Physico-chemical and color parameters to distinguish cow ghee from buffalo ghee

Abstract

Ghee production form one of the largest segments of the milk consumption and utilization pattern in India. Recently, cow ghee has become more popular and fetching premium over buffalo ghee as there are innumerable health benefits credit to cow ghee since they contain an important array of nutrients and therapeutic principles. Therefore, the present investigation was conducted to differentiate and characterize cow ghee from buffalo ghee using physico-chemical parameters viz. BR reading, RM value, Polenske value, Kirschner value and different color values. Pure cow and buffalo ghee samples were prepared using creamery butter method. Pure ghee samples (cow and buffalo) and cow ghee samples admixed with buffalo ghee @ 5%, 10%, 15% and 20% were analyzed for above mentioned physico-chemical parameters and different color parameters. The results revealed that BR reading, RM value, Polenske value and Kirschner value of pure cow ghee ranged from 41.87–43.62, 27.5–31.13, 1.30–1.90 and 20.74–24.14 and in buffalo ghee these values ranged from 40.01–43.23, 31.91–39.99, 1.10–1.50 and 26.84–33.96, respectively. The color values i.e. lightness (L), redness(a), yellowness (b), yellowness index (Y) and whiteness index (W) of pure cow ghee ranged from 70.17–81.56, − 14.04 to − 28.96, 59.68–79.31, 74.25–88.92 and 16.07–28.85 and of buffalo ghee ranged from 71.89–83.71, − 1.07 to – 11.92, 1.39–9.61, 5.21–22.46 and 68.74–84.61. BR reading, RM value, Polenske value and L, a, b and Y of cow ghee adulterated with buffalo ghee up to 20% falls within the range of different pure cow ghee samples but whiteness index (W) and Kirschner value of admixed cow ghee (23.91 and 34.86) were having significantly higher values than the pure cow ghee (21.07 and 25.45, respectively). Kirschner Value and whiteness index (W) can be used to distinguish cow ghee from buffalo ghee.

Introduction

India is the largest producer of milk in the world, producing 187.7 million tones according to the latest estimates for 2018–19 (NDDB 2020). Cows and buffaloes are the predominant dairying species and their milks are traditionally by far the most produced types of milk (Fox and McSweeny 1998). Cow milk represents ~ 81% of the global milk production, whereas buffalo milk comprises ~ 15% (FAO 2020). In India Buffalo contribute 53.4% whereas cows contribute 43.1% to the total milk production (Ramesha and Divya 2014). Ghee production forms one of the largest segments of the milk consumption and utilization pattern in India. It is the second largest consumed dairy product in India after liquid milk. The ghee production amounted to almost 171 thousand metric tons in India during fiscal year 2018 (Statista 2020). Ghee is clarified butter and by far the most important product widely consumed in the Indian sub-continent since time immemorial. Its image as a natural product and its organoleptic attributes, nutritional value, and functional properties make it suitable for numerous food applications. It is prepared from buffalo or cow milk or combination thereof. Now a days, more emphasis is laid on the cow ghee. Niche brands are selling organic ghee which are made from milk of cows that are fed organic fodder and desi cow ghee made from the milk of Indian cow breeds. At present cow ghee has become very popular among the consumers due to its publicity as healthier option as well as its color, aroma and taste. Hence, these days, cow ghee is being sold at higher premium. This phenomenon is creating a situation, where in ghee producers are admixing buffalo ghee in cow ghee and selling the products at higher cost. In order to ensure a genuine product to consumer, the Government of India has prescribed the compositional standards for ghee (Table 1), under FSSR (2011) and AGMARK rules (1981). However, there have been no standards prescribed separately for cow and buffalo ghee. Keeping in mind the present investigation was carried to characterize cow ghee and to differentiate cow ghee from buffalo ghee using different physico-chemical and color parameters.

Table 1.

