Escolar Documentos
Profissional Documentos
Cultura Documentos
CAMPINAS
2018
KLEIDSON BRITO DE SOUSA LOBATO
CAMPINAS
2018
Agência(s) de fomento e nº(s) de processo(s): CAPES, 3300301702P1
ORCID: https://orcid.org/0000-0003-4619-027
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Engenharia de Alimentos
Márcia Regina Garbelini Sevillano - CRB 8/3647
Título em outro idioma: Infrared spectroscopy in UHT milk and freeze-dried açai pulp :
quality parameters, detection and identification of adulterants
Palavras-chave em inglês:
Milk - Composition
Chemometrics
UHT milk
Açai
Milk - Adulteration
Área de concentração: Ciência de Alimentos
Titulação: Doutor em Ciência de Alimentos
Banca examinadora:
Juliana Azevedo Lima Pallone [Orientador]
Ádria de Sousa Bentes
Daniela Souza Ferreira
Douglas Fernandes Barbin
Jesuí Vergílio Visentainer
Data de defesa: 28-02-2018
Programa de Pós-Graduação: Ciência de Alimentos
Aos meus pais Antonio Messias Brito Lobato, Marlene Farias de Souza Lobato, meus irmãos
Kleiton Brito de Souza Lobato e Karla Priscila de Souza Lobato e sogros Roberto de Freitas
Neves e Elisa Cristina Andrade Neves por todo o carinho e suporte.
À Prof. Dra. Juliana Azevedo Lima Pallone pela excelente orientação no doutorado.
À prof. Dra. Márcia Miguel Castro Ferreira por toda a ajuda e ensinamento em Quimiometria.
Aos amigos da Célula Mansões Santo Antônio e aos líderes do Ministério Nova Vida da
Igreja Bola de Neve Campinas.
Ao Eduardo Adilson Orlando, técnico do Laboratório de Análise de Alimentos II, pelos seus
serviços prestados com muita responsabilidade, agilidade e sabedoria.
À Maria de Fátima Martins Pinhel e Lívia Cavaletti Corrêa da Silva por terem acreditado na
proposta do trabalho e por todo o suporte na realização das análises Físico-Químicas e
utilização do espectrômetro no Infravermelho Próximo no LANAGRO-Campinas.
Ao Prof. Dr. Ronei de Jesus Poppi por permitir a utilização dos Espectrômetros no
Infravermelho Próximo e Médio.
RESUMO
O leite UHT tem propriedades diferentes em relação ao leite cru e é amplamente consumido
no Brasil. Vários adulterantes já foram encontrados no leite, sendo a adição de água
considerada como a forma de adulteração mais frequente, que tem por objetivo principal
aumentar o volume do produto final e consequentemente os lucros. Açai é o fruto da palmeira
Euterpe oleracea Martius, conhecida como a mais produtiva do estuário da Amazônia. A
polpa de açaí é comumente adulterada com materiais de menor valor para também aumentar
os lucros. Este é o primeiro trabalho que investigou o uso de espectroscopia no infravermelho
próximo (NIR) e médio (MIR) na determinação de componentes, detecção e identificação de
adulterantes em leite UHT integral, e o uso de NIR na detecção e identificação de adulterantes
em polpa açaí liofilizada. Amostras de leite UHT integral foram analisadas pelos métodos de
referência para a quantificação dos principais componentes do leite e para a detecção de
adulterantes. As amostras de leite UHT isentas de adulterantes foram adulteradas (água, água
+ uréia, água + amido, água + sacarose, água + uréia + amido, água + uréia + sacarose e água
+ uréia + amido + sacarose) em diferentes níveis (1- 20 %). Os espectros de transflectância na
região do NIR e os espectros de transmissão na região do MIR de todas as amostras de leite
UHT foram utilizados no desenvolvimento de modelos de regressão (PLS) e classificação
(PLS-DA). As amostras de polpa de açaí foram adulteradas com amido de milho, polpa de
beterraba, suco de uva, amido de mandioca e maltodextrina em diferentes níveis (1- 50%), e
liofilizadas. Os espectros de reflectância na região do NIR das amostras de polpa de açaí
liofilizada (incluindo as amostras adulteradas) foram utilizados no desenvolvimento de
modelos de classificação (k-NN, SIMCA e PLS-DA). Os modelos PLSdesenvolvidos com
dados no NIR para determinação dos componentes do leite UHT foram desenvolvidos usando
2-9 variáveis latentes, sendo obtidos baixos valores de RMSEC (0,0065- 0,1550%), RMSEP
(0,0110- 0,1049%), sensibilidade (0,0099- 2,0651%), limite de detecção (0,01- 0,57%), limite
