Patent Grant
11931388
The present disclosure relates to a postbiotic extract and a process of preparing the same. The present disclosure also relates to use of this postbiotic extract to improve gut health and to inhibit biofilm formation.
Probiotics are resident normal flora of the intestinal tract and believed to play important roles in regulating proper intestinal immunity and digestion by balancing intestinal microflora. These beneficial microorganisms are widely used as live microbial dietary supplement and can help restoring intestinal microfloral balance. Many species of lactic acid bacteria (LAB), such as Lactobacillus spp., Lactococcus spp., Streptococcus spp., Enterococcus spp., and Bifidobacterium spp., are generally considered as probiotics. Furthermore, some of Bacillus spp. and some yeasts and Saccharomyces spp. have also been found as suitable candidates.
Probiotics are viable by definition, and their stability and viability are considered to be crucial for their health benefits. Paraprobiotics or postbiotics have emerged to denote that non-viable microbial cells, microbial fractions, or cell lysates might also offer physiological benefits to the host by providing additional bioactivity. Postbiotic efficacy is based on the microbial metabolites, proteins, lipids, carbohydrates, vitamins, organic acids, cell wall components, or other complex molecules that are generated in the matrix that is fermented. These postbiotics have drawn attention because of their clear chemical structure, safety dose parameters, long shelf life and the content of various signaling molecules which may have anti-inflammatory, immunomodulatory, anti-obesogenic, antihypertensive, hypocholesterolemic, anti-proliferative, and antioxidant activities.
Postbiotics can be obtained using cell disruption methods, which include heat, enzymatic treatment, solvent extraction, and sonication. However, these methods usually have low extraction efficiency and use large amounts of solvents.
Thus, there is still a need to develop a new method for efficiently producing a postbiotic from a probiotic microorganism.
Accordingly, in a first aspect, the present disclosure provides a process for producing a postbiotic extract, including the steps of:
In a second aspect, the present disclosure provides a postbiotic extract which is prepared by a process as described above.
In a third aspect, the present disclosure provides a food product including a postbiotic extract as described above.
Ina fourth aspect, the present disclosure provides a method for inhibiting biofilm formation, which includes applying a postbiotic extract as described above onto an object.
In a fifth aspect, the present disclosure provides a method for improving gut health, which includes administering to a subject a postbiotic extract as described above.
The above and other objects, features and advantages of the present disclosure will become apparent with reference to the following detailed description and the exemplary embodiments taken in conjunction with the accompanying drawings, in which:
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Taiwan or any other country.
For the purpose of this specification, it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described.
The present disclosure provides a process for producing a postbiotic extract, comprising the steps of:
According to the present disclosure, the probiotic microorganism may be selected from the group consisting of Bacillus spp., Streptococcus spp., Lactococcus spp., Abiotrophia spp., Aerococcus spp., Carnobacterium spp., Enterococcus spp., Lactobacillus spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Tetragenococcus spp., Vagococcus spp., Weissella spp., Bifidobacterium spp., Saccharomyces spp., Kluyveromyces spp., Staphylococcus spp., Pediococcus spp., Propionibacterium spp., and combinations thereof.
According to the present disclosure, the Lactobacillus spp. may be selected from the group consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus paracasei, and combinations thereof.
According to the present disclosure, the Bifidobacterium spp. may be selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, and combinations thereof.
According to the present disclosure, the Bacillus spp. may be selected from the group consisting of Bacillus coagulans, Bacillus subtilis, Bacillus clausii, and combinations thereof.
According to the present disclosure, the probiotic microorganism may be alive or dead, concentrated or non-concentrated, or in the form of a liquid, a paste, a semi-solid, or a solid (e.g. a pellet, a granule, or a powder), and may be heat-inactivated, frozen, dried, or freeze-dried (for example, may be in a freeze-dried form or a spray/fluid bed dried form). In an exemplary embodiment, the probiotic microorganism is heat-inactivated and is in a spray-dried powder form.
According to the present disclosure, the heat inactivation of the probiotic microorganism may be conducted at 60° C. to 140° C. for 1 second to 30 minutes. In an exemplary embodiment, the heat inactivation is conducted at 73±2° C. for 15 seconds.
