Ann Microbiol (2012) 62:1573–1578

DOI 10.1007/s13213-011-0412-5

ORIGINAL ARTICLE

Fortification and fermentation of fruit juices

with probiotic lactobacilli
Ravinder Nagpal & Ashwani Kumar & Manoj Kumar

Received: 27 July 2011 / Accepted: 20 December 2011 / Published online: 17 January 2012
# Springer-Verlag and the University of Milan 2012

Abstract In present investigation, two Lactobacillus iso- Introduction

lates, viz. L. plantarum and L. acidophilus, were observed
to be able not only to survive but to utilize fruit juices The growing demand for ‘healthy’ foods is stimulating
for their cell synthesis, as indicated by a decrease in innovation and new product development internationally.
fruit sugar and pH, and increase in acidity. L. acidophilus was The food industry plays a central role in facilitating healthier
found to consume the sugar at a faster rate than L. plantarum, eating practices through the provision and promotion of
although the fall in sugar and pH and increase in acidity healthy foods. The functional food concept has, in recent
was faster during the first 24 h and became a little years, moved progressively towards the development of
slower during the next 48 h, which could be due to dietary supplements that can affect the intestinal microbial
the accumulation of too much acid during the initial composition and activities. Probiotics are live microbial feed
24 h of fermentation. Still, both cultures were found supplements that enhance the health of consumers by improv-
to be able to survive in fermented juices with high ing the balance of microflora in the gut when ingested live in
acidity and low pH. Therefore, it could be concluded sufficient numbers (Fuller 1989). Probiotic lactobacilli are
that such probiotic-fortified fruit juices could certainly claimed to provide a number of health benefits, including
be exploited as a medium for the delivery of probiotics, antimicrobial effects against pathogens, anti-tumor effects,
and could be used as a functional healthy beverage to anti-cholesterol, immuno-modulation, anti-diabetic, and treat-
promote better health and nutrition of the population, espe- ment of diarrhea and lactose intolerance (Roberfroid 2000;
cially for those who are allergic or intolerant to milk-based Ouwehand et al. 2002; Salminen et al. 2005; Kumar et al.
products. 2009, 2010, 2011a, b; Nagpal et al. 2007, 2010, 2011). Pro-
biotic strains can be incorporated successfully into highly
Keywords Fortification . Fruit juice . Functional food . acceptable food products where they can retain their viability.
Lactobacillus . Probiotic Currently, industrial demand for technology for probiotics
provides promising prospects for improved culture perfor-
mance; due to survival and colonization difficulties, a ‘syn-
R. Nagpal : A. Kumar (*)
biotic’ (probiotic + prebiotic) approach offers an attractive
Department of Biotechnology, alternative (Nagpal et al. 2007; Nagpal and Kaur 2011).
JMIT Institute of Engineering and Technology, Consequently, consumer demand exists for new food products
Radaur, 135133, Yamuna Nagar, Haryana, India containing such beneficial microbes. Probiotics are a perfect
e-mail: [email protected]
fit for dairy-based beverages, as dairy is an excellent vehicle in
M. Kumar which to deliver beneficial bacteria such as Bifidobacterium
Department of Microbiology, National Institute of Nutrition, and Lactobacillus species. Consumption of probiotics with
Hyderabad, India dairy foods such as milk, yogurt and cheese can be an excel-
R. Nagpal
lent vehicle, since these foods buffer the stomach acid and
Shaheed Udham Singh College of Research & Technology, increase the chance that the bacteria will survive in the intes-
Mohali, Punjab, India tine. However, there are many members of society who
1574 Ann Microbiol (2012) 62:1573–1578