de quantificação (0,03- 1,74%) e bias (4,76x10-3- 3,64x10-2). Os modelos MIR foram
desenvolvidos com 6-8 variáveis latentes e também apresentaram baixos valores de RMSEC
(0,0061- 0,0969%), RMSEP (0,0099- 0,0836%), sensibilidade (0,0004- 0,0522%), limite de
detecção (0,06- 0,90%), limite de quantificação (0,17- 2,72%) e bias (-2,89x10-4- 2,51x10-2).
Ao considerar as figuras de mérito (R2cal, R2pred, sensibilidade, seletividade, limites de
detecção e quantificação), observou-se que os melhores resultados para a quantificação de
sólidos totais, lactose, gorduras, proteínas e cinzas em leite integral UHT foram desenvolvidos
utilizando os dados no NIR. De modo geral, a análise de componentes principais mostrou boa
distinção entre as amostras de leite UHT integral autênticas e adulteradas. Este estudo
mostrou que tanto o NIR como o MIR tem potencial para detectar amostras de leite integral
UHT adulteradas. Por outro lado, apenas a espectroscopia na região do NIR permitiu a
identificação do tipo de adulterantes nas amostras de leite integral UHT. Dentre os modelos
de classificação testados para detecção e identificação dos adulterantes em polpa de açaí
liofilizada, os modelos dek-NN mostraram que é possível detectar as amostrasadulteradas e
identificar o tipo de adulterante em cada. Este estudo mostrou que a espectroscopia no
infravermelho tem potencial para a quantificação dos componentes de leite integral UHT e
para a detecção de amostras adulteradas. Aespectroscopia no infravermelho próximo pode ser
aplicada na quantificação dos componentes de leite UHT integral, na detecção e identificação
de adulterantes em leite integral UHT integral e polpa açai liofilizada.
Palavras chave: Leite- composição, Quimiometria, Leite UHT, açaí, Leite- Adulteração
ABSTRACT
The UHT milk has different properties compared to raw milk and is largely consumed in
Brazil. Several adulterants have been found in milk, being the addition of water considered as
the most frequent, which is characterized by a fraud with the main purpose to increase the
volume of the final product and consequently the profits. Açai is the fruit of the palm tree
Euterpe oleracea Martius, known as the most productive of the Amazon estuary. Açai pulp is
commonly adulterated with less expensive materials to increase the profit margins. This is the
first work that investigated the use of near-(NIR) and mid-infrared (MIR) spectroscopy in the
determination of UHT whole milk components and the detection and identification of
adulterants, and the use of NIR in the detection and identification of adulterants in freeze-
dried açaí pulp. UHT whole milk samples were analyzed by reference methods for
quantification of the main components and for the detection of adulterants. The UHT whole
milk samples free of adulterants were adulterated (water, water + urea, water + starch, water +
sucrose, water + urea + starch, water + urea + sucrose and water + urea + starch + sucrose) in
different levels (1- 20%). The NIR transflectance spectra and the MIR transmittance spectra
of all UHT milk samples were used in the development of regression (PLS) and classification
(PLS-DA) models.The açaí pulp samples were adulterated with cornstarch, beet pulp, grape
juice, cassava starch, maltodextrin in different levels (1- 50%), and freeze-dried. The NIR
reflectance spectra of the freeze-dried açai pulp samples (including the adulterated ones) were
used in the development of classification models (k-NN, SIMCA and PLS-DA). The PLS
models were developed using 2- 9 latent variables, obtaining low values of RMSEC, RMSEP,
Limits of detection and quantification. The PLS models developed with NIR data had 2-9
latent variables and presented low values of RMSEC (0.0065- 0.1550%), RMSEP (0.0110-
0.1049%), sensitivity (0.0099- 2.0651%), limit of detections (0.01- 0.57%), limit of
quantification (0.03- 1.74%) and bias (4.76x10-3- 3.64x10-2). The PLS models developed with
MIR data had 6-8 latent variables and also presented low values of RMSEC (0.0061-
0.0969%), RMSEP (0.0099- 0.0836%), sensitivity (0.0004- 0.0522%), limit of detection
(0.06- 0.90%), limit of quantification (0.17- 2.72%) and bias (-2.89x10-4- 2.51x10-2).
