Individual and combined effect of Nisin and Natamycin on sensory attributes Of Shrikhand

Introduction

Milk is an ideal food consumed by human since ancient times. Practices of converting milk into various milk products have been developed years ago. Shrikhand is an indigenous ethnic fermented milk food that assumes special importance due to its pleasant taste. Shelf life is defined as the number of days a product can be stored at a certain temperature and can be consumed safely, without deterioration of sensory characteristics such as taste, colour and texture. The shelf life is therefore determined by organoleptic quality. Biopreservation is defined as the extension of shelf life and enhanced safety of foods by the use of natural or controlled microbiota and/or antimicrobial compounds (Ananou et al. 2007) [2] . Nisin is a natural antimicrobial peptide produced by strains of Lactococcus lactis subsp. lactis that effectively inhibits Gram-positive bacteria and also the outgrowth of spores of Bacilli and Clostridia. Additionally, it has been used as a biopreservative and a potential agent in pharmaceutical, veterinary and health care products (Arauz et al., 2009) [3] . Natamycin is a polyene macrolide natural anti-fungal agent produced by fermentation of the bacterium Streptomyces natalensis. Natamycin (previously known as pimaricin) has an extremely effective and selective mode of action against a very broad spectrum of common food spoilage yeast and mould, with most strains being inhibited by concentrations of 1 to 15 ppm of natamycin (Hondrodimou et al., 2011) [8] .

Materials and Methods

Materials

The fresh, clean whole buffalo milk was procured from local market of Rahuri, Ahmednagar (MS). The freeze-dried culture of LF-40 was procured from National Collection of Dairy culture (NCDC) unit, NDRI, Karnal (Haryana) and used @ 1 per cent of milk for preparation of dahi for every trial. All chemicals required for the analytical work were used of analytical (AR) or guaranteed reagent (GR) grade manufactured by Merk, India Ltd / Glaxo India Ltd. Nisin and Natamycin a biopreservatives were obtained as a complimentary sample from Danisco Ingredients, Danisco India Pvt. Ltd., DLF Corporate Park, Gurgaon, Haryana, India.

Sensory Evaluation of Shrikhand

Sensory evaluation of both fresh and stored control and experimental shrikhand samples was carried out by a semitrained panel of five judges from the Department of Animal Husbandry and Dairy Science. The samples were assessed for colour and appearance, body and texture, flavour, and overall acceptability using a 9-point Hedonic scale according to Amerine et al. (1965) [1] .

Statistical Analysis

Data generated during the course of investigation was analyzed with the help of statistical design namely Factorial Completely Randomized Design (FCRD) as described by Snedecor and Cochran (1967) [29] .

