INTRODUCTION:

The genus Lactobacillus belongs to the large group of lactic acid bacteria (LAB) which are all Gram-positive organisms which produce lactic acid by fermentation. Genera of LAB include, among others, Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus, Leuconostoc and Lactobacillus [1]. With over 100 species and subspecies, the genus Lactobacillus represents the largest group within the family Lactobacillaceae. Members of the genus are rod-shaped, often organized in chains. They are strictly fermentative and aero tolerant, but grow well under anaerobic conditions. There are two groups of species depending on the ability to ferment sugars: homofermentative species, converting sugars mostly into lactic acid, and heterofermentative species, converting sugars into lactic acid, acetic acid, ethanol and CO₂. Because the main catabolite is lactic acid, lactobacilli prefer relatively acidic conditions (pH 5.5 - 6.5).

Bacteria belonging to the genus Lactobacillus can be found in a variety of ecological niches such as plants, animals and raw milk[1]. In addition, Lactobacilli can be found in insects. The ability to colonize such a variety of habitats is a direct consequence of the wide metabolic versatility of this group of LAB. Hence, Lactobacilli have been used for decades in food preservation, as starters for dairy products, fermented vegetables, fish and sausages as well as silage inoculants.

Amino acids

The body has twenty different amino acids that act as building blocks. Some amino acids are classiﬁed as essential (indispensable) because these amino acid are amino acids that cannot be synthesized by humans and hence must be provided in the diet or parenteral solution. Non-essential amino acids can be synthesized from other amino acids or from other precursors. Some amino acids are categorized as semi- essential. These amino acids can be synthesized from other amino acids but their synthesis is limited under certain circumstances [2,3].

Phytochemicals

Phytochemicals are bioactive non-nutrient chemical compounds found in plant foods, such as fruits, vegetables, grains and other plant foods. They can be categorized into various groups, i.e., polyphenols, organ sulfur compounds, carotenoids, alkaloids, and nitrogen-containing compounds. The polyphenols are some of the most studied compounds and can be further divided into flavonoids (including flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavones) phenolic acids, stilbenes, coumarins, and tannins [4].

Numerous epidemiological studies suggest that diets rich in phytochemicals and antioxidants execute a protective role in health and disease. Frequent consumption of fruits and vegetables is associated with a lowered risk of cancer, heart disease, hypertension and stroke [5]. In the present study an attempt has been made to inhibit curd Lactic acid bacteria growth and thereby to control fermentation process by specific amino acids and phytochemicals. The control on the fermentation process and Lactic acid bacteria may have implications on improved food preservation.

MATERIALS AND METHODS:

Bacteria isolated from 24 h old curd sample were checked for Gram’s reaction. The species identified as Lactobacillus delbrueckii based on morphological and biochemical characteristics by catalase, oxidase, methyl red and Voges-Proskauer tests.

Preparation of cell suspension

A small fraction of selected LAB cells in their exponential growth phase were aseptically taken to prepare a suspension in 1mL sterile saline.

Inhibition of LAB by different amino acids by disc diffusion method

The cell suspension was aseptically spread onto MRS plates. Discs separately charged with varying concentrations (250 ppm, 500 ppm and 1000 ppm) of 20 amino acids were placed on LAB seeded MRS plates and incubated for 24 h at 32 °C. After incubation plates were observed for appearance of zones of inhibition.

Inhibition of LAB by different phytochemicals by disc diffusion method

The cell suspension was aseptically spread onto MRS plates. Discs separately charged with varying concentrations (250 ppm, 500 ppm and 1000 ppm) of phytochemicals were placed on LAB seeded MRS plates and incubated for 24 h at 32 °C. After incubation plates were observed for appearance of zones of inhibition.

Inhibition of LAB by direct introduction of selected amino acids and phytochemicals in curdling

5 ml of each amino acid or phytochemical at 250 ppm concentration was directly introduced into 50 ml of fresh curd and shift in pH (due to lactic acid production) was checked subsequently for 5 days to study the influence of these compounds. Growth inhibition could be indicated by detectable increase in pH due to the inhibition of LAB growth resulting into blockage of lactic acid production.

Statistical analysis

All quantitative measurements were expressed as means ± SD for control and experimental curd sample. The data were analyzed using one way analysis of variance (ANOVA) on SPSS and the group means were compared by Duncan’s Multiple Range Test (DMRT). The results were considered statistically significant if the p value is less than 0.05.