Considering the figures of merit (R2cal, R2pred, sensitivity, selectivity, limits of detection and
quantification) it was observed that the best results for the quantification of total solids,
lactose, fats, proteins, and ash in UHT whole milk were developed using the NIR data. In
General, the PCA has shown good distinction between the authentic and adulterated UHT
whole milk samples. This study showed that both NIR and MIR feature potential to detect
adulterated UHT whole milk samples. On the other hand, only the NIR spectroscopy has
enabled the correct identification of adulterants in UHT whole milk samples. Among the
classification models developed for the detection and identification of adulterants in freeze-
dried açai pulp, the k-NNmodels showed that it is possible to detect adulterated samples and
to identify the type of adulterant in each one. This study showed that spectroscopy has great
potential for the quantification of the UHT whole milk components and for detection of
adulterated samples. Near-infrared spectroscopy can be applied for the quantification of UHT
whole milk components and for the detection, and identification of adulterants in UHT whole
milk and in freeze-dried açai pulp.
INTRODUÇÃO GERAL
ocorre durante todo o ano, com um período de maior produção entre Julho e dezembro
(Rogez, 2000). Consequentemente, a comercialização é diretamente afetada pela safra, que
implica em escassez de produto e aumento dos preços na entressafra. De modo a garantir o
fornecimento durante o ano inteiro, a polpa de açaí pode ser congelada, desidratada por
secagem ou liofilizada (Schauss, 2009).
A fraude em alimentos se caracteriza por qualquer substituição, adição,
adulteração ou falsificação de alimentos, ingredientes alimentares ou embalagens. Essa
prática visa principalmente ocultar ou mascarar a má qualidade dos produtos, de modo a
atribuir-lhes qualidades ou requisitos que não possuem para o ganho econômico(Moore et al.,
2012). Isto ocorre, principalmente, em alimentos ou ingredientes alimentícios largamente
consumidos ou de grande valor agregado (Manley et al., 2008). Portanto, não é surpreendente
as tentativas de alguns fornecedores de maximizar os lucros através das práticas de
falsificação e adulteração, que são consideradas fenômenos recorrentes no comércio de
alimentos(Schieber, 2008).
Por ser um alimento bastante comercializado, o leite é um dos alimentos mais
fraudados e o 2° alimento com maior número de resultados de busca sobre adulteração (143
resultados) em bases de dados de pesquisa (Web of Science, Pubmed e Google
Scholar)(Moore et al., 2012). Podem ser citados dois motivos principais explicam o grane
número de casos de adulteração do leite. O primeiro está relacionado ao aumento dos lucros
através do aumento do volume de produção pela adição de água. O segundo está relacionado à
adição de adulterantes para evitar perdas por deterioração causada pela ação de
microrganismos (Faostat, 2016; Smit, 2003). A polpa de açaí liofilizada é um produto de alto
valor agregado, que pode ser adulterada através da adição de materiais espessantes, como
farinha de trigo, amido de milho e maltodextrina; e corantes artificiais ou derivados de outras
frutas e vegetais (uva e beterraba)(Binois, 2012; Soares, 2014).