Preparation of Shrikhand

Receiving of fresh buffalo milk

Colour and appearance

The colour and appearance scores of shrikhand stored at 5±1 °C showed a steady decline across all treatments (Table 1), reflecting progressive physicochemical and microbial changes during storage. The control (T 0 ) deteriorated most rapidly, with scores dropping from 8.75 on day 0 to 6.02 by day 35, after which the product was considered spoiled. This confirmed that the absence of biopreservatives accelerated microbial growth and pigment degradation. (-) Indicates that the product was spoiled and no further analysis was carried out Samples treated with nisin alone (T 1 and T 2 ) retained colour and appearance slightly better than the control, with mean scores of 7.83 and 7.78. Although nisin slowed bacterial spoilage, scores approached 6.00 by day 42, indicating limited protection over longer storage. Treatments containing only natamycin (T 3 and T 4 ) followed a similar pattern, with mean scores of 7.73 and 7.72, reflecting its stronger action against yeasts and mould than bacteria. A clearer improvement was seen in treatments combining nisin and natamycin (T 5 to T 8 ). T 5 (nisin 400 IU/g + natamycin 10 ppm) achieved the highest mean score (7.91), followed by T 6 , T 7 and T 8 . The superior retention of visual quality in T 5 suggests a synergistic antimicrobial effect, where nisin controlled bacterial spoilage and natamycin inhibited yeasts and mould, together reducing pigment breakdown and surface discoloration. The moderate concentrations in T 5 also appeared optimal, avoiding potential preservative interactions seen at higher levels. Overall, colour and appearance scores declined significantly (p < 0.05) over time, and the combined use of nisin and natamycin, particularly in T 5 , was most effective in maintaining the visual quality of shrikhand during refrigerated storage. These findings are consistent with earlier studies showing that nisin and natamycin help retain colour and appearance in a wide range of dairy products during storage. Kamble (2023) [13] reported a steady decline in colour and appearance scores in custard apple shrikhand, with slower deterioration in nisin treated samples. Similar improvements in visual quality with nisin have been documented in fermented milk products such as nono (Olasupo et al., 1996) [20] , salted sorghum lassi (Shaikh, 2008) [28] , mango lassi (Khurana, 2006) [15] and lassi fortified with different nisin levels (Chaure, 2010) [4] . Studies on pasteurized milk (Radha, 2014) [21] and paneer (Kankhare, 2005; Ranmode, 2010; Das, 2017) [14,23,6] also confirm that nisin slows sensory degradation by reducing microbial and oxidative changes. Natamycin has shown comparable effects. Treated yoghurt maintained stable appearance scores throughout refrigerated storage (El-Diasty et al., 2009) [7] , while khoa and cheese exhibited higher score retention due to reduced fungal growth (Vaquil, 2018; Jalilzadeh et al., 2020) [31,9] . Combined treatments of nisin and natamycin have further strengthened visual quality preservation in processed cheese sauces (Saad et al., 2015) [25] , strained yoghurt (Misirlilar et al., 2012) [2] , and Doda burfi under MAP conditions (Chawla et al., 2021) [5] . Several researchers have also documented a steady decline in the colour and appearance of control shrikhand during storage, confirming the pattern observed in the present study.

Body and texture

The body and texture scores of shrikhand stored at 5±1 °C decreased steadily across all treatments (Table 2). This decline reflects moisture migration, whey separation and microbial or biochemical changes that gradually weaken firmness and smoothness during storage. The control (T 0 ) showed the fastest deterioration, with scores falling from 8.00 on day 0 to 6.03 by day 35, after which the product was spoiled. Nisin treated samples (T 1 and T 2 ) retained higher scores than the control, with mean values of 7.42 and 7.37. Nisin slowed the activity of Gram-positive bacteria and helped maintain structural stability, although its effect weakened after extended storage. Natamycin-only treatments (T 3 and T 4 ) showed mean scores of 7.31 and 7.30. These samples benefited from reduced yeast and mould activity but lacked antibacterial protection, making them slightly less effective than the nisin treatments. (-) Indicates that the product was spoiled and no further analysis was carried out The combined treatments (T 5 to T 8 ) performed best. T 5 (nisin 400 IU/g + natamycin 10 ppm) achieved the highest mean score (7.54), followed by T 6 , T 7 and T 8 . The improved texture stability in T 5 indicates complementary antimicrobial activity that limited acid production, proteolysis and whey expulsion. The moderate concentrations used in this treatment also supported preservation without negative sensory effects. Body and texture scores declined significantly (p < 0.05) with increasing storage time in all samples. However, the combined preservative system in T 5 was most effective in maintaining desirable texture attributes and extending the physical stability of shrikhand during refrigerated storage. The body and texture scores of shrikhand declined steadily with increasing storage time, mainly due to moisture loss and microbial activity. These observations agree with earlier studies. Kamble (2023) [13] reported a rapid decline in control samples of custard apple shrikhand, while nisin treated samples showed slower deterioration, indicating improved stability. Similar findings were reported for nono by Olasupo et al. (1996) [20] , who noted that low nisin levels enhanced consistency, whereas excessive concentrations weakened texture. Nisin also extended the acceptable consistency period of nono during refrigerated storage. Studies on fermented milk products support this trend. Chaure (2010) [4] , Shaikh (2008) [28] and Khurana (2006) [15] showed that nisin effectively slowed texture degradation in lassi, salted sorghum lassi and mango lassi, extending the acceptable storage period. Positive effects were also reported in quarg cheese (Kadiya, 2009) [10] and paneer, where nisin helped preserve texture quality throughout storage (Ranmode, 2010; Kankhare, 2005; Das, 2017) [23,14,6] . Natamycin has shown similar benefits. Jalilzadeh et al. (2020) [9] demonstrated improved cheese texture at 8±1 °C with natamycin, and Vaquil (2018) [31] observed better body and texture in natamycin treated khoa due to reduced yeast and mould activity. Combined treatments have provided even greater protection. Misirlilar et al. (2012) [18] found that both nisin and natamycin maintained the consistency of strained yoghurt during 28 days of storage. Chawla et al. (2021) [5] and Saad et al. (2015) [25] reported superior texture retention in Doda burfi and processed cheese sauces when both preservatives were used together. A similar pattern of decline in untreated shrikhand has been reported by several researchers.