Ghee Standards as per FSSAI

Parameter	Ghee
Moisture, maximum, %, (m/m)	0.5
Milk fat, minimum, %, (m/m)	99.5
Butyro-refractometer Reading at 40 ˚C	40.0–44.0
Reichert Meissl Value, minimum	4.0
Polenske Value	0.5 -2.0
FFA as Oleic Acid, maximum, %	2.0
Peroxide Value (Milli-equivalent of Oxygen/Kg fat), maximum	–
Baudouin Test	Negative
Iodine Value	25–38
Saponification value	205–235

Material and methods

Chemicals and reagents

Potassium hydroxide pellets, Sodium hydroxide pellets and Sulphuric acid (AR grade, Qualigens fine chemicals, Mumbai, India), Oxalic acid (Glaxo Laboratories Ltd, Mumbai, India), Barium hydroxide and Silver Sulfate (AR grade Sisco Research Laboratories Pvt. Ltd., Maharashtra, India), Glycerol (AR grade, Ranbaxy Laboratories Ltd., Punjab, India), Phenolphthalein (AR, s.d. fine-chem Ltd., Mumbai, India).

Preparation of ghee samples

Samples of cow/buffalo ghee were prepared by creamery butter method (De 2010). Cow and buffalo milk used for the preparation of respective ghee samples were collected bimonthly up to complete eight months (August–September, October–November, December-January and February–March) from the Livestock Research Centre of National Dairy Research Institute (NDRI). Soon after the collection of milk samples, these were warmed to 40 °C and separated into cream using mechanical cream separator. The cream was pasteurized at 77˚C for 5 min, cooled to room temperature and it was separated into two batches. One batch was ripened using the culture M-167 taken from National Collection of Dairy Cultures (NCDC), NDRI at 21˚C for 14 h and second batch of cream was kept in a refrigerator (5 to 10˚ C) for few hours (3 to 5 h) for aging. Butter was prepared from both the batches of cream under standard conditions (9 °C in summer and 13 °C in winter) by churning of cream using hand operated butter churn. The butter (ripened and unripened) was then heated on direct flame in a stainless-steel vessel and clarified into ghee with continuous stirring at two different temperatures of 110 °C/flash and 130 °C/flash. Ghee was then filtered through 6–8 fold muslin cloth followed by further filtration by using Whatman No.4 filter paper in glass vacuum assembly and finally filled in plastic bottles, cooled to room temperature and kept in a refrigerator (5 to 10 °C) till further analysis.

Preparation of admixed ghee samples

For the preparation of admixed ghee samples, cow and buffalo ghee prepared from ripened cream and clarified at 110 °C in the month (Dec-Jan) were used. Before mixing the cow and buffalo ghee samples, these are heated at 65–70 °C for the 10 min. The buffalo ghee was then added to cow ghee at @ 5%, 10%, 15% and 20% (w/w) basis.

Physico-chemical constants of ghee

The physico-chemical constants such as Reichert-Meissl (RM) value, Polenske value (PV) and Butyro-refractometer reading of above said samples (Pure cow ghee samples, Pure buffalo ghee and admixture of cow ghee with buffalo ghee) were determined by the methods as described in IS3508:1966 (2018) and Kirschner Value (K) was determined by the method of Ghatak and Bandyopadhya (2007).

Color values

Color values (L, a, b, yellowness index (Y) and whiteness index (W) of ghee samples were measured by Machine vision system Colordesk D1, designed and developed in Dairy Engineering Division, ICAR-NDRI, India. The color measuring system is based on Scilab 5.4, 64 bit platform. Before determining the color value of samples, lights of the instrument were switched on and then molten ghee (40 °C) samples were placed inside the instrument. Images of the samples were taken by the instrument camera and then digital image processing was done by the system and color values of the ghee samples were recorded.

Statistical analysis

The data obtained in the present study was subjected to one-way analysis of variance (ANOVA) for the significant difference in the samples via Duncan’s Multiple Comparison test performed at 95% confidence interval with SAS software (version 9.3 for windows), San Diego California, USA.

