Experimental Research on Candidate Probiotics: From Strain Identification and Mechanistic Evaluation to Product Selection

1. The Starting Point of Candidate Probiotic Research: Distinguishing Fermentation Microorganisms, Live Microorganisms, and Probiotics

Probiotics are not all live microorganisms, nor are they all microorganisms found in fermented foods. The internationally used definition describes probiotics as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. For scientific experiments, this definition reminds researchers that evaluating a strain requires simultaneous attention to strain identity, viability status, dosage, safety, and verifiable evidence of effect.

When a microorganism participates in food fermentation, it may affect food flavor, acidity, or texture. When it remains viable in a product, it shows a certain level of survivability. However, these pieces of information are not sufficient to demonstrate that it has probiotic properties. A candidate strain must be linked to a defined strain identity, dosage, evaluation endpoint, and experimental results. Only after safety, stability, and functional characteristics have been verified can its suitability for probiotic-related research be assessed.

Concept	Key Point for Evaluation	Explanation
Fermentation microorganism	Whether it participates in the fermentation process	It may improve food flavor, acidity, or texture, but participation in fermentation alone is not enough to call it a probiotic
Live microorganism	Whether it remains viable	The presence of live microorganisms does not necessarily mean probiotic action, nor does it necessarily indicate clear experimental evaluation value
Candidate probiotic	Whether it has a defined strain identity, viability status, and verifiable evidence of potential action	Refers to strains that are still in the stages of screening, cultivation, stability testing, safety evaluation, and functional assessment
Probiotic	Whether it produces a clear benefit when administered in adequate amounts	Requires defined strain identity, dosage, safety, application context, and evidence of effect
Prebiotic	Whether it is selectively utilized by host microorganisms and produces a clear benefit	It is not a live microorganism; experimentally, the focus is on substrate utilization, microbiota response, and metabolic output
Postbiotic	Whether it is derived from a processed non-viable microbial preparation and has clear evidence of effect	A postbiotic is not simply a “dead bacterium.” It is usually a preparation composed of inactivated microbial cells, cellular components, and related metabolites; ordinary fermentation broth, fermentation supernatant, or a single purified metabolite cannot be directly called a postbiotic

2. The Core of Probiotic Research Is the Strain

The functions of probiotics are usually strain-specific. Even within the same species, different strains may differ in acid tolerance, bile salt tolerance, adhesion ability, metabolic products, immunomodulatory capacity, and safety.

The assessment of probiotic action cannot remain at the genus or species level. To evaluate whether a strain has research value, the observed effect should be linked to a specific strain or fixed strain combination, dosage, experimental conditions, and evaluation endpoints. In other words, it is not enough to know which genus or species it belongs to; researchers also need to know the exact strain, the amount used, the conditions under which it was used, and the results observed.

Information Level	Question to Answer	Role
Strain identity	Which exact strain is it?	Determines whether experimental results can be reproduced and compared
Dosage	How many viable cells are used per dose or per day?	Determines the intensity of experimental treatment and the comparability of results
Duration of use	How long is it used?	Affects strain survival, metabolite accumulation, and host or model responses
Research object or application context	Is it used in a culture system, cell model, animal model, food matrix, or formulation system?	Determines the scope of applicability of the experimental conclusion
Evaluation endpoint	Is the observation focused on strain growth, substrate utilization, metabolites, barrier function, inflammatory markers, or stability?	Determines whether the research conclusion is clear

3. Possible Pathways Through Which Probiotics Affect the Intestinal Environment and Host Responses

Probiotics do not act through a single mechanism. Different strains may affect the intestinal environment, microbiota, metabolite composition, barrier function, and immune responses through different pathways. The value of mechanistic research lies in helping determine which type of pathway a strain may act through and in guiding subsequent experimental design. Common mechanisms of probiotic action include competition with pathogens, regulation of short-chain fatty acids and the metabolic environment, modulation of the intestinal barrier and immune homeostasis, and changes in bile acids and microbiota-associated enzyme activities.

Mechanistic Pathway	What May Occur	Common Observation Indicators	Key Points for Experimental Interpretation
Competition with pathogens or opportunistic pathogens	Through acid production, antimicrobial substances, nutrient competition, or competition for adhesion sites, the strain may reduce the growth or attachment opportunities of certain pathogens	Co-culture inhibition assays, adhesion assays, organic acid detection, detection of bacteriocins or bacteriocin-like inhibitory substances	Suitable for pathogen inhibition, adhesion competition, and microbiota disturbance models, but the results should be interpreted in relation to the target strain, culture conditions, and model system
Alteration of short-chain fatty acids and the metabolic environment	The strain may directly produce some metabolites, or indirectly alter metabolites such as acetate, propionate, and butyrate by affecting the existing intestinal microbiota and substrate utilization	Detection of short-chain fatty acids in culture systems, in vitro fermentation systems, or intestinal simulation systems; microbiota structure analysis; substrate consumption assays	The total amount of short-chain fatty acids alone is insufficient; interpretation should combine substrate consumption, bacterial growth, pH changes, microbiota structure, and functional model results
Regulation of the intestinal barrier and immune homeostasis	The strain may affect epithelial permeability, tight junction proteins, the mucus layer, and local immune responses	Epithelial permeability, tight junction proteins, mucus layer indicators, cytokines, immune cell changes	Changes in barrier integrity, tight junction-related proteins, mucus layer indicators, and inflammatory factors can be used to assess the effects of strain treatment on the intestinal epithelial barrier and local immune homeostasis
Effects on bile acids and microbiota-associated enzyme activities	Some strains have bile salt hydrolase activity and may also affect the transformation of glycosidic substances, conjugated metabolites, and nitrogen-containing compounds	Bile salt hydrolase, β-glucosidase, β-glucuronidase, urease, bile acid profiles	Suitable for mechanistic research and metabolic capacity evaluation, but should be interpreted together with strain tolerance, bile acid composition changes, and functional indicators