remain devoid of probiotics, most likely because of lactose in MRS broth at 37°C for 18 h and subsequently har-
intolerance problems, milk allergy or high cholesterol/ fat vested. The bacterial cell pellet was washed twice with
content; therefore, there is a need for a suitable carrier of PBS buffer at 10,000 rpm for 10 min, and then resus-
probiotics for such people. To the remedy this problem, fruit pended in PBS buffer. The absorbance was adjusted to
juices could be exploited as an ideal carrier or medium for 0.8 at 600 nm using a spectrophotometer (UV–vis
probiotics. It is well established that fruits are very healthy, double-beam 2203, Systronics, New Delhi, India). Bac-
having a high content of antioxidants/ vitamins, minerals, terial suspension (3 ml) was added to acid-washed test
dietary fibers and many other beneficial nutrients, and fruits tubes followed by addition of 1 ml hexadecane and then
are totally free from milk allergens or milk sugar; therefore, incubated at 37°C for 10 min. The two phases became
fruit juices could prove to be an ideal candidate for the separated and were again mixed well. The mixed phases
delivery of probiotics to the large segment of the population were again kept at 37°C for 1 h for phase separation.
that cannot or does not want to have milk products in their The aqueous phase was taken out gently with the help of
diets. In this context, an effort was made to fortify various fruit a micropipette and the absorbance of the aqueous phase
juices, viz. orange, apple, grapes and tomato, with selected was measured at 600 nm. The fraction of adherent cells
Lactobacillus isolates, and to check their ability to survive in was taken as percent decrease in absorbance of the
or ferment these juice products. aqueous phase after mixing and phase separation relative
to that of original suspension:

Materials and methods Surface Hydrophobicity ð%Þ ¼ ðAbsFinal Þ=ðAbsInitial Þ  100

Isolation and identification of lactobacilli Inoculation and fermentation of fruit juices

Lactobacillus isolates LH1 (L. plantarum) and LH2 (L. acid- Various fruit juices, viz. tomato, orange and grape, were
ophilus) were isolated previously from human milk samples freshly prepared and filtered repeatedly to obtain clear pulp-
collected from different areas of Yamuna Nagar, Kurukshetra, free juice, and were finally pasteurized. A 100 ml sample of
Ladwa and Radaur, using serial dilution in phosphate buffer pasteurized juice was collected into sterile Erlenmeyer flasks.
saline (pH 6.8), and inoculation on deMan Rogosa and Sharpe Each flask containing 100 ml juice was inoculated using 1%
(MRS, Hi-Media, Mumbai, India; Composition: proteose pep- culture each of LH1 and LH2 under aseptic conditions. No
tone 10 g/L; beef extract 10 g/L; yeast extract 5 g/L; dextrose culture was added to the flasks labeled as control. The flasks
20 g/L; polysorbate-80 1 g/L; ammonium citrate 2 g/L; sodium were then incubated at 37°C. Every 24 h, 10 ml juice was
acetate 5 g/L; magnesium sulphate 0.1 g/L ; manganese sulfate taken out from each flask and tested for biochemical param-
0.05 g/L; dipotassium phosphate 2 g/L; agar 12 g/L; final eters, viz. pH, acidity and sugar concentration. The pH of each
pH 6.5±0.2: agar plates (Awan and Rahman 2005; Nagpal juice sample was measured using a pH meter (μ pH Systems
et al. 2010). 361, Systronics, New Delhi, India) after proper calibration.
Total acidity, expressed as percent lactic acid, was determined
Acid tolerance by titrating with 0.1 N NaOH to pH 8.2, using Phenolphtha-
lein as an end point indicator. The sugar content was estimated
The two isolates LH1 and LH2 were tested for their ability in terms of glucose (mg/ml) by the phenol sulfuric acid
to tolerate varying concentration of acids. The pH of the method, as described by Dubios et al. (1956). Viable cell
MRS broth was adjusted to 1.5, 2.5, 4.5 and 6.5 with 1 N counts (CFU/ml) were determined by the standard plate count
HCl. Overnight grown cultures of lactobacilli were inocu- method using MRS agar after 48 h incubation at 37°C.
lated in MRS broth at different pH and incubated at 37°C.
Inoculated culture (1 ml) was taken at 1, 2, 3 and 4 h
intervals, and again inoculated into fresh broth tubes having Results and discussion
adjusted the pH to 6.8. These broth tubes were incubated at
37°C for 12–14 h and the cell count was measured at Probiotic lactobacilli are gaining enormous attention because of
600 nm. their established health effects such as anti-diarrheal, anti-
pathogenic, anti-diabetic, anti-cholesterol and anti-cancer ac-
Cell surface hydrophobicity tivities, etc. (Roberfroid 2000; Ouwehand et al. 2002; Salminen
et al. 2005; Nagpal et al. 2007, 2010, 2011). Fruit juices are also
The method of Rosenberg et al. (1983) was adopted with extremely healthy, having a high content of antioxidants,
slight modification to measure cell surface hydrophobic- vitamins, minerals, dietary fiber and many other beneficial
ity (Nagpal et al. 2010). The bacterial cells were grown nutrients, and hence could serve as a good medium for
Ann Microbiol (2012) 62:1573–1578 1575