Results and discussion

Physico-chemical constants of pure ghee samples

Physico-chemical constants such as BR reading at 40 °C, RM value, Polenske value and Kirschner value have been depicted in Table 2. It is evident from the data that B.R. reading was 41.87–43.62 for cow ghee and 40.01–43.23 for buffalo ghee (Table 2). Hence, it was difficult to fix the specific B.R. reading for cow and buffalo ghee separately. Similarly, RM value and Polenske value of both type of ghee were very close to each other hence, could not be possible to specify a range separately for cow and buffalo ghee (Table 2). However, Kirschner value (KV) showed that buffalo ghee samples had all time high values in comparison to cow ghee (Table 2) as buffalo ghee contains slightly higher amount of butyric acid as compared to cow ghee (Kehar 1956). Even the lower range of Kirschner value in buffalo ghee was well above the higher range obtained for cow ghee in the present investigation. Therefore, it can be concluded that there is a possibility of specifying the specific range of Kirschner value for cow and buffalo ghee. Hence, Kirschner value can be used to distinguish cow ghee from buffalo ghee after studying wide range of samples including different clarification temperature, ripening conditions and feeding practices.

Table 2.

Physico-chemical constants of pure cow and buffalo ghee

Physico-chemical constant	Cow ghee		Buffalo ghee
	Range	Average	Range	Average
B.R. reading	41.87 – 43.62	42.55 ± 0.052	40.01 – 43.23	41.48 ± 0.105
RM Value	27.56 – 31.13	29.12 ± 0.109	31.91 – 39.99	34.42 – 0.174
Polenske Value	1.30 – 1.90	1.58 ± 0.017	1.10 – 1.50	1.28 ± 0.011
Kirschner Value	20.74 – 24.14	22.12 ± 0.106	26.84 – 33.96	28.34 ± 0.190

Data presented is mean ± SE of 24 determinations

Effect of admixing buffalo ghee with pure cow ghee on Physico-chemical constants

Admixing buffalo ghee with pure cow @ 5%, 10%, 15% and 20% showed decrease in B.R. readings of cow ghee (Table 3). Analysis of variance of the data revealed that B.R. readings of pure cow ghee and cow ghee samples admixed with buffalo ghee @ 5%, 10%, 15% and 20% differed significantly (P < 0.05). It can be concluded that higher the adulteration of buffalo ghee in cow ghee more was the decrease in BR reading as buffalo ghee contains lesser amount of unsaturated fatty acids and higher amount of saturated fatty acids compared to cow ghee (Blasi et al. 2008; Menard et al. 2010). Though there was decrease in the B.R. reading of samples adulterated with buffalo ghee, but values obtained even in 20% adulterated pure samples were within the range obtained for cow ghee in the present study (Table 2). Therefore, B.R. reading could not help to distinguish cow ghee from buffalo ghee.

Table 3.

Physico-chemical constants of pure cow and cow ghee admixed with buffalo ghee @ 5%, 10%, 15% & 20%

Samples	B.R. reading	R.M. value	Polenske value	Kirschner value
PCG	42.84 ± 0.004a	28.18 ± 0.229e	1.73 ± 0.041a	21.07 ± 0.234d
CG + BG (5%)	42.35 ± 0.004b	29.11 ± 0.062d	1.67 ± 0.041b	21.57 ± 0.072c
CG + BG (10%)	42.21 ± 0.008d	29.57 ± 0.186c	1.67 ± 0.041b	22.26 ± 0.176b
CG + BG (15%)	42.22 ± 0.008c	30.86 ± 0.083b	1.53 ± 0.041c	23.63 ± 0.132a
CG + BG (20%)	42.15 ± 0.004e	31.38 ± 0.097a	1.53 ± 0.041c	23.91 ± 0.198a
CD value (P ≤ 0.05)	0.014	0.368	0.102	0.399
Range of PCG	41.87–43.62	27.56–31.13	1.30–1.90	20.74–24.14
Reference value of PBG	41.29 ± 0.004	34.21 ± 0.146	1.23 ± 0.041	29.34 ± 0.056

PCG = Pure Cow Ghee PBG = Pure Buffalo Ghee CD = Critical Difference

Data presented is mean ± SE of three determinations

Values bearing different superscripts (a to e) in each column differ significantly

Similarly, RM value and Polenske value of admixed samples also remained within the range of pure cow ghee and addition of buffalo ghee in cow ghee could not be detected even at 20% level of admixing (Table 3).

