Introduction
Tulsi, mint and spinach all are herbs and very important as well as very beneficial for overall health and well-being. However, sometimes they contain some harmful bacteria and parasites that may cause food borne infections. Tulsi is sometimes used as divine offering or Prasad. Not many studies have tried to find out the load of pathogenic bacteria, parasitic cysts and ova causing foodborne infections, in these leafy vegetables. In this study we will try to find the pathogenic microorganisms present on these herbs and their detrimental effects on the health of consumers.
Tulsi
Holy basil (Ocimum sanctum) is commonly known as Tulasi in Sanskrit or Tulsi in Hindi.1 Holy basil is a perennial flowering plant from the family called Lamiaceae. While this plant is native to the Indian subcontinent, it may also grow throughout Southeast Asia.2 Tulsi has a special place in Ayurveda as well as the homes of Hindus in India. It is considered sacred by Hindus and worshipped by them and is also used medicinally to relieve symptoms like cough and cold. Three main types of Tulsi are seen growing in India:
Bright green leaves called Ram Tulsi. Purplish green leaves called Krishna Tulsi. Common wild variants are called Vana Tulsi.
Tulsi has used as prasad or offering to God and also proved to be highly effective in protecting our body from various infections and diseases of the liver, skin, kidney, respiratory tract and other sites.
Tulsi is known to have many medicinal properties like:
It might be an antipyretic agent
It might have anti-inflammatory activity
It might have an antiemetic action
It might help lower blood sugar (antidiabetic effect
It might act as a hypotensive ( can lower blood pressure
It might have hypolipidemic activity
It might act as an analgesic
It might have anti-asthmatic activity
It might be an hepatoprotective (liver-protective) agent
It might help reduce stress (antistress)
It might be a potent expectorant (expels mucous)
It might have anticancer potential
It might be a diaphoretic (induces sweating).
Owing to its numerous health benefits, Tulsi is rightly called the ‘Queen of Herbs’.3
In spite of having several beneficial roles, Tulsi may contain several harmful bacteria like- Enterobacterales (a previous large family of Gram-negative bacteria that includes a number of pathogens such as Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Salmonella spp., Shigella spp., Proteus spp., Serratia spp. and other species), Pseudomonas spp.4 and coccidian parasites like- Cyclospora spp.5
Pudina
Mint, scientifically known as Mentha spicata, is an aromatic herb belonging to the family Lamiaceae. It is called Spearmint in English and Pudina in Hindi. It is widely used in Indian cuisines for its characteristic flavour. The herb originated in Europe.6 Fresh and dried leaves and essential oils derived from Pudina are widely used in the food, cosmetic, confectionery, chewing gum, toothpaste, and pharmaceutical industries.7 It is grown worldwide for commercial uses as a flavouring agent for food and to be used in medicines.8
The properties of Pudina are given below:
It may act as carminative (for removal of gas from the stomach)
It may show antispasmodic property (used to relieve muscle spasms)
It may show diuretic activity (causes the kidneys to make more urine)
It may show antibacterial and antifungal effects.
It may show antioxidant agent and minimize the damage caused by oxidants.7
It may show anti-diarrhoeal effects and also potent anti-asthmatic activity.
It may help to reduce cold & fever.
It may help to manage weight
It may act as an antidote to counteract some particular poisons.8
There is still no information about the presence of any pathogenic bacteria and parasites on or in pudina leaves, and people are searching for it.
Spinach
Spinach (Spinacia oleracea) is a green leafy flowering plant native to central and western Asia. It belongs to the family Amaranthaceae. It is consumed either fresh, after cooking, or after storage using preservation techniques by canning, freezing or dehydration. It may be eaten cooked or raw, and the taste differs considerably. The high oxalate content may be reduced by steaming.9
The beneficial properties of spinach are as follows:-
High in antioxidants, and scavenges free radicals and prevents oxidative stress18
Although spinach is quite beneficial for our health, a variety of bacteria can be present in it, like Salmonella spp. and Shigella spp.19, 20, 21 Even parasites like- Ascaris lumbricoides eggs, Entamoeba histolytica, Giardia lamblia cysts may be present in it.22, 23, 24, 25
Tulsi, pudina and spinach are all used as food as well as for medicinal purposes. Leafy vegetables like spinach, lettuce and pudina are good sources of dietary fiber and vitamins like Vitamin K. Tulsi (Ocimum sanctum) is also used as food rarely and also commonly as offering to God in temples for Prasad. Leafy vegetables have many antimicrobial properties and are also good source Vitamin K, Dietary fibers, mineral and Vitamin K. However, they can also contain many pathogenic microorganisms which can infects us by feco-oral route. Many researchers have been able to detect parasitic eggs and cysts along with pathogenic bacteria in leafy vegetables. High concentrations of thermotolerant coliforms (ThC), intestinal helminth eggs, and protozoa have been found in a wastewater-fed lake where water spinach was grown. Water spinach samples can contain Cyclospora (8%), Giardia cysts (56%), Cryptosporidium (17%).26 Many workers have detected Cyclospora in vegetables in the local agricultural markets in the Central Valley of Costa Rica. The highest proportion of faecal coliforms was identified during the rainy season. Cyclospora spp. have been identified only in lettuce during the dry season.27
In other studies, protozoa Giardia duodenalis cysts have been found on lettuce.27 In fact, fresh produce, in particular, as it is eaten with minimal preparation, is a very strong vehicle for transmission of bacterial and parasitic infections, and G. duodenalis cysts have been detected in such produce in many countries.27
VTEC (Verotoxigenic Escherichia coli) strain, E. coli O157:H7 ‘Sakai,’ has also been found to colonize the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum), and prickly lettuce (Lactuca serriola).28 This contamination is related to irrigation pattern, use of portable toilets and the field worker personal hygiene. various factors like contaminated soil, fertilizer (manure/compost), wildlife, and irrigation water also contribute to presence of Escherichia coli in produce.29 This can be a dangerous pathogen as it may also cause Hemolytic uremic syndrome (HUS). High burden of Salmonella contamination has been seen in leafy vegetables, including mint leaves, parsley and lettuce.30 Moreover these leaves are consumed raw or by making sauce (e.g. Mint leaves).