According to the present disclosure, the first material may be selected from the group consisting of nonfat dry milk, casein, whey proteins, soybean proteins, pea proteins, egg proteins, rice proteins, hydrolyzed proteins, corn proteins, wheat proteins, barley proteins, gelatin, collagen, amino acids (for instance, branched chain amino acids), chitosan, chitin, and combinations thereof. In an exemplary embodiment, the first material is a whey protein.
According to the present disclosure, the second material may be selected from the group consisting of sodium alginate, agar, carrageenan, pectin, arabic gum, xanthan gum, locust bean gum, starch (such as modified starch), trehalose, dextrin (such as resistant maltodextrin), syrup, guar gum, konjac powder, vegetable fiber, synthetic fiber, semi-synthetic fiber, and combinations thereof. In an exemplary embodiment, the second material is dextrin.
In an exemplary embodiment of the present disclosure, the first isoelectric point and the second isoelectric point have a pH difference of 0.8.
According to the present disclosure, the precipitate may be obtained by methods well known in the art, including but not limited to centrifugation, filtration, and gravity settling. In an exemplary embodiment, the precipitate is obtained by filtration.
As used herein, the terms “isolation treatment” and “extraction treatment” can be used interchangeably, and mean that a cell wall component or a microbial metabolite is to be separated from a cell wall, in which it was originally present, through a treatment.
According to the present disclosure, the procedures and conditions of the cell wall isolation treatment are within the expertise and routine skills of those skilled in the art (for example, see Pei-Jun Tian et al. (2015), Int. J. Mol. Sci., 16(8): 20033-20049).
The present disclosure also provides a postbiotic extract which is prepared by a process as described above.
The present disclosure also provides a food product, which includes a postbiotic extract as described above. According to the present disclosure, for preparing the food product, the postbiotic extract may be incorporated into an edible material using a standard technique well known to one of ordinary skill in the art. For instance, the aforesaid postbiotic extract may be directly added to the edible material, or may be utilized for preparing an intermediate composition (e.g., a food additive or a premix) suitable to be subsequently added to the edible material.
According to the present disclosure, the food product may be in the form of fermented foods, processed foods, health foods, or dietary supplements.
The food product according to the present disclosure may further include at least one probiotic microbe. As used herein, the terms “probiotic microbe” and “probiotic” are used interchangeably, and refer to preparations microorganisms of live microorganisms. These microorganisms may remain and survive in the gastrointestinal tract after ingested by an animal, and can exert a desired effect (e.g. gut microbiota modifying effect, preventive or therapeutic effect, etc.).
Probiotic microorganisms suitable for use in the present disclosure include, but are not limited to, a Lactobacillus sp., a Enterococcus sp., a Streptococcus sp., a Pediococcus sp., a Bacillus sp., a Bifidobacterium sp., yeasts, and their combinations.
According to the present disclosure, the food product may further comprise an additional food additive selected from the group consisting of starch, dextrin, lactose, maize flour, rice flour, tricalcium phosphate, silicon dioxide, magnesium stearate, calcium carbonate, glucose, sucrose, fructose, sugar alcohol, oligosaccharides, sugar substitutes, fruit juice powder, yeast powder, nonfat dry milk, casein, whey proteins, amino acids, citric acid, citrate, lactic acid, lactate, nucleotides, and their combinations.
In addition, the postbiotic extract according to the present invention may be prepared in the form of a pharmaceutical composition.
According to the present disclosure, the pharmaceutical composition may be formulated into a suitable dosage form for parenteral, oral or topical administration using technology well known to those skilled in the art. The suitable dosage form includes, but is not limited to, sterile powder, tablets, troches, lozenges, pellets, capsules, dispersible powder or granules, solutions, suspensions, emulsions, syrup, elixir, slurry, external preparations, and the like.
The pharmaceutical composition according to the present disclosure may further include a pharmaceutically acceptable carrier widely employed in the art of drug-manufacturing. For instance, the pharmaceutically acceptable carrier may include one or more of the following agents: solvents, buffers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption delaying agents, liposomes, and the like. The choice and amount of the aforesaid agents are within the expertise and routine skills of those skilled in the art.
The present disclosure also provides a method for inhibiting biofilm formation, which includes applying a postbiotic extract as described above onto an object.
As used herein, the term “biofilm formation” refers to the attachment of microorganisms to surfaces and the subsequent development of multiple layers of cells.
