Characterization of physiochemical parameters & their effect on microbial content of smokeless tobacco products marketed in north India

The use of smokeless tobacco (SLT) products is one of the serious public health hazards globally. The recent global surveillance report showed that consumption of SLT is accountable for nearly 6,50,000 deaths worldwide, of which 88 per cent of the burden is borne by the South-East Asian region, with the majority occurring in India (74%)1. India has the largest number of SLT users among 140 countries that use SLT across the world1. Of the 346 million global consumers, India alone has 80.8 million SLT consumers. As per the Global Adult Tobacco Survey-2 report, 21.4 per cent (199.4 million) of all adults comprising 29.6 per cent of men and 12.8 per cent of women currently consume SLT all over India. The low cost of manufacturing and lower taxation rates on SLTs could be responsible for its very high consumption in India. Further, inadequate manufacturing standards lead to marketing of harmful SLTs as safe substitutes for smoking2.

In India, there are several types of oral SLT products available for chewing, including khaini, gutka, pan masala with tobacco and gul, whereas snuff, a fine tobacco mixture, is inhaled through nostrils. The various ingredients in SLT products are formulated to enhance the tobacco addictiveness; however, the complex mixture culminates in increasing their toxic and carcinogenic potential. The major alkaloids in the tobacco leaves are nicotine, nornicotine, anabasine and anatabine, out of which nicotine comprises 90-95 per cent of the total alkaloids and is responsible for enhancing the addictive profile of tobacco. Myosmine, nicotyrine, isonicotenine, N-methyl anabasine and cotinine are minor alkaloids that have also reported to have pharmacologically relevant properties3. Even though the use of SLT does not involve burning, it exposes the users to a highly carcinogenic mixture (more than 30 in number) of tobacco-specific N-nitrosamines (TSNAs), nitrates, nitrites and heavy metals such as chromium, nickel and cadmium4. Hence, consumption of SLT can cause cancers of oral cavity, oesophagus and pancreas. Other adverse health effects of SLT products are dental caries, gingival recession, tooth attrition, oral submucous fibrosis (areca nut–tobacco products), cardiovascular disease, diabetes, hypertension, reproductive health problems and overall increased mortality5. Moreover, a negative correlation between the complete haemogram and SLT consumption has been reported in the blood profile of SLT users when compared to non-users such as lower levels of mean serum haemoglobin, mean serum total red blood cells, mean serum total platelets and mean serum packed cell volume6.

The pH of the SLT product appears to be a significant factor that determines the amount of un-ionized nicotine absorbed by its user7. Moreover, nitrate–nitrite ratio is also an important factor which affects absorption. The nitrate present in SLT is metabolized by oral bacterial enzymes in the saliva to nitrite, an acutely toxic and powerful oxidizing agent that influences the formation of methaemoglobin in blood8.

Moisture content in the SLT product is one of the key factors promoting the growth of microbes, which influences the conversion of nitrites and alkaloids to TSNAs. The presence of bacterial and fungal species can increase the toxicity profile of the SLT by enriching it with endotoxins, mycotoxins and pro-inflammatory metabolites like lipopolysaccharides. Several species can be pathogenic to human and their ingestion can enhance disease susceptibility. Hence, microbiological contamination is an additional risk factor for altering the constituents of SLT products9.

Richter et al analyzed the pH, moisture and nicotine content in popular brands of SLT available in the United States10. This study demonstrated that pH is the key determinant of the total unprotonated nicotine, the most readily absorbable form of the alkaloid. A similar study was conducted in SLT samples collected from markets in south India; however, the nicotine content was not estimated11. Thus, there is a scarcity of systematic studies that describe the physical, chemical and microbiological characteristics of the most commonly used commercially available SLT products in India. The present study would be the first of its kind to characterize the physiochemical properties of such a broad array of SLTs and correlate the microbial diversity in these samples with their pH and moisture content. Nevertheless, this study has at least two limitations. First, the study comprises 37 SLT products that were procured during the COVID-19-related nationwide travel restrictions, and hence were purchased in the National Capital Region and surrounding districts. This restricted geographical location cannot represent the nation as a whole. Moreover, several brand names or varieties of SLT exist, and varied climatic conditions across the country may also influence the pH and moisture of these products during processing and storage. Second, the study did not collect data on the market share of the products; therefore, the varieties selected for testing may not have been the most popular or highly selling member of the SLT type.