Short-chain fatty acids and bile salt hydrolase in the table are two types of indicators that are easily misinterpreted in probiotic research. Common short-chain fatty acids include acetate, propionate, and butyrate. Acetate can participate in the regulation of intestinal pH and can also be further utilized by other microorganisms. Propionate is related to the regulation of energy metabolism. Butyrate is an important energy source for colon epithelial cells and is closely associated with barrier function and inflammation regulation.

It should be noted that many common probiotics do not necessarily directly produce large amounts of butyrate. Some strains may promote cross-feeding by producing lactate, acetate, or by altering substrate utilization, thereby supporting other butyrate-producing bacteria. Therefore, experiments should not focus only on an “increase in bacterial count.” They should also examine whether the substrate is utilized, whether metabolites change, and whether these changes correspond to barrier function, inflammatory markers, or other functional model results.

Bile salt hydrolase activity should not be simply interpreted as “the higher, the better.” Bile salt hydrolase may help some strains adapt to bile salt environments and may also affect bile acid transformation. Different strains may differ in bile acid deconjugation capacity and substrate preference, and the physiological significance still needs to be analyzed in a specific system. When studying bile salt hydrolase, researchers should simultaneously examine strain tolerance to bile salts, changes in bile acid composition, and related functional indicators, rather than judging strain action using a single enzyme activity result.

4. How to Complete Experimental Validation of a Candidate Probiotic Strain

Experimental validation of candidate probiotics should focus on strain identity, safety, viability maintenance, functional characteristics, and formulation compatibility. The validation process usually begins with strain identification, followed by preliminary safety screening, stability evaluation, and in vitro functional studies. This sequence helps answer several questions step by step: “What strain is this?”, “Can it survive stably?”, “What functional characteristics does it have?”, and “Is it suitable for subsequent application development?”

These experiments can establish the identity, safety, stability, and mechanistic clues of a candidate strain. However, if clear benefits to human health are to be demonstrated, evidence from human studies or clinical research in the target population is still required.

Validation Step	Experimental Purpose	Common Methods or Indicators	Experimental Output
Strain identification	Confirm strain identity and traceability	Colony morphology, microscopic observation, biochemical identification, 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing, whole-genome sequencing, strain number, or deposit information	Genus and species identification results, strain number, genomic characteristics, deposit or source information
Preliminary safety screening	Identify application-related risk factors	Virulence-related genes, transferable antibiotic resistance genes, antimicrobial susceptibility testing, hemolysis risk, biogenic amine production capacity, harmful metabolite detection	Antibiotic resistance profile, virulence-related gene screening results, hemolysis results, biogenic amine or harmful metabolite detection results
Viability and stability	Evaluate viability retention during processing, storage, and simulated gastrointestinal conditions	Acid tolerance assays, bile salt tolerance assays, simulated digestive fluid assays, freeze-drying damage evaluation, encapsulation protection evaluation, viable count after storage	Viable count after processing, viable count after storage, survival rate after simulated digestion, stability trend
In vitro functional screening	Establish functional characteristics and mechanistic clues of the strain	Antimicrobial assays, epithelial barrier models, immune cell models, short-chain fatty acid detection, prebiotic utilization assays, bile salt hydrolase assays	Antimicrobial activity, barrier-related indicators, inflammation-related indicators, metabolite changes, substrate utilization capacity, bile salt-related enzyme activity
Formulation and delivery compatibility	Evaluate compatibility of the strain in application systems	Freeze-drying protection, encapsulation systems, food matrix compatibility, stability of multi-strain combinations, storage condition validation	Formulation survival rate, release characteristics, matrix compatibility, stability of combined systems, suitability of storage conditions

1) Strain Identification Is the Starting Point of Experimental Validation

Traditional morphological observation, biochemical reactions, and selective cultivation can be used for preliminary screening. 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing is commonly used for genus- and species-level identification. Whole-genome sequencing can be used for strain identification, safety analysis, and functional gene screening. For candidate strains that require a traceable evidence chain, strain number, source records, and deposit information should also be organized at the same time.

2) Preliminary Safety Screening Should Precede Functional Evaluation

Even if a candidate strain comes from food, fermented samples, or intestinal-source samples, it still needs to be tested for virulence-related genes, transferable antibiotic resistance genes, hemolysis risk, biogenic amine production capacity, and other harmful metabolites. Safety results are used to determine whether the strain is suitable for subsequent functional research and application development.