As used herein, the term “inhibition” or “inhibiting” refers to a decrease of biofilm associated microorganism formation and/or growth. The microorganisms may include gram-positive or gram-negative bacteria, yeasts, and fungi.
According to the present disclosure, the object may be a medical device, a medical instrument, a dressing, a bandage, a food preparation surface, a food packaging surface, a manufacturing surface, a consumer good, a water treatment system, a water delivery system, or a ventilation system.
In certain embodiments, the object may be selected from the group consisting of a denture, a mouth guard, a dairy line, a water line, an adhesive bandage, a component of an HVAC (heating, ventilation, and air-conditioning) system, a component of a water treatment facility, a component of a vacuum or a vacuum cleaner, a vacuum cleaner bag, a vacuum cleaner filter, an air filter, a component of a cooling tower, a toy, a window, a door, a window frame, a doorframe, a medical instrument, a dental instrument, a bathroom tile, a kitchen tile, food industry processing instruments, hospital tables and beds, an animal water dish, a washing machine, a dish washer, a towel, a dish, a bowl, a utensil, a cup, a glass, a cutting board, a dish drying tray, a whirlpool bathtub, a sink, a toilet, a toilet seat, a swimming pool, a birdbath, a planter, a garden hose, a fish pond, an oil pipe, a gas pipe, a dairy line filter, a line used in food and beverage manufacturing, a cosmetic container, an outdoor pond liner, a tap and water spout, a humidifier, a humidifier filter, a bathroom tile, a bathroom fixture, a toilet lid, a swimming pool liner, a swimming pool skimmer, a swimming pool filter, a hot tub line, a hot tub filter, a washing machine liner, a dishwasher liner, an animal water dish, a food storage container, a beverage storage container, a plate, a cup, a fork, a knife, a spoon, a garbage bag, and a counter top.
According to the present disclosure, biofilm formation may be caused by a microbe selected from the group consisting of Campylobacter spp., Clostridium perfringens, Escherichia coli, Listeria monocytogenes, Vibrio cholerae, Salmonella spp., Staphylococcus spp., and combinations thereof.
Examples of the Staphylococcus spp. include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus warneri, Staphylococcus hominis, Staphylococcus simulans, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus pasteuri, Staphylococcus cohnii, Staphylococcus xylosus, and Staphylococcus saccharolyticus.
The present disclosure also provides a method for improving gut health, which includes administering to a subject a postbiotic extract as described above.
As used herein, the term “improving gut health” means that an individual, upon treatment with the postbiotic extract, exhibits a healthy gut/intestinal microbiota, which is beneficial for human or animal health and suitable for a maintenance and/or an improvement of the digestion of said individual. Such a healthy gut/intestinal microbiota is ultimately linked to proper nutrient absorption, adequate growth, less colic, less infection, less diarrhea, and the best gut health.
The disclosure will be further described by way of the following examples. However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the disclosure in practice.
General Experimental Materials:
Lactobacillus spp.
Lactobacillus
plantarum CB102
Lactobacillus
acidophilus JCM1132
Lactobacillus
casei JCM1134
Bifidobacterium spp.
Bifidobacterium
bifidum JCM1255
Bifidobacterium
lactis JCM10602
Bifidobacterium
longum CB108
Bacillus spp.
Bacillus
coagulans CB106
The TGF-β content was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cat. No. 559119, BD Biosciences) in accordance with the manufacturer's instructions.
A respective one of Lactobacillus plantarum CB102 (deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH (Inhoffenstraße 7B, 38124 Braunschweig, Germany) under an accession number DSM 33894 on 6 Sep. 2021 in accordance with the Budapest Treaty), Lactobacillus acidophilus JCM1132, Lactobacillus casei JCM1134, Bifidobacterium bifidum JCM1255, Bifidobacterium lactis JCM10602, and Bifidobacterium longum CB108 (deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH (Inhoffenstraße 7B, 38124 Braunschweig, Germany) under an accession number DSM 33895 on 6 Sep. 2021 in accordance with the Budapest Treaty) was inoculated in a Lactobacilli MRS broth (BD DIFCO, Cat. No. DF0881-17-5), and was then cultivated in an incubator (37° C., 5% CO2) for 16 hours. The respective resultant culture was inactivated by high-temperature short-time (HTST) pasteurization (73±2° C., 15 seconds), followed by centrifugation at 10,000 rpm and 25° C. for 15 minutes. The resultant cell pellet was collected, followed by spray-drying. A portion of the respective dried bacterial cell powder was subjected to a pretreatment as follows.