Material & Methods

The study was conducted in ICMR-National Institute of Cancer Prevention & Research, Noida, Uttar Pradesh, India after obtaining ethics clearance from the institute ethics committee.

Sample collection: Thirty-seven SLT products of various brands of eight different categories were procured from retail locations in the metropolitan areas of Delhi, Noida and Ghaziabad during December 2019-August 2020. These SLT products are most commonly and widely used in this region. Most of the products are packaged and branded; only two variants of the local product Mainpuri kapoori procured from Mainpuri district in Uttar Pradesh were available in unlabelled pouches. The SLT products characterized according to the product type and brands are schematically represented in Fig. 1. These tobacco samples are common in this region and abundantly available in local shops. The specific product type is most often mentioned on the packaging label under the brand name. If much information about consumption of a product was not available, it was collected from local vendors, and the packets were opened to check the physical state of the content and its appearance for confirmation. Each category of the product has at least two different variants. The unopened packages weighed around 3-5 g and were stored at room temperature as in retail settings, before analysis.

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Fig. 1

Schematic representation of the categorization of smokeless tobacco products sampled for this study.

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Sample characterization: For each sample, the physical analysis [colour, sample state (solid/liquid), smell, pH and moisture content] and chemical analysis (total alkaloid and nicotine content) were performed. Two of the qiwam category products were very sticky in nature; therefore, the estimation of moisture could not be performed. The colour and sample state were tested by visual inspection.

pH measurement: The pH of SLT samples were measured on Eutech 700 pH meter (Thermo Fisher Scientific, Bengaluru) by employing the World Health Organization (WHO) TobLabNet SOP 1412. Before measuring the pH, the electrode was calibrated using at least three pH buffers (4, 7 and 10) to produce a three-point calibration that will cover the pH range of the product tested. Further, 2 g of homogenous tobacco product was suspended in 20 ml de-ionized water and stirred for 30 min. The resulting solution was then filtered through Whatman filter paper, and the pH of each sample was measured to a precision of at least two decimal places. The electrode was rinsed with de-ionized water, before and after each measurement.

Estimation of moisture: The moisture content of SLT samples was performed by using WHO TobLabNet SOP 1313. The oven was set at 100°C±1°C, and the temperature was allowed to stabilize for an hour. The empty pan and a pan with 2 g homogenous tobacco sample were weighed. The tobacco samples with pan were heated for 3 h, and thereafter, the pan with the sample was allowed to cool in an airtight desiccator. The pan with the sample was weighed on an analytical balance, and the loss in weight was used to calculate the percentage moisture content of tobacco samples.

Estimation of total alkaloid content: The analysis of total alkaloid in an SLT sample was measured on a continuous flow autoanalyzer using Flow View Solution 3700 analyzer (Xylem South Asia, Gurugram, Haryana). The analysis was performed by the methods provided by OI Analytical, USA14 with detection limit of 1.19 mg/l.

Estimation of nicotine content: The nicotine level in SLT samples were estimated by gas chromatography using flame ionization detector. The analysis was performed by adopting WHO TobLabNet SOP 1215 with LOD of the method being 2 ppm.

Calculation of bioavailable or free nicotine for immediate absorption: The free nicotine, i.e. the nicotine content available for absorption, was estimated using Henderson–Hasselbalch equation16.

where pKa = 8.02 for nicotine, [B] = amount of absorbable free nicotine and [BH⁺] = amount of ionized nicotine.

Estimation of bacterial and fungal diversity: As described in the previous studies performed, the V₃-V₄ region of 16S rDNA and the ITS1 amplicon-sequencing techniques were used to identify the bacterial and fungal diversity by estimating operational taxonomic units (OTUs), respectively, of 22 SLT products171819.

Data analysis: The one-way ANOVA was used to compare variance in pH, moisture, nicotine levels and microbial richness within different SLT types. Spearman correlation coefficient (r) was calculated to measure association between any two variables. The assumptions for linear regression were met, including constant variance, normal distribution and independent of one another (n=19). All statistical analyse were performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA).

