aloe vera ( av ) is a cactus - like plant which has been used widely in herbal medicines for millennia . the compounds in fleshy leaves , for example , aloe emodin , aloin , aloesin , saponins , terpenoids , and polysaccharides , possess the wound healing , anti - inflammatory , immunity , antidiabetic , antioxidant , laxative , antibacterial , antifungal , antiviral , and antitumor effects [ 26 ] . now , av has also been widely used as cosmetic - moisturizers , tooth pastes , food flavoring compounds , and preservative in the pharmaceutical and food fields . probiotics are defined as  live microorganisms which when administered in adequate amounts , confer a health benefit on the host . previous studies indicated that probiotics conferred miscellaneous health benefits include an improvement in lactose intolerance , an increase in natural resistance to infectious disease in the gastrointestinal tract , the suppression of cancer , an antidiabetic agent , and a decrease in serum cholesterol levels [ 79 ] . in recent decades , probiotics are the hotspot for microbiologist and nutritionists [ 10 , 11 ] , and the health awareness and busy lifestyle have created a strong and dynamic probiotics market which offers good prospects for consumers . among the probiotics , lactobacillus plantarum is pointed out as an industrially important microorganism that can be found and isolated from dairy products , fermented foods ( sauerkraut , sourdough , sausages , cheeses , wines , olives , and pickled vegetables ) , environments ( cow - dung , silage , and sewage ) , human mouth , intestinal tract , and stools [ 13 , 14 ] , and this strain had been widely used for the development of functional foods and potential oral vaccines . in our previous work , l. plantarum hm218749.1 was isolated from human intestinal , which could grow better at 37c in an anaerobic environment and could resist harsh conditions of gastrointestinal tract ( ph 2.5 and 0.30% bile salt ) . moreover , this strain could strongly adhere to ht-29 cells and reduce the adhesion of shigella dysenteriae 2457 , staphylococcus aureus cowan1 , enterobacter sakazakii 45401 , and escherichia coli 44102 to ht-29 cells , and its long - term residence in mice intestine increased the number of lactobacilli and decreased the number of enterococci . in the present study , the tested probiotics ( e.g. , bifidobacterium longum atcc15708 , l. plantarum hm218749 , l. rhamnosus gg , and streptococcus thermophilus g1 ) grew well in av substrate and conferred av sound antioxidant and antibacterial effects , and the l. plantarum hm218749.1 was finally chosen for its popularity in food fields and perfect beneficial effects verified in our previous studies , and the cell experiment was carried out to further verify the anti - inflammatory effects of the aloe fermentation supernatant . the av leaves were freshly harvested by cutting from the bottom and were washed thoroughly . the edges were removed and the leaves were peeled to obtain the slimy and transparent gel contents . the gel was then mashed using a pulper , which was then centrifuged at 4000  g for 10  min to obtain the av extract . the probiotic bifidobacterium longum atcc15708 ( isolated from the milk ) , l. plantarum hm218749 ( isolated from human intestine ) , l. rhamnosus gg ( isolated from the milk ) , and streptococcus thermophilus g1 ( isolated from the milk ) were cultured in de man rogosa sharpe ( mrs ) medium ( for lactobacilli and bifidobacterium ) and brain / heart infusion ( bhi ) medium ( for streptococcus thermophilus g1 ) for 24  h , and 10  cfu / ml of the strains was used as an inoculum to be added to aloe vera , aloe vera + 5% glucose , or aloe vera + 5% glucose + 5% skimmed milk for preparing the aloe fermentation supernatant , which was incubated for 15 to 18  h at 37c or 42c . finally , the aloe fermentation supernatant and av were freeze - dried using vacuum freeze drier under with the following conditions : freeze drying at 0.2  mbar vacuum , 40c for 48  h. the lyophilized powder was stored at 20c for further use . diluted aliquots were replica - plated onto bhi agar and mrs agar and then incubated anaerobically in the anaerobic system ( full of 85% n2 , 5% h2 , and 10% co2 ) at 37c or 42c for 24~36  h followed by counting colonies from plates showing 25250 colonies . for preparation of the aloe fermentation supernatant , 10% of the probiotics were added to av extract and fermented for 15 to 18  h at 37c , and then the aloe fermentation cultures were centrifuged at 4000  g for 10  min , and the pellets in the bottom of the centrifuge tubes ( containing insoluble materials or / and probiotics ) were deemed as the residual substance , and the clear water insoluble substance was deemed as supernatant , and the av extract