A suitable amount of whey protein having an isoelectric point of 4.4 (NZMP, Cat. No. WPC80) was dissolved in water, and the resultant 10% whey protein solution (w/v, g/L) was adjusted to pH 7.5 through addition of sodium carbonate, followed by adding a suitable amount of a respective one of the six dried bacterial cell powders obtained above under agitation to reach a final concentration of 5% (w/v, g/L). Thereafter, dextrin having an isoelectric point of 5.2 (ZHUCHENG DONGXIAO, Cat. No. Maltodextrin DE8-10) was slowly added into the resultant mixture to reach a final concentration of 6% (w/v, g/L), followed by adjusting the dextrin-added mixture to a pH of 4.8 using lactic acid, so that a precipitate was formed due to charge neutralization. Filtration was conducted using a filter paper having a pore size of 25 μm, so as to obtain the precipitate. The precipitate was subjected to a spray drying treatment, thereby obtaining a pretreated bacterial cell powder.
The isolation and extraction of the cell wall of each pretreated bacterial cell powder was conducted using a method slightly modified from that described by Pei-Jun Tian et al. (2015), Int. J. Mol. Sci., 16 (8): 20033-20049. Briefly, 50 mg of the respective pretreated bacterial cell powder was mixed with 1 mL of 10% lactic acid, followed by heating in a water bath incubator (80° C.) for 60 minutes. After centrifugation at 10,000 g for 15 minutes, the resultant pellet was collected and was mixed with 1 mL of a solution containing a 0.5 M citrate solution and ethanol (4:10, v/v, pH 4.6), followed by incubation overnight. After centrifugation at 10,000 g for 20 minutes, the pellet thus obtained was washed with 95% ethanol, followed by heating in a dry bath incubator (80° C.) for 40 minutes to remove ethanol. Thus, a postbiotic extract was obtained. The resultant postbiotic extract was used for the following example and is referred to as “the postbiotic extract of the present disclosure” hereinafter.
In addition, for the sake of comparison, another portion of the respective one of the six dried bacterial cell powders, which was not pretreated according to the procedures described above, was subjected to the same isolation and extraction processes. The postbiotic extract thus obtained was used for the following example and is referred to as “the postbiotic extract of the prior art” hereinafter.
In order to determine the extraction yield and the protein content, the postbiotic extract of the present disclosure and the postbiotic extract of the prior art obtained in Example 1 were subjected to the following analyses.
A. Determination of Extraction Yield
The weight of each of the twelve postbiotic extracts was recorded. The extraction yield (%) of the respective postbiotic extract was calculated using the following Equation (I):
A==(B/50)×100 (I)
where A==extraction yield (%)
The result is shown in Table 2 below. It can be seen from Table 2 that the extraction yields of the six postbiotic extracts of the present disclosure were significantly higher than those of the six postbiotic extracts of the prior art, indicating that the process of the present disclosure can effectively produce postbiotic extracts from probiotics.
Lactobacillus
plantarum CB102
Lactobacillus
acidophilus JCM1132
Lactobacillus
casei
Bifidobacterium
bifidum JCM1255
Bifidobacterium
lactis JCM10602
Bifidobacterium
longum CB108
B. Determination of Protein Content
The respective postbiotic extract was dissolved in a phosphate buffer solution (containing 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, and 0.24 g/L KH2PO4, and having a pH of 6.2), followed by determining the protein content with Pierce™ BCA Protein Assay Kit (Thermo Scientific, Cat. No. 23225) according to the manufacturer's instructions.
The result is shown in Table 3 below. It can be seen from Table 3 that the protein contents of the six postbiotic extracts of the present disclosure were significantly higher than those of the six postbiotic extracts of the prior art.
Lactobacillus
plantarum CB102
Lactobacillus
acidophilus JCM1132
Lactobacillus
casei
Bifidobacterium
bifidum JCM1255
lactis JCM10602
Bifidobacterium
longum CB108
C. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis
1 g of the respective postbiotic extract was dissolved in 20 mL of water, followed by performing SDS-PAGE analysis using an electrophoresis system (Bio-Rad).