cultivating probiotics (Mattila-Sandholm et al. 2002; Luckow binding of a probiotic microorganism in the gut. Adhesion and
and Delahunty 2004). Therefore, in the present investigation, colonization are prerequisite events for a probiotic organism,
an attempt was made to test the fortification of fruit juices with and may be an important component in the complex interplay
probiotic lactobacilli. Out of seven Lactobacillus isolates iso- between several factors that enables a microorganism to bind
lated from mother’s milk, only two isolates, i.e., LH1 and LH2 and colonize the host gut for successful propagation. In present
were chosen for present investigation on the basis of their high study, the hydrophobicity of isolates LH1 and LH2 was ob-
pH tolerance ability. Since most fruit juices, especially tomato, served to be 40.52% and 43.62%, respectively, i.e., much
grapes, and orange, have a high acid content and low pH, any higher than that of Escherichia coli, which was observed to
culture used to fortify juice should be able at least to survive, if be only 1.72%. The LH2 isolate (L. acidophilus) showed high
not grow in the juice. Moreover, in order to become established acid tolerance and adhesiveness properties, which may enable
in the gut and be nominated as a probiotic, a strain must be able it to successfully pass the low pH of the stomach and bind in the
to withstand the acidic conditions in the stomach or intestine. gut of the host, which is needed for successful colonization and
Therefore, all the seven isolates were tested for their ability to propagation for expression of its health-promoting effects.
tolerate varying concentration of acids. Though, all seven Many members of society have problems with lactose
isolated cultures were able to withstand pH values of 5.5 and intolerance, milk allergy or high cholesterol/ fat content;
4.5, but only LH1 (L. plantarum) and LH2 (L. acidophilus) these people are devoid of probiotics as most of the pro-
were able to survive at pH 3.5 for up to 4 h (data not shown). biotic products available on the market are dairy-based.
Therefore, these two cultures were finally selected for fortifi- Therefore, fruit juices could be exploited as a suitable carrier
cation or fermentation of different fruit juices. Moreover, of or medium of probiotics for such people (Mattila-Sandholm
these two cultures, LH2 could survive at pH 2.5 for up to 2 h et al. 2002), since fruits are totally free of milk allergens or
but failed to survive thereafter, whereas LH1 showed no sur- milk sugar (Luckow and Delahunty 2004). In the present
vival at pH 2.5. An important aspect widely considered in investigation, the two isolates L. plantarum (LH1) and L.
bacterial adhesion is cell surface hydrophobicity. Hydrophobic acidophilus (LH2) were tested for their ability to utilize fruit
strength could be involved in bacteria–epithelial cell interac- sugar and produce lactic acid without any additional nutrient
tions, and therefore serves as a good index of the potential addition or pH adjustment. When pasteurized grape juice

Fig. 1 Changes in sugar,

acidity and pH during lactic
acid fermentation of grape
juice (LH1: Lactobacillus
plantarum; LH2: Lactobacillus
acidophilus). Values are
mean of three replicates
1576 Ann Microbiol (2012) 62:1573–1578

