronic acid were the smallest substrates that could be degraded by AlyM, with unsaturated trisaccharide/tetrasaccharide (40.61%/44.42%) and disaccharide/trisaccharide (10.57%/83.85%) as the main products, respectively. The action modes of enzymes remain unaffected after conserved domain reconstruction, but the affinity of AlyMΔ58C toward polyM increased. This study provides a new strategy for rational modification of alginate lyase based on conserved domain reconstruction.

Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium Arthrobacter sp. CS01.

Chondroitinase (ChSase), a type of glycosaminoglycan (GAG) lyase, can degrade chondroitin sulfate (CS) to unsaturate oligosaccharides, with various functional activities. In this study, ChSase AC II from a newly isolated marine bacterium Arthrobacter sp. CS01 was cloned, expressed in Pichia pastoris X33, purified, and characterized. ChSase AC II, with a molecular weight of approximately 100 kDa and a specific activity of 18.7 U/mg, showed the highest activity at 37 °C and pH 6.5 and maintained stability at a broad range of pH (5⁻7.5) and temperature (below 35 °C). The enzyme activity was increased in the presence of Mn2+ and was strongly inhibited by Hg2+. Moreover, the kinetic parameters of ChSase AC II against CS-A, CS-C, and HA were determined. TLC and ESI-MS analysis of the degradation products indicated that ChSase AC II displayed an exolytic action mode and completely hydrolyzed three substrates into oligosaccharides with low degrees of polymerization (DPs). All these features make ChSase AC II a promising candidate for the full use of GAG to produce oligosaccharides.

Evaluation of Prebiotic Potential of Three Marine Algae Oligosaccharides from Enzymatic Hydrolysis.

Alginate oligosaccharides (AlgO), agarose oligosaccharides (AO), and κ-carrageenan oligosaccharides (KCO) were obtained by specific enzymatic hydrolysis method. The molecular weight distributions of the three oligosaccharides were 1.0⁻5.0 kDa, 0.4⁻1.4 kDa, and 1.0⁻7.0 kDa, respectively. The culture medium was supplemented with the three oligosaccharides and fermented by pig fecal microbiota in vitro, for 24 h. Each oligosaccharide was capable of increasing the concentration of short-chain fatty acids (SCFAs), especially butyric acid, and altering the microbiota composition. Linear discriminant analysis effect size (LEfSe) analysis results showed that the opportunistic pathogenic bacteria Escherichia, Shigella, and Peptoniphilus, were significantly decreased in AlgO supplemented medium. AO could improve the gut microbiota composition by enriching the abundance of Ruminococcaceae, Coprococcus, Roseburia, and Faecalibacterium. Besides, KCO could increase the abundance of SCFA microbial producers and opportunistic pathogenic flora. Therefore, these results indicate that AlgO and AO can be used as gut microbial regulators and can potentially improve animal/human gastrointestinal health and prevent gut disease, whereas the physiological function of KCO needs further evaluation.