Referring to
Experimental Procedures:
Staphylococcus aureus was inoculated into a tryptic soy broth (TSB) (BD Bacto™, Cat. No. DF0370-17-3), followed by cultivation in an incubator (37° C., 5% CO2) for 16 hours. The resultant culture was divided into 17 groups, including one control group, eight comparative groups (i.e., comparative groups L-1 to L-4 and B-1 to B-4), and eight experimental groups (i.e., experimental groups L-1 to L-4 and B-1 to B-4). The culture of each group was incubated in a respective well of a 96-well culture plate at a cell number of 1×1010 CFU/L. Thereafter, each of the cultures of the eight comparative groups and eight experimental groups was treated with the respective test postbiotic extract so that the culture of each group had a final concentration of the respective test postbiotic extract as shown in Table 4. The culture of the control group received no treatment.
plantarum CB102
Lactobacillus
plantarum CB102
longum CB108
Bifidobacterium
longum CB108
Each group was cultivated in an incubator (37° C., 5% CO2) for 24 hours. The liquid in each well was removed, followed by washing with phosphate-buffered saline (PBS). Thereafter, 100 μL of 95% ethanol was added into each well, followed by incubating at room temperature for 10 minutes, and the resultant fixed cells were then stained with 100 μL of 0.1% crystal violet for 15 minutes. The liquid in each well was removed, followed by washing with PBS. Thereafter, 200 μL of 10% acetic acid was added into each well, followed by incubating at room temperature for 10 minutes, so as to solubilize crystal violet. The resultant biofilm contained in each well was subsequently subjected to determination of absorbance at a wavelength of 590 nm by a spectrophotometer.
The inhibition rate (%) was calculated using the following Equation (II):
C=(1−D/E)×100 (II)
Table 5 shows the inhibition rate of each group. It can be seen from Table 5 that the inhibition rates determined in the experimental groups L-1 to L-4 were respectively higher than those determined in the comparative groups L-1 to L-4, and the inhibition rates determined in the experimental groups B-1 to B-4 were also respectively higher than those determined in the comparative groups B-1 to B-4. Therefore, the applicant contemplates that the postbiotic extract of the present disclosure is effective in inhibiting biofilm formation.
Lactobacillus
plantarum
Lactobacillus
plantarum
Bifidobacterium
longum
Bifidobacterium
longum
Experimental Procedures:
Staphylococcus aureus and a respective one of Lactobacillus plantarum CB102, Lactobacillus acidophilus JCM1132, Lactobacillus casei JCM1134, Bifidobacterium bifidum JCM1255, Bifidobacterium lactis JCM10602, Bifidobacterium longum CB108, and Bacillus coagulans CB106 were inoculated in a TSB, followed by cultivation in an incubator (37° C., 5% CO2) for 8 hours. The respective resultant co-culture was divided into 4 groups (including one control group, one comparative group, and two experimental groups), and each group was treated with a test sample as shown in Table 6.
Staphylococcus
aureus and
Lactobacillus
acidophilus
from Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Staphylococcus
aureus and
Lactobacillus
casei JCM1134
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Staphylococcus
aureus and
Lactobacillus
plantarum CB102
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Staphylococcus
aureus and
Bifidobacterium
lactis JCM10602
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Staphylococcus
Bifidobacterium
bifidum JCM1255
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Staphylococcus
aureus and
Bifidobacterium
longum CB108
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Staphylococcus
aureus and
Bacillus
coagulans CB106
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
All the groups were cultivated in an incubator (37° C., 5% CO2) for 8 hours. Thereafter, a suitable amount of the respective resultant co-culture was coated onto a MRS agar plate using spread plate technique, followed by cultivation in an incubator (37° C., 5% CO2) overnight. The number of colonies of probiotic microbes on the MRS agar plate of each group was counted, and the log value of the colony forming unit (CFU/mL) was further calculated and the viable cell count was indicated by log CFU/mL.