3) Viability and Stability Determine Whether a Candidate Strain Can Move from the Culture System into Practical Application Systems

Viable cell counts are usually expressed as “colony-forming units,” which reflect the number of culturable viable microorganisms capable of forming colonies under specific culture conditions. When evaluating strains or formulations, researchers should focus on changes in viable counts after processing, after storage, and after passage through simulated gastrointestinal environments.

4) In Vitro Functional Screening Is Used to Establish the Functional Characteristics of Candidate Strains

For example, antimicrobial assays can be used to observe the effects of a strain on target pathogens or opportunistic pathogens. Epithelial barrier models can be used to evaluate permeability and tight junction-related indicators. Immune cell models can be used to observe changes in inflammation-related factors. Short-chain fatty acid detection, prebiotic utilization assays, and bile salt hydrolase assays can be used to analyze metabolic and adaptive characteristics.

5) Formulation and Delivery Compatibility Are Better Placed in the Later Stages of the Validation Process

Probiotic research does not focus only on the strain itself; it also needs to examine the state of the strain in freeze-dried powders, capsules, fermented milk, beverages, encapsulation systems, or multi-strain systems. The same strain may show different survival rates, release states, and storage stability in different carriers. Therefore, formulation conditions are also important variables in the experimental validation of candidate strains.

6) Quality Control Before Application Should Be Recorded Simultaneously

Before candidate strains enter formulations, fermented foods, or functional evaluation systems, testing methods, storage conditions, and contamination control results should also be recorded. Culture counting, selective media, molecular detection, and strain differentiation methods should match the target strain. During storage, attention should be paid to the effects of temperature, moisture, oxygen, packaging, and time on viable counts. Finished products or experimental samples should also be tested for contaminating microorganisms, unwanted microbial contamination, and batch stability. These quality control results help determine whether the experimental data come from the target strain itself and prevent storage loss, contaminating microorganisms, or differences in detection methods from being misinterpreted as functional differences between strains.

5. Key Questions to Check in Probiotic Experimental Design

After a candidate probiotic has completed basic identification and functional screening, the experimental design still needs to further check several issues that can easily affect result interpretation. The focus is to determine whether a clear correspondence can be established among the strain, substrate, metabolites, bile salt response, and combined formulation.

5.1 Can the Strain Utilize the Target Prebiotic Substrate?

Different strains differ in their ability to utilize oligosaccharides and dietary fibers. Some Bifidobacterium and lactic acid bacteria-related strains can utilize fructo-oligosaccharides, inulin, lactulose, stachyose, raffinose, or certain human milk oligosaccharide components. However, complex polysaccharides such as pectin and arabinogalactan often need to be evaluated in microbiota co-fermentation systems; it should not be assumed that a single strain can effectively utilize them. This capacity must be confirmed experimentally.

In research, whole-genome sequencing can first be used to analyze glycoside hydrolases, transporters, and sugar utilization-related genes. This can then be combined with in vitro fermentation experiments to detect substrate consumption, bacterial growth, pH changes, and short-chain fatty acid production. In this way, researchers can determine whether a specific strain is suitable for combined research with a particular prebiotic substrate.

It should be noted that the presence of sugar utilization-related genes in the genome only suggests potential capacity. Whether a strain can actually utilize a certain substrate still needs to be judged in combination with culture conditions, substrate structure, strain activity, and fermentation results.

5.2 Do Changes in Short-Chain Fatty Acids Correspond to Functional Indicators?

Short-chain fatty acid detection should not focus only on changes in total amount. The ratio of acetate, propionate, and butyrate, substrate source, culture time, sample type, and detection method can all affect result interpretation. The following items can be tested simultaneously in experiments:

Detection Item	Main Role
Ratio of acetate, propionate, and butyrate	Determines whether the composition of fermentation metabolites has changed
Substrate consumption	Determines whether metabolite changes are related to substrate utilization
Bacterial growth	Determines whether the substrate supports proliferation of the target strain
Culture medium pH	Reflects acid production during fermentation and environmental changes
Barrier- or inflammation-related indicators	Determines whether metabolic changes correspond to functional model results

If only changes in short-chain fatty acids are observed without combining them with strain growth, substrate consumption, and functional indicators, it is difficult to determine whether those changes have clear experimental significance.

5.3 How Should Bile Salt Hydrolase Activity Be Interpreted Together with Bile Acid Changes?

Bile salt hydrolase activity may help some strains adapt to bile salt-containing environments and may also affect bile acid composition. This indicator should not be interpreted alone as a positive function.

In research, three types of results should be examined simultaneously. The first is the survival of the strain under bile salt conditions. The second is the compositional change in conjugated and unconjugated bile acids. The third is whether bile acid changes correspond to barrier-, inflammation-, or metabolism-related indicators. This makes it possible to distinguish between two different questions: “Can the strain tolerate bile salts?” and “Can the strain alter bile acid composition?”