Spinach leaf has also been found to contain harmful oocysts of coccidia like Cryptosporidium parvum.31 Many foodborne illnesses in the United States are caused by germs on vegetables and fruits that people eat raw. Harmful germs sometimes found on leafy greens include E. coli, norovirus, Salmonella, Listeria, and Cyclospora.32
So keeping these things in mind, it can be surmised that are lots of pathogenic bacteria and parasites found in these leaves which can infect us.
In a study, fifty-eight leaf samples (58) were collected by us from the markets of different zones in Kolkata (east, west, north & south) and also other places of West Bengal.
Samples were collected in sterile universal container or if packed, then in packet. Then samples were transported to the laboratory for testing. Samples will be weighed, and specific weight or volume were cultured on the following media -------
Mac Conkey agar with neutral red as pH indicator (Peptone, Neutral red, agar, Lactose, Sodium taurocholate, deionized water) for culturing aerobic LF or NLF bacteria.
Xylose Lysine Deoxycholate (XLD) media {Agar, Sucrose, Lactose, Sodium thiosulphate, L-Lysine, NaCl, xylose, Yeast extract, Sodium deoxycholate, Ferric ammonium citrate} for culturing aerobic bacteria like Shigella and Salmonella spp.
Robertson’s cooked meat medium (RCM) {Cooked meat medium, Peptic digest of animal tissue, Dextrose, Sodium chloride, yeast extract, Iron filings, Hemin, Vitamin K} for culturing anaerobic bacteria. RCM were incubated for 2 days and then Gram stain was done to find anaerobic bacteria.
Selenite F broth for enrichment and later after 1 day of incubation at 37 degree C, 1 loopful was subcultured from it on to XLD agar and MacConkey agar again. This will ne done to find Shigella spp. and Salmonella spp.
Normal saline and Lugol’s Iodine were used to detect the trophozoites and cysts of protozoa, and egg & larvae of helminths by making wet mount.
One gram of dry sample was mixed in 1 ml normal (0.9%) saline in a staerile universal container, vortexed and transferred to 10 ml test tube. Then it was centrifuged at 5000 rpm for 2 minutes, and with deposit, saline mount, Lugol’s Iodine mount and Gram stain were done. Modified ZN stain was carried out with 4% sulphuric acid as decolorizer. Also culture on the aforesaid media were performed from the deposit. The colonies of the bacteria that appeared on the culture media after overnight incubation, were identified by Gram stain, Catalase, Oxidase and other standard biochemical tests.
We studied 58 samples. Among them 27 samples were of Tulsi, 18 were Mint and 13 were Spinach.
Cryptosporidium oocysts were present in 2 spinach samples.
Commonest bacteria in the leaves were:
In Tulsi- Klebsiella aerogenes, Enterobacter cloacae, K. pneumoniae and Bacillus cereus.
In Pudina- E. cloacae, Klebsiella aerogenes, Pseudomonas aeruginosa and Klebsiella oxytoca.
In Spinach- Aeromonas schuberti, K. aerogenes, Pseudomonas aeruginosa, Klebsiella oxytoca and Non-EHEC 5157:H7 Escherichia coli.
Clostridium spp. grew on 2 tulsi leaves on RCM.
Most leaf washings had pH 6. Figure 1 below shows image of tulsi leaf.
Discussion
Hence researchers have seen that leaves like Tulsi, Pudina and spinach do contain harmful bacteria and parasites which can cause gastroenteritis in the consumers. Protozoan parasites like Giardia duodenalis, Cryptosporidium parvum and Cyclospora cayetanensis have led to such vegetable or fruit-borne outbreaks of gastroenteritis. We also saw that edible leaves like Tulsi and Pudina did harbour pathogenic microbes like Bacillus cereus and Staphylococcus aureus. Even Clostridium spp. were present. Such spore bearing bacteria may not be killed even by cooking and may cause gut infection. Edible leaves can thus harbour many pathogens which colonize them, and hence one should be very cautious while consuming them. These pathogens come on the leaves mainly from soil, water and air. These are somewhat neglected areas of public health and nutritional science. More studies are needed in these aspects.
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