On the contrary, statistical analysis of the data for Kirschner value (Table 3), revealed that in pure cow ghee and cow ghee samples adulterated with buffalo ghee @ 5%, 10%, 15% and 20% there was a statistical significant (P < 0.05) difference in the values. Perusal of the data revealed that higher the admixture of buffalo ghee in cow ghee higher was the increase in Kirschner value. However, the Kirschner value (KV) of samples adulterated with buffalo ghee to the tune of 20% was within the range obtained for pure cow ghee in the present study (Table 2). But, data presented in Table 3 clearly indicated that KV for cow ghee adulterated with buffalo ghee @ 20% was very close to the upper range of KV for pure cow ghee. This lead to the assumption that on admixing buffalo ghee to cow ghee above 20% level will result into KV higher than the upper limit obtained from pure cow ghee. Hence, it may be inferred from the results that admixing of buffalo ghee in cow ghee > 20% could be detected using Kirschner value as one of the parameters. However, limitation of KV parameter is that with addition of small amount of vegetable oil with buffalo ghee may reduce KV to a normal value indicating a false negative analysis (Sen-Gupta, 1943).

Color parameters of cow and buffalo ghee

Color value of different ghee samples were measured by Machine Vision system Color Desk D1 instrument. The result of color value (CV) of ghee samples is described in terms of L, a, b, Y and W;

L = difference in lightness and darkness (+ = lighter, – = darker).

a = difference in red and green (+ = redder, − = greener).

b = difference in yellow and blue (+ = yellower, − = bluer).

Y = yellowness index.

W = whiteness index.

Color parameters of pure cow and buffalo ghee such as L, a, b, Y and W have been depicted in Table 4. It is evident from the data that except the L value all other parameters a, b, Y and W were different for cow and buffalo ghee (Table 4). Color value a (redder) and Whiteness Index (W) was more for buffalo ghee whereas color value b and Yellowness Index (Y) was more for cow ghee. This is due to the yellow color of cow ghee, which is contributed by fat soluble carotenoids (Nozière et al. 2006) on the contrary high value of a and Whiteness Index (W) of buffalo ghee was due to the absence of carotenoids and possibly presence of biliverdin and bilirubin which gave a greenish tint. (Suwarat and Tungjaroenchai 2013; Daniel 1977; Rao and Dastur 1984). Therefore, different parameters such as a, b, Y and W may be used to distinguish the cow ghee from buffalo ghee.

Table 4.

Color parameters of pure cow and buffalo ghee

Color parameters	Cow ghee		Buffalo ghee
	Range	Average	Range	Average
CV(L)	70.17–81.56	77.92 ± 1.065	71.89–83.71	78.86 ± 2.92
CV (a)	− 14.01 to − 28.96	− 21.71 ± 2.22	− 1.07–11.92	− 5.49 ± 4.33
CV (b)	59.68–79.31	71.57 ± 3.43	1.39–9.51	5.23 ± 1.51
Yellowness Index (Y)	74.25–88.92	82.15 ± 1.47	5.21–22.46	9.88 ± 1.54
Whiteness Index (W)	16.07–28.85	21.28 ± 1.64	68.74–84.61	76.48 ± 3.49

CV = Color Value

L = difference in lightness and darkness (+ = lighter, −  = darker)

a = difference in red and green (+ = redder, −  = greener)

b = difference in yellow and blue (+ = yellower, −  = bluer)

Data presented is mean ± SE of 24 determinations

Effect of admixing buffalo ghee with pure cow ghee on Color parameters

Admixture of buffalo ghee with pure cow ghee @ 5%, 10%, 15% and 20% showed increase in L and a value of cow ghee (Table 5). Analysis of variance of the data revealed a significant (P < 0.05) difference in L and a values of pure cow ghee and cow ghee samples admixed with buffalo ghee @ 5%, 10%, 15% and 20% (Table 5). It can be concluded that higher the adulteration of buffalo ghee in cow ghee more was the increase in L and a value. Though the L and a values of cow ghee samples admixed with buffalo ghee were higher than the pure cow ghee but these were within the range obtained for cow ghee in the present study. Even in samples containing buffalo ghee to the tune of 20% the values were falling within the range obtained for cow ghee. (Table 4). Therefore, L and a value could not be used as a parameter to distinguish cow ghee from buffalo ghee (Table 5).

Table 5.