RESULTS AND DISCUSSION:

Inhibition of Lactobacillus by different amino acids using disc diffusion method:

Plates were observed for the formation of ‘Zones of Inhibition’ for 20 amino acids (Table 1 and Figure 1, 2, 3, 4 and 5). The test organism showed sensitivity to only 4 amino acids (alanine, leucine, glutamic acid and valine) and monosodium glutamate. The remaining amino acids showed no activity.

Table 1. Inhibition of Lactobacillus by different L-amino acids using disc diffusion method.

Amino acid	250 ppm	500 ppm	1000 ppm
Alanine	13mm	9mm	4mm
Leucine	12mm	8mm	5mm
Glutamic acid	11mm	9mm	4mm
Valine	12mm	7mm	6mm
Monosodium L- glutamate	18mm	11mm	7mm
Asparagine	NA	NA	NA
Aspartic acid	NA	NA	NA
Cysteine	NA	NA	NA
Glutamine	NA	NA	NA
Glycine	NA	NA	NA
Proline	NA	NA	NA
Serine	NA	NA	NA
Tyrosine	NA	NA	NA
Arginine	NA	NA	NA
Histidine	NA	NA	NA
Isoleucine	NA	NA	NA
Lysine	NA	NA	NA
Metheonine	NA	NA	NA
Phenyl alanine	NA	NA	NA
Threonine	NA	NA	NA
Tryptophan	NA	NA	NA

NA-no activity

Figure 1. Zones of inhibition of alanine against Lactobacillus

Figure 2. Zones of inhibition of leucine against Lactobacillus

Figure 3. Zones of inhibition of glutamic acid against Lactobacillus

Figure 4. Zones of inhibition of valine against Lactobacillus

Figure 5. Zones of inhibition of monosodium glutamate against Lactobacillus

While the acid produced by the Lactic streptococci lowers the pH of the growth medium to inhibitory levels, little is known of other metabolites which might also be auto-inhibitory. It has been reported that certain Lactic streptococci can produce an auto-inhibitor, D-leucine, during growth at controlled pH. The inhibition of microorganisms by the isomers of certain amino acids is well documented in the literature [6]. It is perhaps significant that the D-amino acid isomers isolated from gramicidin have been those of leucine, valine and alanine which have been obtained from gramicidin of the levo configuration only [7]. In the present study L-amino acids are investigated on their influence on Lactobacillus growth. Out of 20 amino acids studied four L-amino acids and sodium salt of glutamic acid showed inhibition on the growth of microorganism. The mechanisms of inhibition of these amino acids are not known. Perhaps the presence of excess amount may interfere with synthesis of protein or expression of mRNA.

Inhibition of Lactobacillus by different phytochemicals using disc diffusion method

Plates were observed for the formation of ‘zones of inhibition’ for 10 phytochemicals (Table 2 and Figure 6, 7). The test organism showed sensitivity to only two phytochemicals (protocatechuic acid and syringic acid). The remaining phytochemicals showed no activity. None of the phenolic compounds assayed seems to inhibit Lactobacillus plantarum growth at the concentrations found in olive food product[8]. There are contradictory data about the inhibition of LAB growth by hydroxytyrosol[8] although recently it has been demonstrated that hydroxytyrosol is not the main antimicrobial compound in olive brines [9]. Bauer et al., (2003) reported that bacteriocins have been described to control the growth of lactic acid bacteria in wine [10].

Figure 6. Zones of inhibition of protocatechuic acid against Lactobacillus

Figure 7. Zones of inhibition of syringic acid against Lactobacillus

Table 2. Inhibition of lactobacillus by different phytochemicals using disc diffusion method.

Phytochemicals	250 ppm	500 ppm	1000 ppm
Protocatechuic acid	16mm	11mm	7mm
Syringic acid	11mm	5mm	NA
Borneol	NA	NA	NA
Vanillic acid	NA	NA	NA
Piperine	NA	NA	NA
Morin hydrate	NA	NA	NA
Ferulic acid	NA	NA	NA
Ursolic acid	NA	NA	NA
Coumarin	NA	NA	NA
Sesamol	NA	NA	NA