Results

Categorization of smokeless tobacco products: The samples were categorized based on their types as mentioned on the packaging label or visual identification upon opening the packets (Fig. 1). Khaini (K) category of SLT products is plain chewing tobacco mixed and consumed with a pack of slaked lime. Eleven different brands (K1-K11) were used for the study. Snus (S) is usually available in pouches and placed on the inner surface of the lips. Four different brands (S1-S4) were used for the study. Moist snuff (MS) is placed between lip and gum. Three different varieties (MS1-MS3) were analyzed in this study.

Four different brands of gul (G1-G4) products available either in plastic sachets or tin boxes were analyzed for the study. Gul is a dry powder like tobacco-containing dentifrice mixed with molasses, lime and red soil. Various brands of pan masala (PM) are widely being used, comprised of areca nut, catechu, flavouring agent, sweeteners, etc., with a separate pouch of sundried tobacco leaves (zarda). Seven different brands of pan masala (PM1-PM7) and three brands of zarda (Z1-Z3) were analyzed in this study.

Mainpuri kapoori (MK) is a local product comprising finely cut betel nut and small pieces of tobacco leaves treated in slaked lime and flavouring agents. It is held in the mouth, chewed and sucked. Two variants were used in the study. Qiwam is available as a thick paste prepared from tobacco leaf extract, flavouring spices and additives such as musk. It is placed in the mouth and chewed. Three varieties of qiwam (Q1-Q3) were analyzed in the present study.

Characterization of smokeless tobacco products: The products examined in this study included khaini, snus, moist snuff, gul, pan masala, zarda, Mainpuri kapoori and qiwam.

Colour: The colour of all the samples of khaini category was from light to deep brown whereas it was dark brown for snus, moist snuff and gul categories. All the samples of pan masala category was silver-grey in colour, whereas zarda products were light brown in colour. The colour of Mainpuri kapoori was light brown, whereas one of the products of qiwam category was dark brown and the rest were light brown.

State: Khaini samples were leafy in nature consisting of either sundried or roasted tobacco leaves. All the samples tested in snus were solid and packed in small pouches, whereas moist snuff used in this study was finely ground moist tobacco. Gul was available as a mixture of powdered tobacco product and ashes of tendu leaves. All the pan masala products used for the study were solid in nature, and zarda products were leafy in nature. Mainpuri kapoori was a mixture of tobacco, betel nut and artificial flavouring agent prepared locally. One of the qiwam category products was semi-solid, and the rest were solid in nature.

Smell/odour: All the pan masala products had flavouring agents. However, the rest of the SLT products studied were having the smell of tobacco.

Solubility: All the SLT products studied were partially soluble in water.

pH: The recorded pH values of the 37 samples were found to range between 5.36±0.06 and 10.12±0.45, with a mean pH of 7.8±0.34 (Fig. 2A). The maximum pH [±standard deviation (SD)] values were recorded for snus products (10.12±0.45), the values being significantly higher than that observed for khaini (P<0.0001), zarda (P<0.0001), Mainpuri kapoori (P<0.001) and qiwam (P<0.0001) (Supplementary Table I). The three moist snuff products also exhibited high pH (±SD) levels of 9.94±0.28. The gul and pan masala brands exhibited a moderate-to-high pH having mean (±SD) values of 9.10±2 and 8.99±0.08, respectively. The products exhibiting lower pH values were khaini (6.40±0.24) and Mainpuri kapoori (7.04±0.43); however, the lowest pH (±SD) values were exhibited by products of qiwam (5.53±0.14) and zarda (5.36±0.06).

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Fig. 2

The pH and moisture content were estimated in 37 smokeless tobacco products. The data of (A) pH and (B) moisture are expressed in the form of bar graph. Data were expressed as mean ± standard deviation, P<0.05 (95% confidence interval) is significant, calculated compared to each group. P^*<0.05, ^**< 0.01, ^***<0.0001.