Devido à complexidade das matrizes alimentares, os métodos analíticos utilizados
na determinação de adulterantes e da composição centesimal são considerados trabalhosos,
demorados, são destrutivos, utilizam grande quantidade de solvente e geram um grande
volume de resíduos (Frankhuizen, 2008; Moore et al., 2012). Visando a obtenção de
resultados mais rápidos com a geração de menos solvente, as técnicas de espectroscopia no
infravermelho próximo (NIR) e médio (MIR) vêm sendo cada vez mais exploradas para a
realização de controle de qualidade e monitoramento de processos na área de
alimentos(Siesler, 2008). Isso se deve ao fato de que praticamente qualquer amostra pode ser
analisada(líquidos, soluções, pastas, pós, filmes, fibras, gases e superfícies), as amostras
14
requerem pouco ou nenhum preparo, após o desenvolvimento dos modelos os resultados são
rapidamente obtidos sem a destruição das amostras (McClure, 2007; Stuart, 2005a).
O espectro no infravermelho está compreendido entre os comprimentos de onda
de 0,78 e 100 µm, sendo dividido em três regiões: o infravermelho próximo (near-infrared)
de 0,78 a 2,5 µm, o infravermelho médio (mid-infrared) de 2,5 a 50 µm e o infravermelho
distante (far-infrared) de 50 a 100 µm (Skoog et al., 2007). A espectroscopia no
infravermelho consiste na capacidade das moléculas de absorverem apenas frequências
(energias) de radiação infravermelha selecionada, o que provoca sua excitação e a vibração
dos seus átomos (Pavia et al., 2009; Stuart, 2005b). Os espectros obtidos nas faixas do
infravermelho próximo e médio apresentam uma grande quantidade de informações.
Entretanto, a interpretação desses espectros é realizada através da utilização de ferramentas de
quimiometria que permitem o desenvolvimento de modelos matemáticos capazes de predizer
propriedades relevantes de amostras desconhecidas (Manley et al., 2008).
15
REFERÊNCIAS
Bichara, C.M.G., Rogez, H., (2011). Açai (Euterpe oleracea Martius), In: Yahia, E.M. (Ed.),
Postharvest biology and technology of tropical and subtropical fruits. . Woodhead
Publishing Ltd, Cambridge, pp. 1-23.
Binois, D., (2012). The obstacles to açaí exportation in Brazil, Gestão Internacional.
Fundação Getúlio Vargas, São Paulo, p. 91.
Fao. (2014). Dairy production and products: Milk and Milk Products. Food and Agriculture
Organization of the United Nations. http://www.fao.org/agriculture/dairy-
gateway/milk-and-milk-products/en/#.U77MJvldWSp. Access: 2014.
Faostat. (2016). Brazilian production of Milk, whole fresh cow in 2006 and 2016. Food and
Agricultural commodities production. The Statistics Division of the FAO- Food and
Agriculture Organization of the United Nations.
http://faostat.fao.org/site/339/default.aspx. Access: 2018.
Frankhuizen, R., (2008). NIR Analysis of Dairy Products, In: Burns, D.A., Ciurczak, E.W.
(Eds.), Handbook of Near-Infrared Analysis, 3 ed. CRC Press, Boca Raton, EUA, pp.
415- 436.
Ibge, (2014). Indicadores IBGE: Estatistica da Produção Pecuária. Setembro de 2014.
Instituto Brasileiro de Geografia e Estatística, pp. 5- 44.
IDF. (2015). The World Dairy Situation. Summit Daily. Vol. 2 (21), pp. 3.