NA-no activity

Table 3. Effect of amino acids on pH during the process of curdling

Phytochemicals	pH
Phytochemicals	0 day	I day	II day	III day	IV day	V day
Control	4.0 ± 0.02^a	3.50 ± 0.06^a	3.45 ± 0.04^a	3.40 ± 0.04^a	3.37 ± 0.05^a	3.23 ± 0.04^a
L-Alanine (250 ppm)	4.0 ± 0.01^a	4.23 ± 0.04^b	4.29 ± 0.04^b	4.45 ± 0.05^b	4.52 ± 0.05^b	4.63 ± 0.05^b
L-Leucine (250 ppm)	4.0± 0.02^a	4.15 ± 0.05^c	4.20 ± 0.09^c	4.32 ± 0.05^c	4.39 ± 0.04^c	4.47 ± 0.05^c
L-Valine (250 ppm)	4.0 ± 0.03^a	4.21 ± 0.04^b,c	4.32 ± 0.04^b	4.35 ± 0.05^c	4.44 ± 0.05^c	4.52 ± 0.05^c
L-Glutamic acid (250 ppm)	4.0 ± 0.01^a	4.30 ± 0.04^d	4.35 ± 0.04^b	4.42 ± 0.04^b	4.47 ± 0.04^b,c	4.52 ± 0.05^c
Monosodium L-Glutamate (250 ppm)	4.0 ± 0.02^a	4.23 ± 0.04^b,d	4.39 ± 0.05^b	4.56 ± 0.04^d	4.79 ± 0.04^d	4.95 ± 0.05^d
L-Asparagine (250 ppm)	4.0 ± 0.03^a	3.92 ± 0.04^e	3.72 ± 0.04^d	3.69 ± 0.03^e	3.55 ± 0.03^e	3.42 ± 0.03^e
L-Aspartic acid (250 ppm)	4.0 ± 0.03^a	3.85 ± 0.04^b,c	3.70 ± 0.04^b	3.62 ± 0.03^b	3.41 ± 0.03^a	3.35 ± 0.03^b
L-Cystein (250 ppm)	4.0 ± 0.05^a	3.79 ± 0.05^b	3.57 ± 0.06^c	3.49 ± 0.05^c	3.27 ± 0.05^b	3.24 ± 0.05^b
L-Glutamine (250 ppm)	4.0 ± 0.01^a	3.87 ± 0.05^c	3.62 ± 0.05^c	3.57 ± 0.04^b	3.37 ± 0.04^a	3.29 ± 0.03^d
L-Glycine (250 ppm)	4.0 ± 0.03^a	3.62 ± 0.05^d	3.57 ± 0.04^c	3.47 ± 0.04^a,c	3.35 ± 0.04^a	3.25 ± 0.04^b
L-Proline (250 ppm)	4.0 ± 0.04^a	3.86 ± 0.05^c	3.59 ± 0.04^c	3.49 ± 0.04^c	3.37 ± 0.04^a	3.23 ± 0.04^a
L-Serine (250 ppm)	4.0 ± 0.03^a	3.65 ± 0.05^b	3.52 ± 0.06^b	3.43 ± 0.04^a	3.27 ± 0.04^b,d	3.25 ± 0.05^b
L-Tyrosine (250 ppm)	4.0 ± 0.02^a	3.40 ± 0.05^c	3.35 ± 0.05^c	3.27 ± 0.05^c,b	3.10 ± 0.05^c	2.89 ± 0.05^c
L-Arginnine (250 ppm)	4.0 ± 0.04^a	3.47 ± 0.05^c,a	3.42 ± 0.04^a,c	3.25 ± 0.04^b	3.17 ± 0.03^b	2.95 ± 0.03^c,d
L-Histidine (250 ppm)	4.0 ± 0.03^a	3.52 ± 0.05^a	3.45 ±0.05^a,b	3.32 ±0.05^d,c	3.29 ± 0.05^d	2.99 ± 0.04^d
L-Isoleucine (250 ppm)	4.0 ± 0.01^a	3.67 ± 0.05^b	3.52 ± 0.05^b	3.37 ± 0.05^d	3.27 ± 0.05^d	3.24 ± 0.04^b
L-Lysine (250 ppm)	4.0 ± 0.02^a	3.72 ± 0.04^b	3.56 ± 0.06^b	3.42 ± 0.04^a	3.39 ± 0.03^a	3.28 ± 0.03^b
L-Methionine (250 ppm)	4.0 ± 0.04^a	3.81 ± 0.06^c	3.72 ± 0.04^c	3.45 ± 0.04^a	3.37 ± 0.05^a	3.27 ± 0.04^a,b
L- Phenylalanine (250 ppm)	4.0 ± 0.01^a	3.67 ± 0.05^b	3.52 ± 0.06^b,a	3.43 ± 0.04^a	3.37 ± 0.03^a	3.27 ± 0.03^a,b
L-Threonine (250 ppm)	4.0 ± 0.03^a	3.71 ± 0.05^b	3.65 ±0.05^c	3.42 ±0.05^a	3.39 ± 0.05^a	3.29 ± 0.04^b
L-Tryptophan (250 ppm)	4.0 ± 0.01^a	3.62 ± 0.05^d	3.57 ± 0.04^c	3.47 ± 0.04^a	3.35 ± 0.05^a	3.25 ± 0.04^b