Bile salt hydrolase activity assays should preferentially use taurine- or glycine-conjugated bile acids as substrates. Cholic acid, deoxycholic acid, lithocholic acid, and their sodium salts are more suitable for bile acid profiling, bile acid treatment studies, or bile salt stress models; they should not be directly treated as bile salt hydrolase substrates.

5.4 Should Multi-Strain Combinations Be Validated Separately?

Multi-strain combinations are not necessarily superior to single strains. Multi-strain combinations may produce synergistic effects, but they may also lead to nutrient competition, mutual inhibition, reduced activity, or poorer stability.

When evaluating multi-strain combinations, the following questions should be emphasized:

1. Can each strain survive stably under the same culture conditions?

2. Is there inhibition or competition among the strains?

3. Are the viable counts of each strain stable after combination?

4. Does the functional change come from a single strain or from the combined effect?

5. Are the formulation ratio, culture conditions, and storage conditions kept consistent?

Multi-strain experiments should, as far as possible, include single-strain controls, pairwise combination controls, and complete combination controls. This makes it possible to determine whether the combined effect truly comes from strain interactions, rather than from the independent action of one strain or from changes in culture conditions.

5.5 Do Differences in Experimental Conditions Affect Result Interpretation?

The same strain may behave differently under different experimental conditions. Medium composition, carbon source type, pH, oxygen conditions, bile salt concentration, culture time, inoculum level, and storage state can all affect strain growth, metabolite production, and functional indicators. Therefore, key conditions should be fixed in probiotic experiments, and the following information should be clearly recorded:

Experimental Condition	Possible Effect
Medium composition	Affects strain growth rate, acid production capacity, and metabolite profile
Carbon source or prebiotic substrate	Affects substrate utilization, short-chain fatty acids, and bacterial proliferation
Oxygen conditions	Affects the survival and metabolism of oxygen-sensitive strains such as bifidobacteria
pH	Affects strain acid tolerance, enzyme activity, and antimicrobial results
Bile salt concentration	Affects bile salt tolerance, bile salt hydrolase activity, and survival rate
Inoculum level and culture time	Affects bacterial quantity, metabolite accumulation, and result comparability
Storage state	Affects initial experimental activity and subsequent functional performance

6. Product Selection Navigation for Probiotic-Related Products: From Strain Cultivation and Substrate Utilization to Mechanistic Evaluation and Safety Verification