Manley, M., Downey, G., Baeten, V., (2008). Spectroscopic Technique: Near-Infrared (NIR)
Spectroscopy, In: Sun, D.-W. (Ed.), Modern Techniques for Food Authentication.
Academic Press, London, pp. 65- 99.
McClure, W.F., (2007). Introduction, In: Ozaki, Y., McClure, W.F., Christy, A.A. (Eds.),
Near-Infrared Spectroscopy in Food Science Technology. Wiley John Wiley & Sons,
Hoboken, EUA, pp. 1- 10.
Menezes, E.M.S., Torres, A.T., Sabaa Srur, A.U. (2008). Valor nutricional da polpa de açaí
(Euterpe oleracea Mart) liofilizada. Acta Amazonica. Vol. 38, pp. 311-316.
Moore, J.C., Spink, J., Lipp, M. (2012). Development and Application of a Database of Food
Ingredient Fraud and Economically Motivated Adulteration from 1980 to 2010.
Journal of Food Science. Vol. 77 (4), pp. R118-R126.
Nogueira, M. (2014). Depois de 2012 difícil, indústria de Leite Longa Vida prevê crescimento
de cerca de 4% em 2013 ABLV. Assosiação Brasileira da Indústria de Leite Longa
Vida. http://www.ablv.org.br/implistcontentint.aspx?id=792&area=imp-not. Access:
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2014.
Pavia, D.L., Lampman, G.M., Kriz, G.S., (2009). Infrared, In: Pavia, D.L., Lampman, G.M.,
Kriz, G.S. (Eds.), Introduction to Spectroscopy, 4 ed. Brooks/ Cole, USA, pp. 15- 104.
Rogez, H., (2000). Açaí: preparo, composição e melhoramento da conservação. EDUFPA.
Schauss, A.G., (2009). Açaí: An Extraordinary Antioxidant-Rich Palm Fruit. Biosocial
Publications.
Schieber, A., (2008). Introduction to Food Authentication, In: Sun, D.-W. (Ed.), Modern
Techniques for Food Authentication. Academic Press, London, pp. 1-17.
Siesler, H.W., (2008). Basic Principles of Near-Infrared Spectroscopy, In: Burns, D.A.,
Ciurczak, E.W. (Eds.), Handbook of Near-Infrared Analysis, 3 ed. CRC Press, Boca
Raton, EUA, pp. 7- 20.
Skoog, D.A., Holler, F.J., Crouch, S.R., (2007). An Introduction to Infrared Spectrometry, In:
Skoog, D.A., Holler, F.J., Crouch, S.R. (Eds.), Principles of Instrumental Analysis, 6
ed. Thomson Brooks/Cole, Belmont, EUA, pp. 430- 452.
Smit, G., (2003). The major constituents of milk, In: Smit, G. (Ed.), Dairy Processing:
Improving Quality. CRC Press LLC, USA, pp. 5- 38.
Soares, K. (2014). Vigilância Sanitária apreende 50 litros de açaí adulterado. ORM News.
Portal ORM. Organizações Rômulo Maiorana.
http://www.ormnews.com.br/noticia/vigilancia-sanitaria-apreende-50-litros-de-acai-
adulterado. Access: 2017.
Stuart, B., (2005a). Introduction, In: Stuart, B. (Ed.), Infrared Spectroscopy: Fundamentals
and Applications. John Wiley & Sons, Hoboken, EUA, pp. 1-14.
Stuart, B., (2005b). Spectral Analysis, In: Stuart, B. (Ed.), Infrared Spectroscopy:
Fundamentals and Applications. John Wiley & Sons, Hoboken, EUA, pp. 45- 70.
Walstra, P., Geurts, T.J., Noomen, A., Jellema, A., van Boekel, M.A.J.S., (1999). Milk:
Compostion, Structure, and Properties., In: Walstra, P., Geurts, T.J., Noomen, A.,
Jellema, A., van Boekel, M.A.J.S. (Eds.), Dairy Technology: Principles of Milk
Properties and Processes. Marcel Dekker, Inc., New York, pp. 3- 26.