was used as a control . then , the clearance of the dpph , o2 , oh , fe chelation , and oxygen - reduction activity were measured just as in . for antimicrobial activity , overnight ( 12  h ) cultures of the pathogenic microorganisms including salmonella typhimurium atcc 13311 , s. enteritidis atcc13076 , s. flexneri atcc 12022 , e. coli 44102 , listeria monocytogenes atcc 19111 , s. dysenteriae 301 , s. aureus cowan1 , and propionibacterium acnes atcc 11827 were spread on the surface of the lb agar plates , and the culture supernatant ( 200  l ) was loaded into an oxford cup ( outer diameter 7.8  0.1  mm , inner diameter 6.0  0.1  mm , and height 10.0  0.1  mm ) , which was placed on the surface of the agar . the size of the inhibition zone was measured until a formation of clear zone around the oxford cup . raw 264.7 cells ( passages 820 ) were maintained in dmem supplemented with 10% fbs and antibiotics ( 100  u / ml of penicillin and 100  g / ml of streptomycin ) at 37c in a 5% co2 incubator . emodin ( wuhan qi boster biological technology co. ltd . ) , polysaccharide ( wuhan qi boster biological technology co. ltd . ) , aloe vera ( lyophilized powder ) , and aloe vera + l. plantarum ( lyophilized powder ) were dissolved in phosphate - buffered saline ( pbs ) solution and their effect on cell viability of raw 264.7 cells was determined by mtt assay . raw 264.7 cells were plated at a density of 10000 per well in 100  l of complete culture medium and treated with designed concentrations of emodin ( 0 , 1 , 2.5 , 5 , and 10  g / ml ) , polysaccharide ( 0 , 2.5 , 5 , 15 , 30 , and 50  g / ml ) , aloe vera ( 0 , 5 , 15 , 30 , 50 , 100 , and 200  g / ml ) , and aloe vera + l. plantarum ( 0 , 5 , 15 , 30 , 50 , 100 , and 200  g / ml ) in 96-well microtiter plates for 36  h at 37c in a humidified incubator . control cells treated with pbs served as the vehicle group . after incubation for specified times , mtt reagent ( 20  l , 5  mg / ml ) was added to each well and incubated for 4  h. after careful removal of the mtt solution , 150  l of dmso was added to each well . the absorbance was recorded on a microplate reader at the wavelength of 570  nm . raw 264.7 cells were seeded into 24-well plates ( 5  10 cells per well ) for 24  h and were then washed with pbs . cells were treated with / without lipopolysaccharide ( lps , 1  g / ml each ) , emodin ( 2.5  g / ml and 10  g / ml ) , polysaccharide ( 2.5  g / ml and 15  g / ml ) , aloe vera ( 5  g / ml and 50  g / ml ) , and aloe vera + l. plantarum ( 5  g / ml and 50  g / ml ) for 24  h , and then the cells and supernatants were harvested for q - pcr and elisa analysis , respectively . for the evaluation of cytokine mrna expression levels , total rnas from the raw 264.7 cells were prepared by adding trizol reagent ( gibco brl , grand island , ny , usa ) according to the manufacturer 's protocol , and 2.4  g of total rna from each group was reverse transcribed to cdna using a commercially available kit ( applied biosystems ) . quantitative real - time pcr was performed with 7900ht fast real - time pcr system ( abi , foster city , ca ) using 2  sybr green master mix ( bio - rad ) . forty cycles were conducted as follows : 95c for 30  s and 60c for 30  s , preceded by 1  min at 95c for polymerase activation with the following primers ( q - pcr il-1 : sense primer 5-gtgtctttcccgtggaccttc-3 , antisense primer 5-tcatctcggagcctgtagtgc-3 : q - pcr tnf- : sense primer 5-gtggaactggcagaagaggca , antisense primer 5-agagggaggccatttgggaac-3 q - pcr il-6 : sense primer 5-ggaaatcgtggaaatgag-3 , antisense primer 5-gcttaggcataacgcact-3 q - pcr gapdh : sense primer 5-ctcgtggagtctactggtgt-3 , antisense primer 5-gtcatcatacttggcaggtt-3 ) . the products of il-1 , tnf- , and il-6 in cell supernatants were determined using the elisa kit for il-1 ( ebioscience ) , tnf- ( ebioscience ) , and il-6 ( ebioscience ) . data are reported as means  sd , and results were analyzed using spss 13.0 software ( spss inc . , chicago , il , usa ) by means of independent one - way anova tests in each sampling point . the differences among the three groups were assessed by means of the least significant difference ( lsd ) multiple comparison test ( p < 0.05 or 0.01 ) . to determine the optimum growth temperature of the selected probiotics , the same initial inoculums of b. longum ( mrs medium ) , l. plantarum ( mrs medium ) , l. rhamnosus lgg ( mrs medium ) , and s. thermophilus ( bhi medium ) were inoculated in the corresponding media and cultured at 25c , 30c , 37c , and 42c , and the results showed that the optimum temperature for b. longum , l. plantarum , and l. rhamnosus lgg was 37c , and s. thermophilus received its maximum yield at 42c ( figure 1(a ) ) . to further investigate the effects of different additives on the