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Moisture content: The analysis showed that the highest moisture (%±SD) content was recorded in moist snuff products (51.75±2.2), the values being significantly higher (P<0.0001) than all other SLTs (Fig. 2B, Fig. 2 Supplementary Table II). Snus exhibited a mean moisture content (%±SD) of 23.14±4.82, followed by khaini brands having a mean moisture content of 17.24±3.1, which was comparable to that observed in zarda products (16.24±0.95). The mean moisture content (%±SD) for gul SLT products (6.79±1.12) and Mainpuri kapoori (12.27±2.3) was slightly higher than those observed for the pan masala brands (4.75±1.01). The moisture content of qiwam could not be determined; however, the physical appearance suggested that it had reasonably high moisture content. The SLT samples showed normal distribution of variables for pH and moisture in each category of SLT product. There was no significant correlation between the pH and moisture of the SLT products (Table I).

Total alkaloid content: Total alkaloid content of the SLT products were estimated. The results indicated that the products in qiwam category recorded the maximum alkaloid content (mg/g±SD) of 61.25±2.12, whereas pan masala products had the least alkaloid content 0.82±0.1.

Nicotine content: Nicotine level of the SLT products were estimated, and the results are summarized in Table II. It was observed that khaini products had the highest nicotine content ranging from 16.05 to 31.934 mg/g; however, one sample (K4-tobacco leaf) reported 51.135 mg/g nicotine which was much higher than the mean level. Zarda products contained nearly 16.7±0.64 mg/g nicotine followed by gul samples that reported 13.9±5.1 mg/g nicotine. The qiwam samples showed low levels of nicotine of around 4.4±1.63 mg/g, whereas the lowest nicotine level was observed in Mainpuri kapoori (2.593 mg/g & 0.663 mg/g, respectively).

Next, with the help of Henderson–Hasselbalch equation, the free nicotine, i.e. the bioavailable nicotine, was calculated for each product (Table II). A graph was plotted between per cent of free nicotine vs. pH of the SLT products which showed that the SLT sample with high pH had higher free nicotine levels (Fig. 3). A highly significant correlation was observed between the pH and the fraction of free nicotine available for absorption from the SLT (Table I).

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Fig. 3

Correlation between per centage of free nicotine and pH of the smokeless tobacco products.

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Bacterial and fungal richness: The number of bacterial OTUs determined per sample was positively correlated with the moisture content of the SLT (r=0.3158; P=0.0235) (Fig. 4A). However, the number of fungal OTUs per sample showed a non-significant relationship with the moisture level (r=0.1604; P=0.5502) (Fig. 4B). Further, the study explored the relationship between the bacterial and fungal populations in the SLT microbiota. Bacterial and fungal alpha diversity values were positively correlated (r_observed = 0.5504; r_chao1 = 0.4914) when assessed by linear regression (p_observed = 0.0146; p_chao1 = 0.0326) (Fig. 4C and D). Furthermore, the study also checked the influence of pH on SLT-associated microbial composition and observed that there was no significant effect of pH on the bacterial as well as fungal OTUs in the SLT (

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Fig. 4

Correlation between moisture content and microbial diversity in smokeless tobacco samples. (A) Bacterial operational taxonomic units (OTUs) vs. moisture; (B) Fungal OTUs vs. moisture; (C) Fungal observed richness vs. bacterial observed richness; (D) Fungal Chao1 diversity vs. bacterial Chao1 diversity.

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Discussion

The WHO Framework Convention on Tobacco Control (WHO FCTC), along with the members, has made several efforts to fight the tobacco epidemic. In India, the omnibus tobacco control law, Cigarettes and Other Tobacco Products Act (COTPA), has been implemented for the prevention and control of tobacco consumption. This legislation also requires the display of tar and nicotine contents along with the maximum permissible limits on tobacco product packaging; however, it has not been brought into effect [Section 7(5)]20. The content of nicotine, an alkaloid in the tobacco leaves, in the SLT is responsible for its addictiveness and consumer appeal21. In the present study, 37 Indian SLT samples, including several popular brands of khaini, snus, moist snuff, gul, pan masala, zarda, Mainpuri kapoori and qiwam, were analyzed. The objective was to provide information on the pH, moisture and nicotine content of the SLT, the major determinants, which influence the bioavailable nicotine levels upon consumption of these products.