Research or Experimental Objective	Recommended Table to Start With	Why Start With This Table	Related Table(s) to Consult	Navigation Guidance
Establish a basic cultivation system for candidate probiotics	Table 1	Table 1 brings together cultivation-condition regulators such as agar, glucose, cysteine, Tween, acetate, magnesium salts, and manganese salts. It can be used first to establish basic conditions for strain isolation, expansion, and cultivation	Table 6, Table 3	First determine whether the target strain requires anaerobic, low-oxygen, acetate, trace metal, or specific carbon source conditions, and then proceed to selective cultivation and metabolic evaluation
Isolate or enrich target microbial groups from complex samples	Table 6	Table 6 includes mupirocin lithium, nalidixic acid, and cycloheximide, which can be used for selective cultivation, contamination control, and isolation of target microbial groups	Table 1	Select inhibitors according to the target microbial group, sample source, and possible contamination types, and then combine them with basic medium conditions for isolation and counting
Compare the growth capacity and cultivation adaptability of different candidate strains	Table 1	Table 1 covers basic carbon sources, reducing components, acidity regulators, and ion supplements, which can be used to establish evaluation systems for growth curves, acid production capacity, and cultivation stability	Table 3, Table 4	First observe whether the strain can proliferate stably, and then test organic acid production, enzyme activity characteristics, acid and bile salt tolerance, and survival during digestion
Evaluate the ability of strains to utilize prebiotic substrates	Table 2	Table 2 brings together fructo-oligosaccharides, inulin, lactulose, stachyose, raffinose, pectin, arabinogalactan, and human milk oligosaccharide components, which can be used for substrate utilization screening	Table 3, Table 1	First determine which oligosaccharides or polysaccharides the strain can utilize, and then measure acid production, short-chain fatty acids, and bacterial proliferation
Design studies on combined use of probiotics and prebiotics	Table 2	Table 2 helps select prebiotic substrates with different structural types and is suitable for establishing strain–substrate matching relationships	Table 3, Table 5	First compare substrate utilization and metabolic output, and then combine barrier, inflammation, or cell models to judge whether the metabolic changes have functional significance
Analyze changes in organic acids and short-chain fatty acids after probiotic fermentation	Table 3	Table 3 includes propionic acid, butyric acid, sodium butyrate, succinic acid, and different lactic acid isomers, which can be used for fermentation metabolite detection and standard system development	Table 2, Table 5	First obtain metabolite changes through substrate fermentation experiments, and then combine cell viability, barrier permeability, and inflammatory indicators to assess functional relevance
Evaluate lactic acid production characteristics and metabolic safety	Table 3	Table 3 includes L-lactic acid, D-lactic acid, and mixed lactic acid, which can be used to distinguish lactic acid isomer production	Table 1, Table 2	First clarify the lactic acid configuration produced by the strain under different media and substrate conditions, and then decide whether further metabolic safety assessment is needed
Screen microbiota-associated enzyme activities and potential metabolic risks	Table 3	Table 3 includes urea, β-glucuronidase substrates, β-glucosidase substrates, and multiple biogenic amine standards, which can be used for enzyme activity and safety indicator evaluation	Table 1, Table 6	First detect target enzyme activities and biogenic amines under stable culture conditions, and then combine strain source, culture conditions, and contamination control to determine the source of the results
Study the stability of probiotics during gastrointestinal transit	Table 4	Table 4 includes pepsin, pancreatin, and various bile acid-related reagents, which can be used to simulate gastric conditions, small-intestinal conditions, and bile salt stress	Table 1, Table 5	First evaluate the survival and stability of strains or formulations under digestive conditions, and then decide whether to proceed to barrier protection or inflammation regulation experiments
Evaluate strain bile salt tolerance and bile acid transformation capacity	Table 4	Table 4 covers conjugated bile acids, primary bile acids, and secondary bile acids, which can be used for bile salt hydrolase activity, bile acid profiling, and tolerance studies	Table 3, Table 5	First distinguish between the two questions of bile salt tolerance and bile acid transformation, and then interpret their biological significance together with metabolites, barrier indicators, and inflammatory indicators
Establish cell- or animal-related evaluation models for probiotic mechanisms of action	Table 5	Table 5 includes lipopolysaccharide, dextran sulfate sodium, fluorescent dextran, thiazolyl blue tetrazolium bromide, and resazurin, which can be used to evaluate inflammatory stimulation, barrier damage, permeability, and cell viability	Table 3, Table 4	First clarify whether the research endpoint is inflammatory response, barrier function, or cell viability, and then select metabolites, bile acids, or simulated digestion results for linked interpretation
Evaluate the cytocompatibility of probiotics, postbiotics, or fermentation supernatants	Table 5	The cell viability and metabolic activity detection materials in Table 5 can be used to determine whether sample treatment affects cell status	Table 3	First confirm that the sample has no obvious interference with cells, and then analyze the relationship between short-chain fatty acids, biogenic amines, lactic acid, or other metabolites and cellular responses
Determine whether probiotic intervention is related to intestinal barrier protection	Table 5	Table 5 can support the establishment of intestinal epithelial permeability, inflammatory stimulation, and barrier injury models	Table 3, Table 4	First detect permeability and cell status, and then combine butyrate, bile acid changes, or digestive stability to determine whether the mechanism is consistent
Evaluate the stability and delivery of probiotic products or formulations	Table 4	Table 4 can be used to simulate gastrointestinal digestion conditions and observe the stability of formulations, encapsulation systems, or fermented matrices during digestion	Table 1, Table 5	First examine viability retention and structural stability during digestion, and then combine culture counting, cytocompatibility, and barrier-related indicators for evaluation
Move from “Can the strain grow?” to “Does it have functional evidence?”	Table 1	Table 1 is used to confirm whether candidate strains can be cultivated and expanded stably, which is the basis for subsequent functional evaluation	Table 2, Table 3, Table 4, Table 5	First establish reproducible culture conditions, and then examine substrate utilization, metabolic output, digestive tolerance, and host-related functional indicators in sequence
Build a complete experimental validation chain for probiotics	Table 1	Table 1 can serve as the starting point for strain cultivation and basic condition establishment	Table 2, Table 3, Table 4, Table 5, Table 6	Organize the experiment in the sequence of “cultivation and isolation—substrate utilization—metabolite detection—digestive tolerance—barrier and inflammation evaluation—selective control” to make result interpretation more coherent

Table 1｜Products Related to Strain Cultivation, Medium Preparation, and Basic Culture Condition Regulation

