Separação e quantificação de proteínas do leite com adição de soro de queijo por <i>lab-on-a-chip</i>

Santos, Alessa Siqueira de Oliveira dos; Pereira, Hyago Passe; Fogaça, Gisele Nogueira; Meurer, Vaneida Maria; Furtado, Marco Antônio Moreira; Borges, Cristiano Amâncio Vieira; Weller, Mayara Morena Del Cambre Amaral; Martins, Marta Fonseca

Alessa Siqueira de Oliveira dos Santos

Universidade Federal de Juiz de Fora, Faculdade de Farmácia, Rua José Lourenço Kelmer, s/nº, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0001-8917-4290

Hyago Passe Pereira

Universidade Federal de Juiz de Fora, Instituto de Ciências Biológicas, Rua José Lourenço Kelmer, s/nº, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0002-8243-4667

Gisele Nogueira Fogaça

Universidade Federal de Juiz de Fora, Faculdade de Farmácia, Rua José Lourenço Kelmer, s/nº, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0001-9478-9708

Vaneida Maria Meurer

Universidade Federal de Juiz de Fora, Faculdade de Farmácia, Rua José Lourenço Kelmer, s/nº, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0002-2974-0825

Marco Antônio Moreira Furtado

Universidade Federal de Juiz de Fora, Faculdade de Farmácia, Rua José Lourenço Kelmer, s/nº, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0002-9766-3040

Cristiano Amâncio Vieira Borges

Embrapa Gado de Leite, Rua Eugênio do Nascimento, nº 610, Dom Bosco, CEP 36038-330 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0001-8120-3288

Mayara Morena Del Cambre Amaral Weller

Universidade Federal do Espírito Santo, Departamento de Zootecnia, Alto Universitário, s/nº, Guararema, CEP 29500-000 Alegre, ES, Brazil

https://orcid.org/0000-0002-7216-4587

Marta Fonseca Martins Corresponding author

Embrapa Gado de Leite, Rua Eugênio do Nascimento, nº 610, Dom Bosco, CEP 36038-330 Juiz de Fora, MG, Brazil

https://orcid.org/0000-0002-4260-5329

Corresponding author

Figuras (3)
Tabelas (4)

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Figure 1
Gel-like images obtained in the microfluidic chip electrophoresis system with proteins extracted from raw (A), pasteurized (B), powdered (C), and UHT (D) milk samples. Cheese whey was added to the samples in increasing concentrations of 0, 1, 2.5, 5, 10, 20, 30, 50, and 100% (v/v). Ladder (molecular weight marker) and SP (standard proteins) were used. Proteins were separated and quantified according to molecular mass and retention time. α-LA, alpha-lactoglobulin; β-LG, beta-lactoglobulin; β-CN, beta-casein; α-CN, alpha-casein; κ-CN, kappa-casein.

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Figure 2
Electrophoretic profile of milk proteins obtained by microfluidic electrophoresis. α-LA, alpha-lactoglobulin; β-LG, beta-lactoglobulin; β-CN, beta-casein; α-CN, alpha-casein; κ-CN, kappa-casein.

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Figure 3
SDS-PAGE electrophoretic profile of milk proteins. A, B, C, and D - samples of raw, pasteurized, UHT, and powdered milk adulterated with whey, respectively. Well 1 - NovexProtein molecular marker. The values from 10 to 260 correspond to the values of the molecular marker in kDa. Wells 2 to 10 - % whey addition. Arrows indicate the main milk proteins. α-LA, alpha-lactoglobulin; β-LG, beta-lactoglobulin; β-CN, beta-casein; α-CN, alpha-casein; κ-CN, kappa-casein.

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Table 1
Estimated values of the molecular mass of milk caseins and whey proteins for samples of raw, pasteurized, UHT, and powdered milk, compared with the values of molecular mass described in the literature using the microfluidic technique.

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Table 2
Mean values of the peak areas of the main protein fractions of samples of milk tupes (powdered, UHT, pasteurized, and raw) with addition of cheese whey in increasing levels obtained with the lab-on-a-chip technique.

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Table 3
Comparison of the significance levels of α-CN, β-CN, κ-CN, β-LG and α-LA proteins, between treatments applied to the four milk types (raw, pasteurized, UHT, and powdered), obtained by Dunnet’s test.

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Table 4
Estimated values of the molecular mass of milk caseins and whey proteins for samples of raw, pasteurized, UHT, and powdered milk, compared with the values of molecular mass described in the literature using the SDS-PAGE technique.

Protein	Bovine milk
Protein	Estimated molecular mass by microfluidic chip (kDa)⁽¹⁾	Reported molecular mass by microfluidic chip (kDa)⁽²⁾	Traditional SDS-PAGE (kDa)⁽³⁾
α-LA	12.5±0.2	12±1	11.03
β-LG	18.5±0.3	18±1	13.47
β-CN	32.5±0.8	29±2	25.90
α-CN	37.8±0.5	37±1	30.66
κ-CN	42.7±0.5	42±2	23.02