Natural tobacco occurs in acidic form, leading to slow release of free-base nicotine unless buffered to alkaline levels21. Earlier findings have demonstrated that the levels of nicotine absorbed by the SLT users are impacted by the nicotine content in the product, pH and behavioural factors such as the rate of expectoration22. The pH (±SD) of SLTs varied in our samples from 5.36±0.06 to 10.27±0.03, and was highest for snus and lowest for qiwam. This variability in pH can be attributed to additives such as ammonium bicarbonate, ammonium carbonate, ammonium chloride, sodium citrate, sodium bicarbonate, calcium bicarbonate, potassium bicarbonate and slaked lime, which are added during the curing process of the product to modulate their acidity levels23. Increasing the alkalinity of SLT enhances the absorption of nicotine and subsequently increases its physiological effects24.

Nicotine pKa (dissociation constant) value is 8.02 and in solution it occurs as ionized and unionized form. Therefore, the pH of SLT is a crucial determinant of nicotine absorption through the nasal and oral mucosal membranes. Nicotine is unionized at alkaline pH and therefore gets rapidly absorbed and transported to the central nervous system, resulting in instant stimulation. Whereas, in acidic (low pH) condition the ionic form of nicotine prevalent, which make it difficult to cross biological membranes25. The results of the current study are in consensus with the above findings, and a positive correlation was observed between pH of the SLT product and its free nicotine levels.

The moisture content is another product characteristic that is regulated by the manufacturer. High moisture content favours the growth of microbes such as bacteria, fungi and viruses192627. The presence of microorganisms and their metabolic activities has been linked to the conversion of nicotine and other tobacco alkaloids to highly carcinogenic TSNAs by nitrosation reaction2829. The microbial growth also exposes the oral cavity of SLT users to harmful microbial metabolites such as endotoxins and mycotoxins, leading to dysbiosis of the oral microbiome or even chronic inflammation resulting in pre-cancerous lesions303132. In the present study, an inverse correlation was observed between moisture and free nicotine content, i.e. high moisture content does not influence the levels of free nicotine. Further, our previous studies determined the presence of a complex diversity of microbes (both bacterial and fungal) in Indian SLT products171819. Interestingly, the SLTs having higher moisture content exhibited elevated levels of microbes, both in terms of diversity and number, which is corroborated with the previous studies33. Therefore, the moisture content can enhance the toxicity profile of an SLT.

All things considered, even though these SLTs do not hamper consumers similar to burnt tobacco, their consumption can deliver a sufficient amount of nicotine to cause addiction and toxic manifestations, including the development of pre-cancerous lesions in the oral mucosa. Therefore, the characterization of SLTs, especially the unpackaged local SLTs that lack in manufacturing and packaging standards, is very crucial, and the present findings have important implications for tobacco control research and policy development.

Consumption of SLT globally poses a significant public health threat, predominantly in South and Southeast Asia. The Article 20 of the WHO FCTC emphasizes the significance of research, surveillance and information exchange to control SLT34. It highlights the need for national scientific research, surveillance mechanisms and comprehensive programmes to tackle SLT’s impact on society, economy and health. In addition, it encourages international cooperation to enhance SLT control measures, with special consideration for developing countries34. As a party to the convention, India’s proactive approach in tackling SLT consumption through legislative measures, participation in regional initiatives and innovative taxation strategies showcases its leadership in the global fight against tobacco-related health hazards. The Government of India has gone beyond COTPA and implemented several additional laws to combat SLT consumption effectively, such as introducing a new Goods and Services Tax structure to significantly increase the taxation on cigarettes, SLT and bidis. Notably, India’s National Tobacco Testing Laboratories in different regions are playing vital roles in advancing SLT control measures. To further enhance these efforts, India has become part of a new initiative called Addressing SLT and Building Research Capacity in South Asia (ASTRA). Through ASTRA, India conducted a comprehensive policy review and analysis to assess existing SLT regulations35.

Overall the outcome of the current study, suggests that the manufacturing of SLT products should be coupled with thorough quality control protocols at all stages including tobacco cultivation, harvesting, curing, storage and mixing with other components during synthesis of SLT. Furthermore, the use of SLT as an alternative to cigarette smoking should be discouraged, and cessation programmes must call attention to their detrimental effects and emphasize the benefits of quitting SLT consumption. Additional studies with a much larger sample size representative of the Indian retail market are warranted, focussing specifically on the levels of potentially toxic constituents of SLT and their microbiological analysis.