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Solid medium matrix	9002-18-0	A501219	YE Agar	Medium for vegetative growth of the fission yeast Schizosaccharomyces pombe	Can be used to establish solid culture systems, observe colonies, and screen microbial culture conditions, supporting strain isolation, purification, and morphological recording. If used for isolation or counting of candidate probiotics such as lactic acid bacteria and bifidobacteria, the medium system should be selected or prepared according to the target strain.
Basic fermentation carbon source	50-99-7	D432810	D-(+)-Glucose	Anhydrous grade, PharmPure™, USP, BP, European Pharmacopoeia (Ph.Eur), ACS	Can be used as a basic fermentation carbon source for studies on strain growth curves, acid production capacity, sugar metabolism, and medium formulation optimization.
Reducing component and low-oxygen culture regulator	7048-04-6	C118575	L-Cysteine hydrochloride monohydrate	Animal-free, PharmPure™, USP, European Pharmacopoeia (Ph.Eur), for cell culture	Can be used as a reducing component in anaerobic or low-oxygen culture systems, suitable for cultivation and viability studies of oxygen-sensitive strains such as bifidobacteria.
Surfactant and fatty acid source	9005-65-6	T485985	Tween® 80	Viscous liquid, preservative-free, low peroxide; low carbonyl	Commonly used in culture systems for lactic acid bacteria and bifidobacteria as a surfactant and fatty acid source, supporting studies on the effects of medium composition on strain growth, membrane stability, and viable counts.
Acetate and selective cultivation component	127-09-3	S118648	Sodium acetate, anhydrous	For cell culture, for insect cell culture, ≥99%	Commonly used in lactic acid bacteria media and acetate environment construction; can be used to examine the effects of acetate on strain growth, acid production, and selective cultivation performance.
Acidity regulator and acid stress reagent	64-19-7	A116166	Glacial acetic acid	Guaranteed reagent, ≥99.5%	Can be used for medium acidity adjustment, acetate buffer systems, and acid stress condition establishment, suitable for evaluating the tolerance of candidate strains to low-acid environments.
Trace manganese source	10034-96-5	M111715	Manganese sulfate monohydrate	For plant cell culture, for cell culture	Can be used as a trace manganese source for lactic acid bacteria medium optimization and is relevant to studies on oxidative stress tolerance, enzyme activity, and growth status.
Magnesium ion and inorganic salt nutrient component	10034-99-8	M434885	Magnesium sulfate heptahydrate	Pharmaceutical grade, PharmPure™	Can be used as a magnesium ion source in medium preparation, supporting microbial enzyme activity, osmotic regulation, and optimization of basic growth conditions.
Citrate and nitrogen source component	3458-72-8	A116440	Ammonium citrate	AR, ≥98.5%	Can be used in lactic acid bacteria medium preparation, providing citrate and nitrogen source conditions and supporting studies on the effects of medium composition on strain proliferation and metabolite formation.

Table 2｜Products Related to Prebiotic Substrates and Fermentable Carbon Sources

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Human milk oligosaccharide substrate	41263-94-9	F130960	2'-Fucosyllactose	≥95%	A typical human milk oligosaccharide component; can be used for studies on infant gut microbiota, bifidobacterial substrate utilization, and host–microbe interactions.
Fructan-type oligosaccharide	308066-66-2	F303397	Fructo-oligosaccharide	Fructo-oligosaccharide content >90.0%	A typical prebiotic oligosaccharide used to examine selective utilization capacity, acid production capacity, and metabolic interactions of bifidobacteria and lactic acid bacteria-related strains.
Fructan-type dietary fiber	9005-80-5	I111088	Inulin	Biochemical reagent	A typical fermentable dietary fiber used to evaluate utilization of fructan substrates, short-chain fatty acid production, and microbiota modulation by probiotics and intestinal microbiota.
Disaccharide-type prebiotic substrate	4618-18-2	L104550	Lactulose	Moligand™, ≥98%	Can be used as a prebiotic-type disaccharide substrate for evaluating bifidobacterial proliferation, intestinal fermentation acid production, and microbiota metabolic regulation.
α-Galactoside oligosaccharide	17629-30-0	R111690	D-Raffinose pentahydrate	For cell culture, ≥99%	Can be used as a raffinose-type oligosaccharide substrate to examine candidate strains’ utilization of α-galactoside carbon sources and fermentation characteristics.
α-Galactoside oligosaccharide	512-69-6	R413236	Raffinose	≥98%	Can be used for oligosaccharide utilization screening, fermentation acid production detection, and carbon source preference studies of bifidobacteria and lactic acid bacteria-related strains.
α-Galactoside oligosaccharide	10094-58-3	S1421262	Stachyose tetrahydrate	≥98%	Can be used as a stachyose-type prebiotic substrate to study candidate strains’ utilization capacity for tetrasaccharide substrates, acid production characteristics, and microbiota proliferation responses.
α-Galactoside oligosaccharide	470-55-3	S353428	Stachyose	≥70%	Can be used in prebiotic formulations, in vitro fermentation, and strain substrate utilization experiments, supporting comparison of microbial metabolic responses in stachyose systems of different purities.
Plant pectin polysaccharide	9000-69-5	P112756	Pectin	Galacturonic acid, ≥74.0% on dry basis	Can be used as a fermentable dietary fiber and intestinal microbiota substrate to study the effects of polysaccharide structure on microbiota fermentation, short-chain fatty acid production, and intestinal barrier-related indicators.
Plant arabinogalactan	9036-66-2	A304929	(+)-Arabinogalactan	From larch wood	Can be used as a plant-derived polysaccharide substrate for in vitro fermentation, microbiota substrate utilization, and studies on the effects of polysaccharides on probiotic proliferation.