growth of probiotics , glucose and skimmed milk were added in the av extract , and the results indicated that all the tested strains grew well in av extract with the initial ph 6.0 ( about 9  lg  cfu / ml , figure 1(b ) ) . it seemed that the addition of 5% glucose and 5% glucose + 5% skimmed milk had significantly enhanced the biomass of b. longum , l. plantarum , and l. rhamnosus gg ( a < 0.01 ) , and the growth promoting effect of the 5% glucose + 5% skimmed milk was much better than 5% glucose ( b < 0.01 ) ( figure 1(b ) ) . to evaluate the additive of the probiotics on the perfect antioxidant and antibacterial activities of av [ 9 , 2022 ] , the av + 5% glucose , which gives us more choices for the future products , was chosen for our next study . compared with the av , the addition of probiotics made little change on o2 clearance , fe chelation , and reduction activity , while the l. plantarum hm218749.1 had significantly ( p < 0.01 ) enhanced the dpph clearance and oh clearance ( table 1 ) . considering the sound probiotic properties of the l. plantarum hm218749.1 , this strain was finally chosen for the antibacterial evaluation . interestingly , no antimicrobial effect was observed using av extract and aloe fermentation supernatant ( fermented by l. plantarum hm218749.1 without additional glucose ) , while the addition of 5% glucose conferred the aloe fermentation supernatant a perfect inhibition to all the tested pathogens , for example , s. typhimurium atcc 13311 ( inhibition zone diameter : 16  mm ) , s. enteritidis atcc13076 ( izd : 15  mm ) , s. flexneri atcc 12022 ( izd : 19  mm ) , e. coli 44102 ( izd : 11  mm ) , l. monocytogenes atcc 19111 ( izd : 20  mm ) , s. dysenteriae 301 ( izd : 21  mm ) , and s. aureus cowan1 ( izd : 20  mm ) ( figure 2 ) . most of all , the aloe fermentation supernatant possessed the best antimicrobial effect on p. acnes atcc 11827 ( izd : 27  mm ) , which leads to an accumulation of the skins natural oils , bacteria , and acne vulgaris . during inflammation , excess levels of cytokines ( il-1 , il-6 , and tnf- ) lead to the damaging of cells and tissues and the activation of macrophages in inflammation - associated diseases ( e.g. , rheumatoid arthritis and chronic hepatitis ) [ 24 , 25 ] . to check the functional components of av and aloe fermentation supernatant on the production of il-1 , il-6 , and tnf- , different concentrations of emodin ( 0 , 1 , 2.5 , 5 , and 10  g / ml ) , polysaccharide ( 0 , 2.5 , 5 , 15 , 30 , and 50  g / ml ) , av extract ( 0 , 5 , 15 , 30 , 50 , 100 , and 200  g / ml ) , and aloe fermentation supernatant ( 0 , 5 , 15 , 30 , 50 , 100 , and 200  g / ml ) were cocultured with raw 264.7 cells to test their toxicity on raw 264.7 cells , and the results indicated that no obvious toxicity was observed for all the tested materials , and the low concentration of emodin ( 1  g / ml ) showed promoting effects on the growth of raw 264.7 cells ( figure 3 ) then , low and high doses of the above drugs were determined to evaluate the anti - inflammatory effects of aloe fermentation supernatant according to the previous studies [ 1 , 20 , 22 , 26 , 27 ] . the real - time pcr results indicated that all the tested emodin , polysaccharide , av extract , and aloe fermentation supernatant had significantly reduced the inflammatory factor of il-1 , il-6 , and tnf- in gene level , except for the polysaccharide at 15  g / ml . in protein level , our elisa results showed that both the av extract and aloe fermentation supernatant presented significantly inhibition on il-1 , il-6 , and tnf- production , and it seemed that the inhibition effect of av extract was better than aloe fermentation supernatant . however , little inhibition effect of emodin and polysaccharide on il-1 and polysaccharide on il-6 was observed ( figure 5 ) . as we all know , the single target drug had been widely used in various human diseases but often failed to cure the metabolic diseases . thus , the whole - body systems biology was proposed and regarded the human and symbiotic microorganisms as a whole [ 28 , 29 ] , which led a more and more reorganization by the scientific community to the chinese herb extracts and the human intestinal probiotics . previously studies showed that both the av [ 2 , 22 ] and probiotics [ 25 , 30 , 31 ] had sound antioxidant and anti - inflammatory effects , so we guessed that the combination of the av and probiotics should possess better antioxidative , antibacterial , and anti - inflammatory effects . first , we optimized the optimum growth temperature of the selected probiotics and found that the probiotics could grew well in av extract without any additives ( figure 1 ) . however , no antimicrobial effects of av and aloe fermentation were observed which was inconsistent