Flavour

The flavour score of shrikhand declined steadily across all treatments during storage at 5±1 °C (Table 3). This reduction was associated with gradual biochemical and microbial changes such as acid development, lipid oxidation and the formation of volatile compounds that affect sensory perception. The control (T 0 ) showed the fastest deterioration, with scores decreasing from 8.02 on day 0 to 6.05 by day 35, after which the product was spoiled. The rapid decline in T 0 reflected uncontrolled microbial growth and associated off flavour formation. Samples treated with nisin alone (T 1 and T 2 ) showed a slower reduction in flavour scores, reaching 6.02 and 6.00 by day 42, with mean scores of 7.46 and 7.40. Nisin helped suppress spoilage bacteria, improving flavour retention compared to the control, although its limited effect on yeasts and moulds still allowed gradual deterioration. Natamycin-only treatments (T 3 and T 4 ) followed a similar trend. Both spoiled by day 35, with mean scores of 7.26 and 7.25. This outcome reflected the selective activity of natamycin against yeasts and moulds, which did not fully prevent bacterial changes responsible for flavour decline. Combined treatments (T 5 to T 8 ) provided the strongest flavour stability throughout storage. T 5 (nisin 400 IU/g + natamycin 10 ppm) recorded the highest mean score (7.55), followed by T 6 , T 7 and T 8 . The improved performance of T 5 suggests a synergistic effect, where nisin controlled Grampositive bacteria and natamycin inhibited yeasts and moulds, together reducing off-flavour development. The moderate preservative levels in T 5 also helped maintain the natural flavour profile. (-) Indicates that the product was spoiled and no further analysis was carried out Overall, the results show that combining nisin and natamycin was more effective than using either preservative alone. Treatment T 5 offered the best protection against flavour deterioration and supported extended storage stability without compromising sensory quality. These observations agree with earlier work showing that nisin helps preserve flavour in a variety of fermented and dairy products during storage. Kamble (2023) [13] reported a faster decline in flavour in control custard apple shrikhand than in the nisin treated sample. Similar benefits of nisin on flavour stability have been documented in nono (Olasupo et al., 1996) [20] , Doogh (Shahbazi, 2016) [27] , lassi (Chaure, 2010; Shaik, 2008; Kakde, 2010) [4,28,11] , carrot-based yoghurt drinks (Rashmi, 2014) [24] , and mango lassi (Khurana, 2006) [15] . These studies consistently show slower deterioration of sensory quality when nisin is used. Improved flavour retention with nisin has also been reported in paneer by several researchers (Kankhare, 2005; Ranmode, 2010; Mane et al., 2007; Das, 2017) [14,23,17,6] , as well as in quarg cheese (Kadiya, 2009) [10] and pasteurized milk (Radha, 2014) [21] . In all cases, nisin delayed microbial spoilage and extended flavour acceptability. Natamycin produced similar results in yoghurt (El-Diasty et al., 2009) [7] , cheese (Jalilzadeh et al., 2020) [9] , and khoa (Vaquil, 2018) [31] , where treated samples maintained better flavour scores than the control during storage. Combined treatments of nisin and natamycin further enhanced flavour preservation in strained yoghurt (Misirlilar et al., 2012) [18] , processed cheese sauces (Saad et al., 2015) [25] , and Galotyri cheese (Kallinteri et al., 2013) [12] . Chawla et al. (2021) [5] also found that their combined use slowed flavour decline in Doda burfi. A progressive reduction in flavour scores of control shrikhand during storage has been reported by numerous authors.