Values are means ± SD for five values

Values not sharing a common superscript between amino acids differ significantly at p< 0.05 (DMRT)

Table 4. Effect of phytochemicals on pH during the process of curdling

Phytochemicals	pH
Phytochemicals	0 day	I day	II day	III day	IV day	V day
Control	4.0 ± 0.02^a	3.50 ± 0.06^a	3.45 ± 0.04^a	3.40 ± 0.04^a	3.37 ± 0.05^a	3.23 ± 0.04^a
Syringic acid (250 ppm)	4.0 ± 0.03^a	4.19 ± 0.04^b	4.25 ± 0.05^b	4.37 ± 0.05^b	4.49 ± 0.03^b	4.62 ± 0.04^b
Protocatechuic acid (250 ppm)	4.0 ± 0.01^a	4.15 ± 0.05^b	4.21 ± 0.05^b	4.35 ± 0.05^b	4.42 ± 0.05^c	4.50 ± 0.04^c
Borenol (250 ppm)	4.0 ± 0.04^a	3.95 ± 0.05^c	3.87 ±0.03^c	3.56 ±0.03^c	3.47 ± 0.05^d	3.41± 0.04^d
Ursolic acid (250 ppm)	4.0 ± 0.02^a	3.72 ± 0.04^d,c	3.65 ± 0.04^c	3.61 ± 0.05^d	3.57 ± 0.06^e	3.53 ± 0.06^e
Vanillic acid (250 ppm)	4.0 ± 0.01^a	3.87 ± 0.04^d	3.71 ± 0.05^d	3.69 ± 0.05^d	3.62 ± 0.04^f	3.50 ± 0.04^c
Piperine (250 ppm)	4.0 ± 0.02^a	3.94 ± 0.04^b	3.85 ± 0.04^b	3.69 ± 0.04^b	3.62 ± 0.04^b	3.56 ± 0.04^b
Ferulic acid (250 ppm)	4.0 ± 0.01^a	3.90 ± 0.03^c	3.82 ± 0.03^c	3.72 ± 0.04^c	3.50 ± 0.04^b	3.43 ± 0.04^c,e
Morin hydrate (250 ppm)	4.0 ± 0.02^a	3.72 ± 0.04^c	3.69 ± 0.04^c	3.65 ± 0.05^c,b	3.57 ± 0.06^b	3.50 ± 0.06^c
Coumarin (250 ppm)	4.0 ± 0.03^a	3.69 ± 0.04^d	3.65 ±0.05^b	3.61 ±0.03^d	3.57 ± 0.06^c	3.52 ± 0.06^d
Sesamol (250 ppm)	4.0 ± 0.01^a	3.73 ± 0.04^c	3.68 ± 0.03^c	3.65 ± 0.05^c,b	3.61 ± 0.03^b	3.58 ± 0.04^e

Values are means ± SD for five values

Values not sharing a common superscript between amino acids differ significantly at p< 0.05 (DMRT)

Inhibition of LAB by amino acids and phytochemicals on pH during the process of curdling

Table 3 shows the effect of amino acids on pH during curdling process. Alanine, leucine, valine, glutamic acid, and monosodium L-glutamate increased the pH due to inhibition of lactic acid production in curdling process whereas other amino acids significantly decreased the pH values and not inhibited the secretion of lactic acid.

Table 4 shows the effect of phytochemicals on pH values during curdling process. Protocatechuic acid and syringic acid significantly increased the pH values, due to the inhibition of lactic acid production during curdling process, whereas all the other phytochemicals did not shows significant changes.

CONCLUSION:

In this study, we investigated the role of amino acids and certain phytochemicals when introduced during curdling process. The lactic acid bacteria showed sensitivity to 4 amino acids, namely, alanine, leucine, valine and glutamic acid and monosodium L-glutamate and two phytochemicals namely, protocatechuic acid and syringic acid. Similar results also observed while curdling process. Thus, the present study shows Lactobacillus growth can be inhibited by amino acids and certain phytochemicals and control the fermentation process.

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