was inoculated with LH1and LH2 cultures, a reduction in again supported by a decrease in pH and increase in juice
sugar content was observed in both cultures, while there was acidity. LH1 and LH2 were able to reduce the pH from 4.4 to
no, or an insignificant, reduction in the case of control 3.8 and 3.6, respectively; and increase the acidity from 0.6% to
(un-inoculated) juice during a 72-h incubation at 30°C 1.16% and 1.25%, respectively (Fig. 3).
(Fig. 1). The initial sugar content of grape juice was 0.00628, The two Lactobacillus isolates—LH1 (L. plantarum) and
which was reduced to 0.00374 and 0.00378 by isolates LH1 LH2 (L. acidophilus)—were also tested for their ability to
and LH2, respectively. The results of decreased pH and in- survive during lactic acid fermentation of fruit juice (Table 1).
creased acidity showed a similar trend (Fig. 1). In the case of The initial cell count in all tested juices was 6.2×108 and 7.7×
freshly prepared orange juice, the original pH 3.9 was reduced 108 CFU/ml for LH1 and LH2 isolates, respectively. How-
to 3.65 and 3.52 by isolates LH1 and LH2, respectively ever, after 72 h incubation, the cell counts of LH1 isolates
(Fig. 2). The decrease in pH was concurrent with the decrease increased to 6.8×108, 6.9×108 and 7.7×108 CFU/ml for
in the sugar content of the orange juice. The original sugar grape, orange and tomato juices, respectively, while in the
content of 0.00528 was reduced to 0.00338 and 0.00315, by case of LH2 isolates, the cell count observed was 8.4×108,
LH1 and LH2, respectively. The results of reduced pH were 8.1×108 and 9.0×108 CFU/ml for grape, orange and tomato
well supported by the similar trend in increased acidity in juices, respectively. These results indicate that the two cultures
samples inoculated with LH1 and LH2 (Fig. 2). In the case were not only able to survive but also utilized and fermented
of tomato juice, a trend similar to that seen in grape and orange the fruit sugar for their cell synthesis and metabolism.
juice was observed (Fig. 3). The original sugar content of Tomato juice contains 93.1% moisture, 4.89% carbohy-
0.00528 was reduced to 0.00317 and 0.00307 by isolates drate, vitamins, and minerals, and is low in protein and fat
LH1 and LH2, respectively, while in the control sample, sugar (Abdel and Abdel 1982), and is well recognized as a healthy
was reduced insignificantly to 0.00514. The sugar results were beverages (Suzuki et al. 2002). Babu et al. (1992) reported

Fig. 2 Changes in sugar,

acidity and pH during lactic
acid fermentation of orange
juice (LH1: L. plantarum; LH2:
L. acidophilus). Values are
mean of three replicates
Ann Microbiol (2012) 62:1573–1578 1577

Fig. 3 Changes in sugar,

acidity and pH during lactic
acid fermentation of tomato
juice (LH1: L. plantarum; LH2:
L. acidophilus). Values are
mean of three replicates

that addition of tomato juice to skimmed milk stimulated the pH, increase in acidity, and an improvement in the digestibil-
growth of L. acidophilus and resulted in higher viable ity of starch and protein. Yoon et al. (2004) also observed
counts, shorter generation time, and improved sugar utiliza- decrease in sugar and pH and increased acidity when tomato
tion with more acid production and lower pH. Sindhu and juice was inoculated and incubated with Lactobacillus del-
Khetarpaul (2001) also reported that probiotic fermentations brueckii, L. acidophilus, L. plantarum, and L. casei. In the
of indigenous food mixtures containing tomato pulp using present investigation, the two Lactobacillus isolates, i.e., LH1
Lactobacillus casei and L. plantarum showed a decrease in (L. plantarum) and LH2 (L. acidophilus), were observed to be
able not only to survive in but also to utilize the fruit juices for
their cell synthesis, as indicated by a decrease in fruit sugar
Table 1 Viable cell counts during lactic acid fermentation of fruit
and pH, and increase in acidity and bacterial counts. However,
juices (LH1: Lactobacillus plantarum; LH2: Lactobacillus acidophilus). isolate LH2 was found to consume the sugar at a faster rate
Values are mean of three replicates than isolate LH1; however, the fall in sugar and pH and
increase in acidity was faster during the first 24 h and slowed
Juice CFU/ml (× 108)
down a little during the next 48 h, which could be due to the
LH1 LH2 extremely low pH and high acidity achieved during the first
24 h of fermentation. Moreover, several other factors that
0h 72 h 0h 72 h could have affected the survival/growth of cells, e.g., accu-
Grape 6.2 6.8 7.7 8.4
mulation of other metabolic end products such as lactic acid
and other organic acids, diacetyl, acetylaldehyde and acetoin,
Orange 6.2 6.9 7.7 8.1
etc., which could reduce culture viability if accumulated in
Tomato 6.2 7.7 7.7 9.0
high amounts (Hood and Zottola 1988; Shah and Jelen 1990;
1578 Ann Microbiol (2012) 62:1573–1578