17
OBJETIVOS
OBJETIVOS GERAIS
Este trabalho teve como principal objetivo o desenvolvimento de modelos de
calibração e classificação utilizando espectroscopia no infravermelho próximo (NIR) e médio
(MIR) para determinação de parâmetros de qualidade e adulterantes em leite UHT integral, e
desenvolver modelos de classificação utilizando NIR para determinação de adulterantes em
polpa de açaí liofilizada.
OBJETIVOS ESPECÍFICOS
CAPÍTULO I
Revisão Bibliográfica
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1. LEITE
total) em rações para animais e fórmulas infantis. Devido o consumo dos alimentos
adulterados, cerca de 1000 animais domésticos morreram nos Estados Unidos. Na China, em
torno de 294 mil foram afetados, sendo que destes, 50 mil apresentaram problemas renais e 6
morreram (Fda, 2009).
Nos últimos 10 anos, vários casos de adulteração em leite no Brasil têm sido
relatados. No ano de 2007, o Ministério Público Federal do Estado de Minas Gerais e a
Polícia Federal iniciaram a Operação Ouro Branco com o objetivo de combater crimes
cometidos por cooperativas suspeitas de praticarem a adulterações em leite(Mpf, 2007).No
ano de 2012, o Ministério Público do Estado do Rio Grande do Sul (MPRS) em parceria com
o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) iniciaram a operação “Leite
Compen$ado” com objetivo deflagrar a adulteração em leite(Mprs, 2013a).
Durante a operação Ouro Branco foram encontradas grandes quantidades de soda
cáustica (hidróxido de sódio) e de peróxido de hidrogênio, além das substâncias que, embora
permitidas, estavam sendo adicionadas em quantidades acima do limite máximo permitido por
lei, tal como o citrato de sódio (Mpf, 2007).Na primeira coleta da operação Leite
Compen$adofoi identificada a presença de formol em amostras de leite em um posto de
resfriamento e durante o cumprimento de mandados de busca e apreensão, foram recolhidos
sacos de uréia, soda cáustica, corantes, coagulantes líquidos e emulsão para obtenção de
consistência, entre outros produtos (Mprs, 2013a).Durante as fases da operação Leite
Compen$ado no estado do Rio Grande do Sul, foi encontrada uma formulação contendo uréia
utilizada para compensar a diluição promovida pela adição de água (Bertão, 2014). Em outra
formulação foi relatada a utilização de bicarbonato de sódio e açúcar (Truda & Colossi,
2013). Em outra operação, foi verificado que o leite estava sendo adulterado com uréia
contendo formol. O produto adulterado foi largamente comercializado, sendo produzido entre
Janeiro e Abril de 2013 em torno de 1.5 milhões de litros (Mprs, 2013b). No mesmo ano, foi
verificado que empresas transportadoras de leite eram responsáveis pela adição de uréia
contendo formol. A quantidade de uréia adquirida pelos fraudadores alcançou mais de 100
toneladas, sendo movimentado no período de 1 ano em torno de 100 milhões de litros de leite
(Mprs, 2017).
Por ser um alimento bastante comercializado, o leite é um dos alimentos mais
fraudados e o 2°alimento com maior número de pesquisas envolvendo a investigação de
adulterantes (143 resultados) em bases de dados de pesquisa (Web of Science, Pubmed e
Google Scholar)(Moore et al., 2012). Assim como em outros tipos de alimentos,
normalmente, a adulteração de leite se dá através da adição de produtos de menor valor, tal
22
como no caso da adição de água, que é considerada o modo mais simples de adulteração de
leite e que tem o objetivo de aumentar seu volume(Nascimento et al., 2017). Isto é motivado
por um fator econômico, visto que no Brasil, por exemplo, no ano de 2012 a comercialização
de leite movimentou em torno de 10 bilhões de dólares (Faostat, 2016).