Table 3｜Products Related to Fermentation Organic Acids, Short-Chain Fatty Acids, Microbiota-Associated Enzyme Activities, and Metabolic Safety Evaluation

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Short-chain fatty acid standard	79-09-4	P110449	Propionic acid	For insect cell culture, ≥99.5%	Can be used as a short-chain fatty acid standard or treatment reagent for fermentation metabolite quantification, propionate production capacity evaluation, and intestinal metabolic environment research.
Short-chain fatty acid standard	107-92-6	B431361	Butyric acid	Moligand™, suitable for synthesis	Can be used as a butyrate detection standard or treatment reagent for studies on intestinal barrier function, inflammation regulation, and short-chain fatty acid-related mechanisms.
Butyrate treatment reagent	156-54-7	S102956	Sodium butyrate	BioReagent, ≥99%	Can be used in cell models to simulate butyrate action and evaluate intestinal epithelial barrier function, inflammatory responses, and effects related to probiotic metabolites.
Fermentation intermediate organic acid	110-15-6	S431422	Succinic acid	PharmPure™, ChP, JP, ACS, NF, crystalline	Can be used as a research material for intestinal microbial intermediate metabolites, supporting studies on fermentation metabolic pathways, cross-feeding, and organic acid profiling.
Lactic acid isomer standard	79-33-4	S485917	(S)-Lactic acid	PharmPure™, BP, European Pharmacopoeia (Ph.Eur)	Can be used to distinguish lactic acid isomer production, evaluate lactic acid bacteria fermentation characteristics, and analyze the effects of lactate metabolism on the intestinal environment.
Lactic acid isomer and safety indicator	10326-41-7	S161106	D-Lactic acid	Moligand™, ≥90% (T)	Can be used for lactic acid isomer detection, strain metabolic safety evaluation, and analysis of D-lactate production risk in fermentation systems.
Mixed lactic acid control	50-21-5	L108839	DL-Lactic acid	AR, 85–90%	Can be used as a mixed lactic acid system control for establishing methods to analyze fermentation acidity, total lactic acid, and different lactic acid isomers.
Urease activity detection substrate	57-13-6	U111904	Urea	For cell culture, ≥99.5% (T)	Can be used as a substrate for urease activity detection to evaluate nitrogen metabolism-related enzyme activity and potential safety indicators in candidate strains or samples.
β-Glucuronidase substrate	10344-94-2	N1500709	4-Nitrophenyl β-D-glucuronide	Moligand™, 10 mM in water	Can be used as a β-glucuronidase substrate for detecting microbiota-associated enzyme activity and studying the risk of reactivation of conjugated metabolites.
β-Glucosidase substrate	2492-87-7	N573315	4-Nitrophenyl β-D-glucopyranoside	≥98%	Can be used as a β-glucosidase substrate to evaluate strain glycoside-hydrolyzing capacity, transformation of plant bioactive compounds, and enzyme activity related to polysaccharide utilization.
Biogenic amine standard	51-67-2	T105543	Tyramine	Moligand™, ≥98%	Can be used as a biogenic amine standard for fermentation safety, strain decarboxylase activity, and biogenic amine risk evaluation in probiotic formulations.
Biogenic amine standard	51-45-6	H111796	Histamine, free base	Moligand™, ≥96%	Can be used to establish biogenic amine detection methods and evaluate fermented product safety, supporting screening of strains or contaminating microorganisms that may produce histamine.
Biogenic amine standard	110-60-1	D110948	1,4-Diaminobutane	Moligand™	Can be used as a putrescine-related standard for biogenic amine monitoring in fermentation systems, strain metabolic safety assessment, and product quality evaluation.
Biogenic amine standard	462-94-2	D283638	1,5-Diaminopentane	≥99.5%	Can be used as a cadaverine-related standard for biogenic amine risk analysis in fermented foods, strain cultures, and probiotic formulations.

Table 4｜Products Related to Gastrointestinal Digestion Simulation, Bile Salt Tolerance, and Bile Acid Transformation

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Gastric-phase digestive enzyme	9001-75-6	rp218612	Recombinant pepsin (MS grade)	Animal-free, carrier-free, bioactive, mass spectrometry grade (MS), ActiBioPure™, EnzymoPure™, for protein sequencing, ≥10,000 U/g enzyme powder; ≥20,000 U/g protein	Can be used to simulate the gastric digestion environment and evaluate the stability of probiotic formulations, encapsulation systems, or postbiotic samples under gastric conditions.
Small-intestinal-phase digestive enzyme	8049-47-6	P755357	Pancreatin from porcine pancreas	Bioactive, ActiBioPure™, natural, high-performance, EnzymoPure™, 8 × USP; protease ≥200 U/mg enzyme powder; amylase ≥200 U/mg enzyme powder; lipase ≥16 U/mg enzyme powder	Can be used to simulate small-intestinal digestion conditions and examine the stability of probiotic delivery systems, food matrices, and encapsulation materials during digestion.
Conjugated bile acid salt and bile salt hydrolase substrate	145-42-6	S100834	Sodium taurocholate	≥95%	Can be used as a taurine-conjugated bile acid substrate for studies on bile salt hydrolase activity, strain bile tolerance, and conjugated bile salt transformation capacity.
Primary bile acid salt and bile salt stress reagent	361-09-1	S161419	Sodium cholate	Moligand™, ≥98%	Can be used for bile salt tolerance, bile acid treatment, and bile acid transformation-related experiments, supporting evaluation of candidate strains’ adaptability to bile acid environments; bile salt hydrolase activity assays should preferentially use conjugated bile acid substrates.
Primary bile acid standard	81-25-4	C103690	Cholic acid, anhydrous	≥98%	Can be used as a primary bile acid control, for bile salt transformation research, and for establishing bile acid profiling methods, supporting analysis of probiotic-related bile acid metabolic changes.
Primary bile acid standard	474-25-9	C104902	Chenodeoxycholic acid (CDCA)	Moligand™, ≥98%	Can be used as a primary bile acid standard or treatment compound to study the effects of strains on bile acid composition, transformation pathways, and host metabolism-related indicators.
Secondary bile acid salt and bile salt stress reagent	302-95-4	S485305	Sodium deoxycholate	Suitable for the manufacture of diagnostic kits and reagents	Can be used in bile salt tolerance experiments and intestinal bile stress simulation to evaluate the survival and adaptability of candidate strains under small intestine-related conditions.
Secondary bile acid standard	83-44-3	D103697	Deoxycholic acid (DCA)	Moligand™, ≥98%	Can be used as a secondary bile acid research material for bile acid profiling, strain bile salt transformation capacity studies, and intestinal metabolic response research.
Secondary bile acid standard	434-13-9	L106779	Lithocholic acid	Moligand™, ≥97%	Can be used as a secondary bile acid research material for studies on bile acid metabolism, microbiota transformation, and mechanisms under intestinal inflammation-related contexts.