Results:
Table 7 shows the viable cell count of probiotic microbes in each group. It can be seen from Table 7 that, for the co-culture of Staphylococcus aureus and Lactobacillus acidophilus JCM1132, the increased viable cell counts of probiotic microbes (i.e., Lactobacillus acidophilus JCM1132) determined in the experimental groups 1-L and 1-B were higher than those determined in the comparative group 1 and the control group 1. Besides, similar satisfactory results were observed with respect to the experimental groups 2-L to 7-L and 2-B to 7-B, indicating that the postbiotic extract of the present disclosure can effectively prevent the imbalance of the gut microbiota, and can eliminate or decrease potential or known pathogenic populations in the gut or intestine, and hence is capable of restoring and maintaining a healthy gut microbiota.
Lactobacillus
acidophilus
Lactobacillus
casei JCM1134
Lactobacillus
plantarum CB102
Bifidobacterium
lactis JCM10602
Bifidobacterium
bifidum JCM1255
Bifidobacterium
longum CB108
Bacillus
coagulans
A. Effect of Postbiotic Extract on TGF-β Content of Caco-2 Cells
Caco-2 cells were divided into 25 groups, including one control group, twelve comparative groups (i.e., comparative groups L-1 to L-6 and B-1 to B-6), and twelve experimental groups (i.e., experimental groups L-1 to L-6 and B-1 to B-6). Each group of the Caco-2 cells was incubated in a respective well of a 96-well culture plate containing 200 μL of DMEM at 1×104 cells/well, followed by cultivation in an incubator (37° C., 5% CO2) for 24 hours. After medium change with a fresh medium, each of the cell cultures of the twelve comparative groups and twelve experimental groups was treated with the respective test postbiotic extract so that the cell culture of each group had a final concentration of the respective test postbiotic extract as shown in Table 8. The cell culture of the control group received no treatment.
Lactobacillus
plantarum CB102
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Bifidobacterium
longum CB108
After cultivation in an incubator (37° C., 5% CO2) for 24 hours, the respective resultant cell culture was subjected to the determination of TGF-β content according to the method described in section 1 of “General Procedures”.
As shown in Table 9 below, the TGF-β contents determined in the experimental groups L-1 to L-6 were respectively higher than those determined in the comparative groups L-1 to L-6, and the TGF-β contents determined in the experimental groups B-1 to B-6 were also respectively higher than those determined in the comparative groups B-1 to B-6. This result suggests that the postbiotic extract of the present disclosure is effective in inducing TGF-β secretion in Caco-2 cells.
B. Effect of Postbiotic Extract on TGF-β Content of Caco-2 Cells in the Presence of Gastric Acid
A suitable amount of a respective one of Lactobacillus plantarum CB102, Lactobacillus acidophilus JCM1132, Lactobacillus casei JCM1134, Bifidobacterium bifidum JCM1255, Bifidobacterium lactis JCM10602, Bifidobacterium longum CB108, the postbiotic extract of the present disclosure from Lactobacillus plantarum CB102, and the postbiotic extract of the present disclosure from Bifidobacterium longum CB108 was mixed with artificial gastric acid (containing 0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, and 0.0018 M NaH2PO4, and having a pH of 2), followed by cultivation in an incubator (37° C., 5% CO2) for 3 hours. The resultant mixtures were used for the following experiment and are referred to as “gastric acid-treated” test samples hereinafter.
In addition, Caco-2 cells were divided into 16 groups, including eight comparative groups (i.e., comparative groups 1-8) and eight experimental groups (i.e., experimental groups 1-8). Each group of the Caco-2 cells was incubated in a respective well of a 96-well culture plate containing 200 μL of DMEM at 1×104 cells/well, followed by cultivation in an incubator (37° C., 5% CO2) for 24 hours. After medium change with a fresh medium, each of the cell cultures of the eight comparative groups and eight experimental groups was treated with the respective test sample so that the cell culture of each group had a final concentration of the respective test sample as shown in Table 10.
Lactobacillus
acidophilus JCM1132
Lactobacillus
acidophilus JCM1132
Lactobacillus
casei
Lactobacillus
casei
Lactobacillus
plantarum CB102
Lactobacillus
plantarum CB102
Bifidobacterium
lactis JCM10602
Bifidobacterium
lactis JCM10602
Bifidobacterium
bifidum JCM1255
Bifidobacterium
bifidum JCM1255
Bifidobacterium
longum CB108
Bifidobacterium
longum CB108
Lactobacillus
plantarum CB102
Lactobacillus
plantarum CB102
Bifidobacterium
longum CB108
Bifidobacterium
longum CB108
After cultivation in an incubator (37° C., 5% CO2) for 24 hours, the respective resultant cell culture was subjected to the determination of TGF-β content according to the method described in section 1 of “General Procedures”.