Overall acceptability

The overall acceptability of all shrikhand samples declined progressively during storage at 5±1 °C (Table 4). (-) Indicates that the product was spoiled and no further analysis was carried out Although initial scores were similar across treatments (8.26 to 8.28), significant differences (p < 0.05) emerged as storage advanced. The control (T 0 ) deteriorated rapidly and spoiled after day 35 due to unchecked microbial activity, which affected flavour, texture and appearance. Samples treated with nisin alone (T 1 and T 2 ) or natamycin alone (T 3 and T 4 ) maintained higher acceptability than the control. Nisin treatments showed mean scores of 7.57 (T 1 ) and 7.52 (T 2 ), while natamycin treatments recorded 7.43 (T 3 ) and 7.42 (T 4 ). These results show that each preservative independently slowed quality loss, although their effectiveness was limited with extended storage. Combination treatments (T 5 to T 8 ) performed consistently better than individual applications. T 5 (nisin 400 IU/g + natamycin 10 ppm) achieved the highest mean acceptability score (7.66), followed by T 6 (7.59), T 7 (7.55) and T 8 (7.54). The combined antimicrobial action of nisin and natamycin likely controlled bacterial and fungal growth more efficiently, helping preserve sensory attributes for a longer period. The superior performance of T 5 reflects an optimal balance of preservative concentrations that protected product quality without affecting flavour. Overall, acceptability scores declined significantly (p < 0.05) with increasing storage time, and the interaction between treatment and storage period was significant. The combination of nisin 400 IU/g and natamycin 10 ppm (T 5 ) was the most effective in maintaining sensory quality and extending the shelf life of shrikhand during refrigerated storage. These results agree with earlier research showing that biopreservatives help slow the decline in overall acceptability of dairy products during storage. Kamble (2023) [13] reported a rapid decrease in acceptability in control custard apple shrikhand, while nisin treated samples deteriorated more slowly. Similar trends were documented in lassi by Chaure (2010) [4] , Shaikh (2008) [28] and Kumar (1993) [16] , who noted that nisin improved flavour and storage stability compared to controls. Paneer studies also support these findings; Ranmode (2010) [23] , Das (2017) [6] and Kankhare (2005) [14] all reported higher acceptability in nisin treated samples throughout storage. Radha (2014) [21] observed that nisin enhanced the shelf life and sensory quality of pasteurized milk. Natamycin has shown comparable benefits in fermented milk and cheese products. El-Diasty et al. (2009) [7] , Misirlilar et al. (2012) [18] and Sara et al. (2014) [26] reported that natamycin treated yoghurt maintained higher sensory scores during extended storage. Similar improvements were noted in paneer (Sreeramaya, 2022) [30] , Iranian UF cheese (Nottagh, 2020) [19] , and various cheese types treated with natamycin (Jalilzadeh et al., 2020; Ramos et al., 2012) [9,22] . Combined treatments provided the best sensory stability. Saad et al. (2015) [25] and Kallinteri et al. (2013) [12] showed that nisin and natamycin together preserved sensory quality more effectively than either preservative alone. Enhanced acceptability with combined treatments was also reported in khoa (Vaquil, 2018) [31] and Doda burfi (Chawla et al., 2021) [5] . A consistent decline in acceptability of untreated shrikhand during storage has been documented by numerous researchers.

Conclusion

From the present study it was concluded that, during storage of shrikhand, the sensory scores for all attributes were decreased significantly (p<0.05) in eight samples. On the basis of sensory evaluation, shrikhand without biopreservatives (T 0 ) and shrikhand treated with natamycin at 10 ppm (T 3 ) and 15 ppm (T 4 ) could be stored for 35 days at 5±1 °C. Shrikhand treated with nisin at 400 IU/g (T 1 ) and 500 IU/g (T 2 ), as well as combinations of nisin and natamycin 400 IU + 10 ppm (T 5 ), 400 IU + 15 ppm (T 6 ), 500 IU + 10 ppm (T 7 ), and 500 IU + 15 ppm (T 8 ) maintained acceptable quality and could be stored for 42 days at 5±1 °C. Treatment T 5 (nisin 400 IU/g + natamycin 10 ppm) demonstrated superior sensory quality, along with an extended shelf life of shrikhand, compared to the control (T 0 ) and other biopreservative treatments throughout the storage period. Hence, the combination of nisin (400 IU/g) and natamycin (10 ppm) is recommended as an effective treatment for the preservation of shrikhand.

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