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ACTA BIOMED 2012; 83: 183-188 © Mattioli 1885

O R I G I N A L A R T I C L E

Effect of Aloe vera juice on growth and activities of

Lactobacilli in-vitro
Ravinder Nagpal1, Varinder Kaur1, Manoj Kumar2, Francesco Marotta3
1
Division of Laboratory for Probiotic Research (Yakult), Juntendo University Graduate School of Medicine, Tokyo, Japan;
2
Department of Microbiology & Immunology, National Institute of Nutrition, Hyderabad, India; 3 Hepato-Gastroenterology
Unit, S. Giuseppe Hospital, Milano, Italy

Abstract. In present investigation, different concentrations of Aloe vera juice incorporated into the growth
media of Lactobacilli were tested to observe the effect on growth and activities of these bacteria. From the
results obtained, it was observed that aloe vera juice at a concentration of 5% v/v was effective in promoting
the growth of L. acidophilus, L. plantarum and L. casei, as evident from the fall in pH and increased acidity,
as well as from the improved generation time. At 15 to 25% concentration, growth was unaffected as com-
pared to the controls; however, concentration higher than 25%v/v discouraged the growth. Overall, it was
concluded that Aloe vera juice or gel at a particular concentration could possibly be used in combination with
probiotic Lactobacillus strain(s) as a combinational therapy for gastrointestinal disorders and cardiovascular
diseases. (www.actabiomedica.it)

Key words: Aloe vera, gastrointestinal diseases, lactobacillus, prebiotics, probiotics, synbiotics

Introduction (16). Today, Aloe vera has attracted significant interest

because of its nutritional and medicinal characteris-
Traditional and modern medicine have suggested tics, and its potential as a generator of economic ac-
to use fermented foods as an alternative for the pre- tivity in arid and semi-arid areas (17). About 320
vention and treatment of malnutrition, chronic diar- species of the genus Aloe have been reported among
rhea, gastrointestinal diseases and other conditions, which the most commonly known is Aloe vera (Aloe
since consumption of these foods provides an entry vera var barbariensis), a species most cultivated in
route for the common biological agents that comprise Mexico (18). Aloe vera contains most of its carbohy-
the beneficial intestinal flora, particularly probiotics drates in the form of mannose polymers (accem-
(1-4). Probiotics are defined as live microorganisms ananos), vitamin A, Vitamin B1, B6 and vitamin C
present in foods which when ingested in sufficient (19). Most important properties of Aloe vera include
quantities can have specific physiological benefits such its anti-inflammatory, antibiotic, regenerative, anti-di-
as anti-pathogenic, anti-diarrheal, anti-carcinogenic, abetic and anti-cholesterolemic (20). In the food in-
anti-diabetic, anti-cholesterolemic etc. to individual dustry, Aloe vera juice has been used for preparation of
health (3-14). An important group of probiotics that soft drinks with healthy nutritional qualities and ton-
are used traditionally are lactic acid bacteria i.e. Lacto- ics containing amino acids and minerals.
bacillus, Streptococcus, Lactococcus, Bifidobacterium. Traditionally, delivery vehicles used for probiotics
Aloe vera pulp, due to its composition, is a food are dairy based; however, there is a large variety of
plant likely to promote the growth of probiotic agents fruits and vegetables available locally that could be
(15). It has long been used for the treatment and pre- feasible to be exploited for probiotics (13). Although,
vention of gastrointestinal diseases in several countries both, Aloe vera as well as probiotic Lactobacilli, have
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184 R. Nagpal, V. Kaur, M. Kumar, F. Marotta