As shown in Table 11 below, the TGF-β contents determined in the experimental groups 1-6 were significantly lower than those determined in the comparative groups 1-6, respectively. However, the TGF-β contents of the experimental groups 7-8 were only slightly decreased as compared to the comparative groups 7-8, respectively, indicating that the postbiotic extract of the present disclosure has an excellent acid tolerance and is able to overcome the environmental pressure posed by the human digestive tract, and hence can reach the intestine (s) and colonize the probiotics therein after ingestion.
C. Effect of the Combination of Postbiotic Extract with Probiotics on TGF-β Content of Caco-2 Cells
Caco-2 cells were divided into 21 groups, including seven comparative groups (i.e., comparative groups 1-7) and fourteen experimental groups (i.e., experimental groups 1-L to 7-L and 1-B to 7-B). Each group of the Caco-2 cells was incubated in a respective well of a 96-well culture plate containing 200 μL of DMEM at 1×104 cells/well, followed by cultivation in an incubator (37° C., 5% CO2) for 24 hours. After medium change with a fresh medium, the culture of each group was treated with probiotics only or additionally with the postbiotic extract of the present disclosure so that the cell culture of each group respectively had final concentrations of the probiotics only or additionally with the postbiotic extract as shown in Table 12.
Bifidobacterium
plantarum
longum CB108
Lactobacillus
acidophilus
Lactobacillus
casei JCM1134
Lactobacillus
plantarum
Bifidobacterium
lactis JCM10602
Bifidobacterium
bifidum JCM1255
Bifidobacterium
longum CB108
Bacillus
coagulans
After cultivation in an incubator (37° C., 5% CO2) for 24 hours, the respective resultant cell culture was subjected to the determination of TGF-β content according to the method described in section 1 of “General Procedures”.
As shown in Table 13 below, the TGF-β contents determined in the experimental groups 1-L to 7-L and 1-B to 7-B were respectively higher than those determined in the comparative groups 1-7, indicating that the combination of a postbiotic extract with probiotics is effective in inducing TGF-β secretion in Caco-2 cells.
Summarizing the above test results, it is clear that the postbiotic extract of the present disclosure has an excellent acid tolerance, and can effectively induce TGF-β secretion in Caco-2 cells, and hence can be used as a food additive for modulating gut immunity.
All patents and references cited in this specification are incorporated herein in their entirety as reference. Where there is conflict, the descriptions in this case, including the definitions, shall prevail.
While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.
This application is a divisional application of U.S. patent application Ser. No. 17/037,090, filed on Sep. 29, 2020, now U.S. patent Ser. No. 11/541,085. The entire content of the prior application is incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20110002902 | Asada et al. | Jan 2011 | A1 |
| 20200093847 | Buck et al. | Mar 2020 | A1 |
| Entry |
|---|
| Zhou, Hai-Lu, “Isolation of Cell Wall Polysaccharides from Probiotics and Studies on Their Bioactivities”, Liaoning University Master's Thesis in Food Engineering, 2017, 2 pages with English translation. |
| Chang, Yi-Chung, “Race on Probiotics in the Intestinal Tract—Unmissable Opportunity”, Mar. 31, 2018, website: https://www.gbimonthly.com/2018/03/21651/, 7 pages with English translation. |
| Search Report appended to an Office Action, which was issued to Taiwanese counterpart application No. 109115724 by the TIPO dated Nov. 11, 2020, 2 pages with English translation. |
| Tian et al., “Extraction of Peptidoglycan from L. paracasei subp. Paracasei X12 and Its Preliminary Mechanisms of Inducing Immunogenic Cell Death in HT-29 Cells”, Int. J. Mol. Sci., 2015, vol. 16, pp. 20033-20049. |
| Number | Date | Country | |
|---|---|---|---|
| 20230285479 A1 | Sep 2023 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17037090 | Sep 2020 | US |
| Child | 18051968 | US |
