Evaluation of the Antioxidative, Antibacterial, and Anti-Inflammatory Effects of the Aloe Fermentation Supernatant Containing Lactobacillus plantarum HM218749.1

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-1β, TNF-α, 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.

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 [7][8][9]. 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 [12].
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 2 Mediators of Inflammation functional foods and potential oral vaccines [13]. In our previous work [15], L. plantarum HM218749.1 was isolated from human intestinal, which could grow better at 37 ∘ C 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 [15].
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 antiinflammatory effects of the Aloe fermentation supernatant.

Preparation of AV Extract and 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 [1].
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 8 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 37 ∘ C or 42 ∘ C. 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, −40 ∘ C for 48 h. The lyophilized powder was stored at −20 ∘ C for further use.

Cultivable Bacterial Counts.
Bacterial counts were enumerated as previously reported [16]. Diluted aliquots were replica-plated onto BHI agar and MRS agar and then incubated anaerobically in the anaerobic system (full of 85% N 2 , 5% H 2 , and 10% CO 2 ) at 37 ∘ C or 42 ∘ C for 24∼36 h followed by counting colonies from plates showing 25-250 colonies [17].

Antioxidative and Antibacterial Activity of AV Extract and
Aloe Fermentation Supernatant. For preparation of the Aloe fermentation supernatant, 10% of the probiotics were added to AV extract and fermented for 15 to 18 h at 37 ∘ C, 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, O 2 •− , • OH, Fe 2+ chelation, and oxygen-reduction activity were measured just as in [18]. 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. The experiment was carried out in duplicate [17].
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 37 ∘ C in a humidified incubator. Control cells treated with PBS served as the vehicle group. Each concentration of drugs was repeated in 5 wells. 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.

Anti-Inflammatory Evaluation of AV Extract and Aloe
Fermentation Supernatant Using RT-PCR and ELISA. RAW 264.7 cells were seeded into 24-well plates (5 × 10 4 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.

Data
Analysis. 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 ( < 0.05 or 0.01).

Exploration of the Fermentation Conditions of Probiotics.
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 25 ∘ C, 30 ∘ C, 37 ∘ C, and 42 ∘ C, and the results showed that the optimum temperature for B. longum, L. plantarum, and L. rhamnosus LGG was 37 ∘ C, and S. thermophilus received its maximum yield at 42 ∘ C (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 ( < 0.01), and the growth promoting effect of the 5% glucose + 5% skimmed milk was much better than 5% glucose ( < 0.01) (Figure 1(b)).

Antioxidative and Antibacterial Activity of Aloe Fermentation Supernatant.
To evaluate the additive of the probiotics on the perfect antioxidant and antibacterial activities of AV [9,[20][21][22], 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 O 2 •− clearance, Fe 2+ chelation, and reduction activity, while the L. plantarum HM218749.1 had significantly ( < 0.01) enhanced the DPPH clearance and • OH clearance (Table 1). Considering the sound probiotic properties of the L. plantarum HM218749.1 [15], 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: Note: data are shown as the mean ± SD. * * < 0.01.

The Anti-Inflammatory Activity of the Aloe Fermentation
Supernatant on Proinflammatory Cytokines. 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].
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).

Discussion
As we all know, the single target drug had been widely used in various human diseases but often failed to cure the metabolic diseases [28]. Thus, the whole-body systems biology was proposed and regarded the human and symbiotic microorganisms as a whole "superorganism" [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 antiinflammatory 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%), O 2 •− (85%), • OH (76%), Fe 2+ 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 ATCC 11827 (IZD: 27 mm). 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 TNFinduced 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) [33] 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 antiinflammatory 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 [34] 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 ( = 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.