Serum addition levels (%)	Mean peak areas of main milk proteins⁽¹⁾
Serum addition levels (%)	α-CN	β-CN	κ-CN	α-LA	β-LG
	Powdered milk
0	773.90	1,141.46	22.78	12.59	16.08
1	727.87	1,075.37	17.38	16.89	11.88
2.5	651.78	989.76	14.27	12.66	12.50
5	592.64	901.33	14.54	16.99	11.68
10	536.19	847.49	14.42	18.98	9.86
20	421.97	673.11	13.24	19.37	9.68
30	355.21	580.01	10.27	18.18	8.55
50	260.64	424.82	7.68	28.22	13.01
100	-	-	-	61.88	25.83
	UHT milk
0	686.60	772.07	47.38	40.87	23.56
1	642.80	733.09	42.81	43.59	23.70
2.5	536.90	614.70	25.17	35.08	23.52
5	481.80	571.32	25.93	32.71	24.37
10	431.90	514.28	19.68	32.01	24.43
20	358.20	432.49	10.50	30.59	26.06
30	305.70	369.76	2.64	30.37	23.84
50	239.30	284.93	2.31	40.49	31.56
100	-	-	-	52.31	38.71
	Pasteurized milk
0	691.41	688.59	37.40	53.17	59.81
1	660.79	670.97	37.30	49.86	50.09
2.5	544.61	569.97	32.40	41.31	43.78
5	494.58	526.11	25.70	41.94	45.94
10	456.50	494.21	23.20	38.30	40.59
20	361.90	397.40	16.10	36.29	34.18
30	293.31	324.04	10.10	36.72	30.69
50	202.72	223.71	7.80	38.88	30.30
100	-	-	-	47.83	24.54
	Raw milk
0	652.62	645.74	34.49	61.37	48.59
1	604.74	609.57	33.38	55.30	47.44
2.5	561.02	574.31	28.01	53.91	42.65
5	519.50	548.10	27.24	49.85	38.77
10	492.72	517.82	23.99	48.22	31.48
20	353.45	383.99	14.86	41.22	30.99
30	333.46	356.57	17.82	44.19	29.46
50	238.59	249.36	13.16	44.41	30.89
100	-	-	-	37.51	20.64

Serum addition levels (%)	p-value for main milk protein⁽¹⁾
Serum addition levels (%)	α-CN	β-CN	κ-CN	β-LG	α-LA
	Raw milk
0	-	-	-	-	-
1	0.0425	0.0316	0.8685	0.8583	0.9998
2.5	< 0.0001	0.0002	0.0670	0.8374	1.0000
5	< 0.0001	0.0013	< 0.0001	0.9767	1.0000
10	< 0.0001	< 0.0001	< 0.0001	0.9576	1.0000
20	< 0.0001	< 0.0001	< 0.0001	0.9997	0.9999
30	< 0.0001	< 0.0001	< 0.0001	1.0000	0.7207
50	< 0.0001	< 0.0001	< 0.0001	1.0000	< 0.0001
	Pasteurized milk
0	-	-	-	-	-
1	0.0410	0.2098	0.1316	0.9995	0.1316
2.5	< 0.0001	0.3140	0.0081	0.8950	0.0081
5	< 0.0001	0.2250	0.0108	0.9996	0.0108
10	< 0.0001	0.0130	0.0095	1.0000	0.0095
20	< 0.0001	0.0525	0.0026	1.0000	0.0026
30	< 0.0001	0.0177	< 0.0001	1.0000	< 0.0001
50	< 0.0001	0.5055	< 0.0001	1.0000	< 0.0001
	UHT milk
0	-	-	-	-	-
1	0.0239	0.0347	0.1871	0.9943	0.9009
2.5	< 0.0001	< 0.0001	< 0.0001	0.0691	0.8860
5	< 0.0001	< 0.0001	< 0.0001	0.0067	0.9441
10	< 0.0001	< 0.0001	< 0.0001	0.0030	0.9474
20	< 0.0001	< 0.0001	< 0.0001	0.0005	0.9907
30	< 0.0001	< 0.0001	< 0.0001	0.0004	0.9119
50	< 0.0001	< 0.0001	< 0.0001	0.8503	1.0000
	Powdered milk
0	-	-	-	-	-
1	0.0310	0.0210	0.0254	0.9995	0.9798
2.5	< 0.0001	< 0.0001	< 0.0001	0.8950	0.8678
5	< 0.0001	< 0.0001	< 0.0001	0.9996	0.9752
10	< 0.0001	< 0.0001	< 0.0001	1.0000	0.9973
20	< 0.0001	< 0.0001	< 0.0001	1.0000	0.9998
30	< 0.0001	< 0.0001	< 0.0001	1.0000	1.000
50	< 0.0001	< 0.0001	< 0.0001	1.0000	1.000

Milk proteins⁽¹⁾	Molecular mass described in the literature (kDa)			Molecular mass of milk (kDa)
Milk proteins⁽¹⁾	Bovine milk			Raw	Pasteurized	UHT	Powdered
α-CN	30±3⁽²⁾	23.6⁽³⁾	29.94⁽⁴⁾	30.66	32.09	31.58	32.41
β-CN	27±4	25.2	28.03	25.90	26.33	26.64	26.29
κ-CN	24±4	19.0	25.72	23.02	23.06	23.45	23.24
β-LG	19±2	18.3	16.63	13.47	13.35	13.59	13.57
α-LA	13±3	14.1	12.56	11.03	10.85	11.04	10.99

[1] Corresponding author

Brasil

Brasil

Separação e quantificação de proteínas do leite com adição de soro de queijo por lab-on-a-chip

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