Table 5｜Products Related to Intestinal Barrier, Inflammatory Response, and Cell Function Evaluation

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Inflammatory stimulation reagent	93572-42-0	L386714	Lipopolysaccharide (LPS)	From Escherichia coli 055:B5, purified by trichloroacetic acid extraction	Can be used to establish inflammatory stimulation models and evaluate the regulatory effects of probiotics, postbiotics, or fermentation metabolites on inflammatory responses in immune cells and intestinal epithelial cells.
Intestinal barrier injury model reagent	9011-18-1	D110733	Dextran sulfate sodium salt (DSS)	Molecular weight 500,000, DNase/RNase/protease-free	Can be used in animal models of intestinal inflammation and intestinal barrier injury, and in some in vitro systems for exploration of barrier injury conditions; dose, molecular weight, treatment duration, and model type can significantly affect result interpretation.
Barrier permeability tracer	60842-46-8	F291064	FITC-dextran	110 kDa	Can be used to detect intestinal epithelial permeability and barrier integrity. This product has a molecular weight of 110 kDa and is suitable for observing leakage of larger molecules across the barrier; interpretation should correspond to the experimental model, cell monolayer status, and detection time.
Cell viability assay reagent	298-93-1	D274386	Thiazolyl blue tetrazolium bromide	Ultrapure grade	Can be used for cell viability and cytocompatibility assays to evaluate the effects of probiotic supernatants, postbiotics, or metabolites on intestinal epithelial cells and immune cells.
Metabolic activity assay reagent	62758-13-8	R105538	Resazurin	≥90%	Can be used for cell metabolic activity, strain activity, and antimicrobial assay readouts, suitable for evaluating probiotic viability, cytotoxicity, and functional screening.

Table 6｜Products Related to Selective Cultivation, Strain Isolation, and Contamination Control

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Selective inhibitor for bifidobacterial cultivation	73346-79-9	M352084	Mupirocin lithium	≥95%	Can be used in selective cultivation systems for bifidobacteria, supporting isolation, counting, and cultivation condition screening of target microbial groups from complex samples.
Selective antibacterial and contamination control reagent	389-08-2	N104920	Nalidixic acid	≥98%	Can be used in selective cultivation systems and contamination control experiments, supporting isolation of target strains from complex samples and reducing interference from sensitive contaminating microorganisms.
Antifungal and eukaryotic cell inhibitor	66-81-9	C729197	3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-carboxyethyl]glutaramide	Moligand™, ≥98%	Can be used to inhibit fungal or eukaryotic cell proliferation, supporting bacterial isolation and cultivation, sample contamination control, and establishment of selective cultivation conditions.

Note: The above products are representative Aladdin products. More product specifications can be searched on the Aladdin official website using the “product name/CAS/catalog number.”

References

[1] Hill C, Guarner F, Reid G, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology & Hepatology, 2014, 11: 506–514.

[2] FAO/WHO. Guidelines for the Evaluation of Probiotics in Food. Food and Agriculture Organization of the United Nations and World Health Organization, 2002.

[3] Guarner F, Sanders M E, Szajewska H, et al. World Gastroenterology Organisation Global Guidelines: Probiotics and Prebiotics. World Gastroenterology Organisation, 2023.

[4] National Institutes of Health, Office of Dietary Supplements. Probiotics: Fact Sheet for Health Professionals. 2025.

[5] Salminen S, Collado M C, Endo A, et al. The International Scientific Association of Probiotics and Prebiotics consensus statement on the definition and scope of postbiotics. Nature Reviews Gastroenterology & Hepatology, 2021, 18: 649–667.

[6] Zheng J, Wittouck S, Salvetti E, et al. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 2020, 70: 2782–2858.

[7] Gibson G R, Hutkins R, Sanders M E, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics consensus statement on the definition and scope of prebiotics. Nature Reviews Gastroenterology & Hepatology, 2017, 14: 491–502.

[8] McClanahan C. Probiotics and Human Health. Sigma-Aldrich Technical Article.

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