with the previous studies [ 2 , 32 ] ( figure 2 ) . we guessed that the lack of glucose , the substrate for probiotics to produce antimicrobial production of lactic acid and acetic acid , was the cause of the antimicrobial failure for aloe fermentation ; therefore we added 5% glucose to av extract and found that the added glucose not only significantly increased the probiotics growth ( figure 1 ) but also conferred the aloe fermentation supernatant sound antibacterial effects on all the tested pathogens ( figure 2 ) . after a series of evaluations , the l. plantarum hm218749.1 was chosen and this strain conferred the aloe fermentation supernatant strong scavenging capacities of the dpph ( 86% ) , o2 ( 85% ) , oh ( 76% ) , fe chelation ( 82% ) , and reducing powers ( 242.5  mg / l ) ( table 1 ) . moreover , the aloe fermentation fermented by l. plantarum hm218749.1 could inhibit all the growth of tested pathogens , especially for the p. acnes therefore , the sound inhibition of the growth of p. acnes atcc 11827 ( causative bacteria for acne vulgaris ) and antioxidant activity made the aloe fermentation supernatant a potential ingredient for cosmetic . as we know , excess levels of il-1 , il-6 , and tnf- induced by activated inflammatory cells ( e.g. , eosinophils , macrophages , mononuclear phagocytes , neutrophils ) will lead to the damage of cells and tissues , which eventually cause the inflammation - associated diseases ; for example , rheumatoid arthritis , chronic hepatitis , and macrophages play an important role in regulating several immunopathological conditions and inducing overexpression of the proinflammatory mediators of the inflammatory process [ 24 , 25 ] . so the raw 264.7 cells were used in the present study to evaluate the anti - inflammatory effect of aloe fermentation supernatant fermented by l. plantarum hm218749.1 . the results indicated that both the emodin ( anticancer activity )   and the polysaccharide ( immunity enhancement and reduction of oxidative injury ) showed weaker anti - inflammatory effects compared to av extract and aloe fermentation supernatant in mrna level and protein level ( figures 4 and 5 ) . interestingly , though the aloe fermentation supernatant possessed a strong antioxidant effect ( figure 2 ) , its anti - inflammatory effect was inferior to the av extract in both gene level and protein level . maybe , some anti - inflammatory ingredients in av extract were consumed by l. plantarum hm218749.1 , and the metabolites of this strain might contribute to the inflammatory response of raw 264.7 cells . moreover , in vivo work is needed because of the shortcomings of cell model ; for example , it could not model the interaction between intestinal cells and microbiota . as potential probiotics , the l. plantarum hm218749.1 could produce bacteriocins offering advantages in colonization and competition in the gastrointestinal tract   and endowed the av extract a strong antioxidative effect and antimicrobial effect on pathogens ( especially for p. acnes ) . in addition , the l. plantarum hm218749.1 could remove the bitter taste of av , and our human results ( n = 15 ) indicated that the aloe fermentation supernatant had shortened the acne cure time and had a perfect whitening effect ( data unshown ) . in conclusion , the aloe fermentation supernatant 's sound antimicrobial effect and anti - inflammatory effect , together with its intestinal health promoting effect when l. plantarum hm218749.1 enters host intestines , indicate its potential use as functional foods or cosmetics to guard human intestinal health , delaying senescence and preventing chronic diseases .
little work is done to develop aloe vera ( av ) using probiotics . to explore the potential benefits , 
 the antioxidant effects and the antibacterial effects on foodborne pathogens of aloe fermentation supernatant were evaluated in vitro . 
 our results indicated that the aloe fermentation supernatant fermented by lactobacillus plantarum hm218749.1 had very strong scavenging capacities of the dpph ( 86% ) , o2 ( 85% ) , oh ( 76% ) , and fe2 + chelation ( 82% ) and reducing powers ( 242.5  mg / l ) , and the inhibition zones for salmonella typhimurium , salmonella enteritidis , shigella flexneri , escherichia coli , listeria monocytogenes , s. dysenteriae 301 , staphylococcus aureus cowan1 , and propionibacterium acnes were 16 , 15 , 19 , 20 , 21 , 20 , and 27  mm . 
 moreover , the low concentration of aloe fermentation supernatant had significantly reduced the production of il-1 , tnf- , and il-6 in both mrna and protein levels ( p < 0.01 ) . 
 therefore , the aloe fermentation supernatant can be used as functional beverage or cosmetic ingredients to guard human intestinal health , delaying senescence , and prevent chronic diseases .