Color Value of pure cow and cow ghee adulterated with buffalo ghee @ 5%, 10%, 15% & 20%

Samples	CV(L)	CV (a)	CV (b)	Yellowness index	Whiteness index
PCG	77.41 ± 1.59e	− 21.88 ± 1.16e	68.95 ± 0.42 a	79.93 ± 1.15 a	25.45 ± 0.26e
CG + BG (5%)	78.35 ± 0.68c	− 21.32 ± 3.06d	68.21 ± 1.29b	78.81 ± 1.80b	27.52 ± 1.99d
CG + BG (10%)	77.87 ± 2.56d	− 20.76 ± 3.11c	66.57 ± 1.77c	78.21 ± 2.69c	29.31 ± 0.97c
CG + BG (15%)	78.59 ± 1.15b	− 20.21 ± 1.12b	63.41 ± 1.63d	77.46 ± 2.01d	31.37 ± 1.46b
CG + BG (20%)	78.90 ± 1.85a	− 18.56 ± 1.33a	61.92 ± 2.24e	76.14 ± 2.41e	34.86 ± 1.39 a
CD value (P ≤ 0.05)	0.361	0.168	0.102	0.399	0.178
Range for PCG	70.17 – 81.56	− 14.01 to − 28.96	59.68 – 79.31	74.25 – 88.92	16.07 – 28.85
Reference value of PBG	78.56 ± 2.92	− 5.72 ± 1.02	7.24 ± 1.37	9.08 ± 2.11	78.76 ± 2.78

PCG = Pure Cow Ghee PBG = Pure Buffalo Ghee CD = Critical Difference

Data presented is mean ± SE of three determinations

Values bearing different superscripts in each column differ significantly

Similarly, b value and Yellowness Index of admixed samples also remained within the range as of pure cow ghee and addition of buffalo ghee in cow ghee could not be detected even at 20% level of admixing (Table 5).

But analysis of variance of the data of Whiteness Index (W) revealed a significance (P < 0.05) difference in pure cow ghee and cow ghee samples adulterated with buffalo ghee @ 5%, 10%, 15% and 20% (Table 5). Perusal of the data revealed that higher the admixture of buffalo ghee in cow ghee higher was the increase in Whiteness Index (W). It is also evident from the data that Whiteness Index (W) of cow ghee adulterated with buffalo ghee @ 10%, was (29.31 ± 0.97) higher than the highest range of Whiteness Index (W) of pure cow ghee (16.07–28.85). Therefore, it may be concluded that adulteration of cow ghee with buffalo ghee @ 10% could be detected using Whiteness Index (W) as one of the parameters. However, upon storage of ghee, degradation of carotene causes reduction on colour and may indicate adulteration. Therefore, to avoid any false negative results, it could be stated that @ 15% addition could be detected using Whiteness Index (W) as one of the parameters. Hence, Whiteness Index (W) can be used as a parameter to distinguish cow ghee from buffalo ghee.

Conclusion

Physico-chemical constants such as B.R. reading, RM value and Polenske Value cannot be specified separately for cow and buffalo ghee as the values for both types of ghee samples overlaps the range obtained. Kirschner value and different color parameters such as a, b, indices like W & Y can be used to characterize cow and buffalo ghee as range for these parameters differ for cow and buffalo ghee. Whiteness index (W) can detect admixing of buffalo ghee at the level of 15%, while kirschner value may be used to detect admixing of buffalo ghee (> 20%) in cow ghee, requires further verification.

Abbreviations

RM

Reichert-Meissl

PV

Polenske value

BR

Butyro-refractometer

PCG

Pure Cow Ghee

PBG

Pure Buffalo Ghee

L

Lightness

a

Redness

b

Yellowness

Y

Yellowness index

W

Whiteness index

Authors’ Contributions

Sonia Mor: formal analysis, data interpretation, writing-original draft. Vivek Sharma: Conceptualization, formal analysis, data interpretation, reviewing and editing. Sumit Arora: reviewing and editing. PS Minz: resources, reviewing and editing.

Funding

No funding was received.

Availability of data and material

All the relevant data available is mentioned in the manuscript and no other supplementary data is provided.

Declarations

Conflict of interest

The authors have declared no conflict of interest for this article.