Post 1996; Dave and Shah 1997). It has been reported that the alleviates disease signs in rats challenged with pathogens. Int J
Probiotics Prebiotics 4:211–218
acid production ability of lactic acid bacteria, especially post-
Kumar M, Kumar A, Nagpal R, Mohania D, Behare P, Verma V,
incubation (post-acidification), affects the cell viability of pro- Kumar P, Poddar D, Aggarwal PK, Henry CJ, Jain S, Yadav H
biotic bacteria, including L. acidophilus and Bifidobacterium (2010) Cancer-preventing attributes of probiotics: an update. Int J
bifidum (Ishibashi and Shimamura 1993; Shah et al. 1995). Food Sci Nutr 61:473–496
Kumar M, Verma V, Nagpal R, Kumar A, Behare PV, Singh B,
Aggarwal PK (2011a) Anticarcinogenic effect of probiotic fer-
mented milk and Chlorophyllin on aflatoxin-B1 induced liver car-
Conclusion cinogenesis in rats. Brit J Nutr. doi:10.1017/S0007114511003953
[Epub ahead of print]
Kumar M, Verma V, Nagpal R, Kumar A, Gautam SK, Behare PV,
The results of the present study demonstrated that both Grover CR, Aggarwal PK (2011b) Effect of probiotic fermented
Lactobacillus cultures were able to survive in fermented milk and chlorophyllin on gene expressions and genotoxicity
juices with high acidity and low pH; therefore, it could be during AFB1-induced hepatocellular carcinoma. Gene 490:54–59
advocated that fruit juices could be exploited as a carrier/ Luckow T, Delahunty C (2004) Which juice is healthier? A consumer
study of probiotic non-dairy juice drinks. Food Qual Prefer
medium for the fermentation and delivery of probiotic lactic
15:751–759
acid bacteria, and these probiotic-fortified fruit products Mattila-Sandholm TP, Myllrinen R, Crittenden G, Mogensen R, Fondn
could be used as a functional healthy beverage to promote M, Saarela (2002) Technological challenges for future probiotic
better health and nutrition of the population, especially those foods. Int Dairy J 12:173–182
Nagpal R, Kaur A (2011) Synbiotic effect of various prebiotics on in-vitro
who are allergic or intolerant to, or prefer not to consume,
activities of probiotic lactobacilli. Ecol Food Nutr 50(1):63–68
milk-based products. However, more extensive in vitro and Nagpal R, Yadav H, Puniya AK, Singh K, Jain S, Marotta F (2007)
in vivo studies are vital in order to authenticate the probiotic Potential of probiotics and prebiotics for synbiotic functional
potential and safety of such cultures and fruit products based dairy foods. Int J Probiotics Prebiotics 2:75–84
Nagpal R, Kumar A, Arora S (2010) In-vitro probiotic potential of
on these beneficial microbes before being endorsed for the lactobacilli from indigenous milk products. Int J Probiotics Pre-
better health and nutrition of society. biotics 5(2):103–110
Nagpal R, Behare PV, Kumar M, Mohania D, Yadav M, Jain S, Menon
Acknowledgment The authors wish to thank the Chairman and the S, Parkash O, Marotta F, Minelli E, Henry CJK, Yadav H (2011)
Director, JMIT, Radaur, for providing funding and facilities necessary Milk, milk products and disease free health: an updated overview.
for this research work. Crit Rev Food Sci Nutr 99999(1):1549–7852. doi:10.1080/
10408398.2010.500231 [Epun ahead of print]
Ouwehand AC, Salminen S, Isolauri E (2002) Probiotics: an overview
of beneficial effects. Antonie Van Leeuwenhoek 82:279–289
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