individually been well-documented for numerous titrating with 0.1 N NaOH to pH 8.2, using Phe-
health benefits; there are scanty of reports available on nolphthalein as an end point indicator. The sugar con-
a combined approach and application. In this milieu, tent was estimated in terms of glucose (mg/ml) by the
a novel effort was made through present investigation phenol sulfuric acid method, as described by Dubios
in order to observe the activities of Lactobacilli in Aloe et al. (22). An aliquot from each treatment was taken
vera juice with a prospective approach to blend probi- at 0 hr and 12 hr and diluted (1:10, v/v) with 0.2%
otics and Aloe vera that could possibly be exploited as (w/v) EDTA (pH 12.0) and turbidity measured at 640
a potential combinational remedy. nm using uninoculated media diluted with EDTA as
a blank. Specific growth rate (µ) for each culture was
calculated using the equation: µ = (lnD2– lnD1)/(t2 –
Materials and methods t1), where D2 and D1 are the cell densities at times t2
and t1, respectively. Mean generation time was calcu-
The three Lactobacillus isolates viz. L. acidophilus lated as Td = ln2/µ (23). All the experiments were car-
NCDC14, L. plantarum NCDC20 and L. casei ried out in triplicates, and the values presented in the
NCDC17 were procured from National Collection of manuscript are mean of three replicates.
Dairy Cultures, National Dairy Research Institute,
India, and were maintained in sterile 10% reconstitut-
ed skim milk. The Aloe vera was collected from home Results and discussion
garden (Rajpura, Punjab, India). Freshly harvested,
matured Aloe vera leaves were cut from the bottom of Probiotic Lactobacilli are gaining enormous at-
the plant with a sharp knife, and washed thoroughly. tention because of their established health effects such
Then, using a sharp knife, the rough edges were re- as anti-diarrheal, anti-pathogenic, anti-diabetic, anti-
moved. The slimy mucilage and transparent gel was cholesterol and anti-cancer activities etc. (3, 4, 6-14,
scooped out with a spoon from the Aloe leaf. It was 24-26). Aloe vera juice is also considered extremely
made sure that the yellow sap exuded from the green healthy because of high content of vitamins, minerals,
part of the leaf was discarded properly. This is called amino acids (essential and non-essential), trace ele-
aloin that causes irritation reactions. Aloe vera gel was ments, antimicrobial agents (anthraquinones) and en-
then macerated in a grinder, and was subsequently fil- zymes (27); and hence could serve as a good medium
tered to separate the fiber and get the juice used in the for cultivating probiotics. Therefore, in present inves-
fermentation. It was preserved in the refrigerator for tigation, effect of Aloe vera juice on growth and activ-
everyday usage. ities of probiotic Lactobacilli was studied in-vitro by
Different concentrations of Aloe vera juice i.e. 5%, incorporating different concentrations of juice in basic
15%, 25%, 50%, 100% were prepared in de Man Ro- media, and incubation for upto 48 hrs. At 0 hr, pH of
gosa Sharpe (MRS) broth (21). Total volume was kept all the samples containing different concentrations of
50 ml. One flask containing MRS was kept as nega- Aloe vera juice was set at 6.7. In case of L. acidophilus,
tive control (uninoculated) and one was kept as posi- it was observed that after 24 hr incubation, pH de-
tive control (inoculated). Overnight grown cultures of creased being highest at 100% (6.2) and lowest at 5%
L. acidophilus, L. plantarum, L. casei (1%) were inocu- (4.6) (Fig. 1). Most of the lactic acid bacteria are able
lated respectively into MRS broth containing different to reduce the pH of the environment by production of
concentrations of Aloe vera juice (5%, 15%, 25%, 50% acetic and lactic acid creating an environment adverse
and 100%), and incubated for 48 hr at 37˚C. Readings to the growth of gastrointestinal pathogens (5, 28).
were taken at 0, 24 and 48 hr for pH, acidity and vi- The decrease in pH also indicates the production of
able counts. The pH of each sample was measured us- other organic acids (24). When compared to pH 4.9 at
ing a pH meter (µ pH Systems 361, Systronics, New 0% concentration, pH at 5% (4.6) and 15% (5) was
Delhi, India) after proper calibration. Total acidity, ex- quite same, suggesting that Aloe vera had no limiting
pressed as percent lactic acid, was determined by effect on the growth of L. acidophilus and the growth
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Effet of Aloe vera juice on growth and activities of Lactobacilli in vitro 185

Figure 1. pH of L. acidophilus fermented MRS containing dif- Figure 2. Acidity (%lactic acid) of L. acidophilus fermented
ferent concentrations of Aloe vera juice (values are mean of MRS containing different concentrations of Aloe vera juice
three replicates) (values are mean of three replicates)

rate was normal at 5% and 15% concentrations. Aloe ity at 5 and 15% was quite same. It could be due to the
vera pulp because of its nutritional composition, presence of saccharides like cellulose, glucose, man-
mainly carbohydrates and vitamins, is a food plant nose, L-rhamniose, aldopentose present in the Aloe ve-
that is expected to promote the growth of probiotic ra juice (27). From this, it was again observed that the
microorganisms (15). This could be the reason for the rate of growth of L. acidophilus was highest at 5% fol-
survival of probiotic bacteria at different concentra- lowed by 15% and slower at 25%, 50% and 100%. The
tions of Aloe vera in present study. In case of 25%, acidity further increased after 48 hrs incubation being
50% and 100% juice concentration, the pH was high- highest at 5% (0.93) followed by 15% (0.86), 25%
er, i.e. 5.4, 5.8 and 6.2 respectively, as compared to pH (0.78), 50% (0.68) and lowest at 100% (0.56), when
4.9 at 0% concentration indicating slow growth of L. compared with 0% (0.87) concentration.
acidophilus. From the above data, it could be analyzed In case of L. plantarum, it was analyzed that after
that the growth of L. acidophilus was highest at 5% and 24 hrs incubation, pH decreased from 6.7 being high-
lowest at 100%. The pH further decreased after 48 hrs est at 100% (5.7) followed by 50% (5.5), 25% (5.2),
incubation, being highest at 100% (5.8) followed by 15% (5) and lowest at 5% (4.6) (Fig. 3). When com-
50% (5.4), 25% (5.1), 15% (4.7) and lowest at 5% pared with 0% (4.9), the growth was found to be near-
(4.4), when compared with 0% (4.7) concentration. ly equal at 5% and 15% concentrations but slower at
Hypothesis thrown in previous studies (15, 18) sug- 25%, 50% and 100% concentrations. The pH further
gest that the acemanane and use of glucomannan from decreased after 48 hrs incubation, being highest at
Aloe vera could increase the growth and production of 100% (5.5) followed by 50% (5.4), 25% (4.8), 15%
some antimicrobial metabolites (short-chain organic (4.5) and lowest at 5% (4.3). When compared with 0%
acids) that could inhibit the bacterial. (4.6), the growth was found to be nearly same at 5%
Acidity of the culture decreases as the pH increas- and 15% concentrations and slower at 25, 50 and
es. In present study, the results of acidity coincided 100% concentrations. The results for acidity also fol-
with that of pH. At 0 hr, the acidity of L. acidophilus lowed the same trend as that for pH. At 0 hr, the acid-
was observed to be 0.2 at all concentrations (0%, 5%, ity of L. plantarum was 0.24 at all concentrations (0%,
15%, 25%, 50% and 100%) of Aloe vera. After 24 hrs, 5%, 15%, 25%, 50% and 100%) of Aloe vera. After 24
acidity was highest at 5% (0.87) followed by 15% hrs incubation, acidity was highest at 5% (0.87) fol-
(0.82), 25% (0.75), 50% (0.62) and lowest at 100% lowed by 15% (0.82), 25% (0.70), 50% (0.68) and low-
(0.54) (Fig. 2). When compared to 0% (0.83), the acid- est at 100% (0.50) (Fig. 4). From this it was analyzed
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186 R. Nagpal, V. Kaur, M. Kumar, F. Marotta

During the growth kinetics for 12 hrs for L. aci-

dophilus, L. casei and L. plantarum using medium with
different concentrations of Aloe vera juice, it was ob-
served that in case of L. acidophilus at different con-
centrations of Aloe vera (0%, 5%, 15%, 25%, 50% and
100%) , the generation time (in minutes) was 53 (0%),
48 (5%), 60 (15%), 92 (25%), 208 (50%), 217 (100%)
supporting the results obtained for pH and acidity
(Table 1). In case of L. plantarum, it was found to be
62 (0%), 59 (5%), 71 (15%), 103 (25%), 203 (50%),
213 (100%); and for L. casei, it was found to be 58
(0%), 61 (5%), 81 (15%), 132 (25%), 196 (50%), 210
Figure 3. pH of L. plantarum fermented MRS containing dif- (100%). The results again supported the observation
ferent concentrations of Aloe vera juice (values are mean of that the viability of L. acidophilus, L. plantarum and L.
three replicates)
casei was promoted at 5% Aloe vera concentration but
started reducing with increased concentration. In case
of L. casei, growth rate was highest at 0% but a slight
reduction was observed at 5%. The generation time
was observed to be reduced further with increased
concentration.
In food industry also, Aloe vera juice has been
used for the preparation of soft drinks with nutrition-
al qualities and tonics containing amino acids and
minerals. Aloe is typically known for easing sunburns,
treating dry skin conditions, and healing wounds.
However, Aloe vera is now being explored as a treat-
ment for gastrointestinal disorders. Aloe vera can pos-
sibly be used as a prebiotic (29) because of its nutri-
Figure 4. Acidity (%lactic acid) of L. plantarum fermented tional composition, particularly acemanane, gluco-
MRS containing different concentrations of Aloe vera juice
mannan, mannose polymers (accemannose), vitamin
(values are mean of three replicates)
A, vitamin B1, B6 and vitamin C etc. Additionally,
Aloe vera has laxative effects that are caused by aloin,
that the rate of growth of L. plantarum promoted was which is found in the sap of the Aloe vera plant. Aloin
normal at 5% and 15%; but slower at 25%, 50% and stimulates colon contractions and decreases water ab-
100%. In case of L. casei also, a same trend of pH and sorption in the intestines, which induces and softens
acidity was observed as that observed in case of L. aci- stools, respectively, helping in alleviating constipation.
dophilus and L. plantarum (data not shown here). Aloe also has anti-inflammatory and analgesic effects,

Table 1. Generation time of L. acidophilus, L. plantarum and L. casei grown at different concentrations of Aloe vera juice (values are
mean of triplicates)
Generation time (min.)
Isolates Aloe vera concentration (%v/v)
0 5 15 25 50 100
L. acidophilus 53 48 60 92 208 217
L. plantarum 62 59 71 103 203 213
L. casei 58 61 81 132 196 210
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Effet of Aloe vera juice on growth and activities of Lactobacilli in vitro 187

which may help lessen other digestive symptoms. 7. Kumar M, Kumar A, Nagpal R, et al. Cancer-preventing
Thus, combining Aloe vera and probiotics could form attributes of probiotics: an update. International Journal of
Food Science & Nutrition 2010; 61 (5): 473-96.
a potential synbiotic (29). It has been hypothesized 8. Kumar M, Verma V, Nagpal R, et al. Effect of Probiotic
that synbiotics may have additive or synergistic effects Fermented Milk and Chlorophyllin on Gene Expressions
on digestive health, meaning there would be more and Genotoxicity during AFB1-induced Hepatocellular
benefits from using both components together than Carcinoma. Gene 2011; 490: 54-9.
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Aloe vera with probiotics may prove to be better for the eases. Experimental Diabetes Research 2012a; (doi: 10.1155/
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