O conhecimento sobre a adulteração do leite é de longa data e sabe-se que a
adulteração pela adição de água não ocorre de forma isolada, conforme mostrado por Mpf
(2007), Mprs (2013a), Bertão (2014) e Truda & Colossi (2013), sendo, portanto, utilizados
outros produtos de baixo valor comercial e de fácil acesso, tal como no caso da uréia, do
amido e da sacarose, que podem ser facilmente encontrados em zonas rurais. A uréia está
presente naturalmente no leite (84- 280 mg/ kg de leite), considerada o componente
majoritário da fração chamada de Nitrogênio não- Protéico (NPN), representando em torno de
50% (Walstra et al., 1999b), sendo adicionada para aumentar o teor dessa fração, de modo a
aumentar a concentração de nitrogênio total (Azad & Ahmed, 2016).O teor de uréia presente
naturalmente no leite é dependente do tipo de alimentação dos animais e da época do ano. O
teor elevado de uréia no leite contribui para o aumento da estabilidade térmica (O'Mahony &
Fox, 2013).
A uréia tem várias aplicações, tais como componente de fertilizantes, ração
animal, aditivo alimentício, agente aromatizante, etc (Epa, 2011). Durante a produção da uréia
polimérica (forma que promove a liberação controlada de nitrogênio no solo) há a reação com
o formaldeído na presença de catalisador, originando um polímero sólido, branco, com teor de
nitrogênio em torno de 38% e teor residual de formaldeído(Guertal, 2009). Isto explica os
resultados positivos para presença de formaldeído em amostras de leite adulteradas com uréia,
que foram coletadas pelo Ministério Público do Estado do Grande do Sul (Mprs, 2013a).
O amido é a reserva predominante de energia de plantas e ocorre na natureza na
forma de grânulos formados, em sua grande maioria (depende da matriz) de dois polímeros-
amilose, uma molécula essencialmente linear formada de unidades de α-D-glicopiranosil
unidas por ligações 1→4 e ligações do tipo 1→6, e amilopectina, uma molécula formada por
unidades de α-D-glicopiranosil unidas por ligações do tipo 1→4 com ramificações do tipo
1→6 a cada 20 a 60 resíduos. As principais fontes de amido são: milho, batata, mandioca,
trigo, arroz, batatadoce etc (Belitz et al., 2009). O leite bovino contém alguns oligossacarídeos
em baixas concentrações (até 150 mg/L) e não contém amido (O'Mahony et al., 2013).
Entretanto, assim, como a sacarose, o amido também tem sido adicionado para aumentar a
densidade do leite que foi previamente adulterado pela adição de água (Sharma, 2006).
23
condições mais severas para remover toda a umidade de um alimento podem simultaneamente
causar a decomposição do produto. Das várias técnicas analíticas para determinar o teor de
umidade, a remoção de umidade ou a secagem é frequentemente aplicada e determinada
gravimetricamente. A matéria residual obtida após a remoção da umidade de uma amostra é
denominada de sólidos totais, um parâmetro de grande importância econômica, visto que a
água não tem valor comercial para o produtor (Park & Bell, 2004).
1.3.2 Carboidratos
1.3.3 Lipídios
1.3.4 Proteínas
abordados de forma mais detalhada por serem métodos instrumentais(Camp & Dierckx,
2004).
A metodologia oficial do (Mapa, 1997) para determinação do teor de proteínas em
leite se baseia no método de Kjeldahl, que consiste na quantificação do nitrogênio total da
amostra na forma de sulfato de amônio, obtido através da digestão com ácidosulfúrico e
posterior destilação com liberação da amônia, que é fixada em solução ácida e titulada. Neste
método são utilizados os seguintes reagentes: Ácido sulfúrico(H2SO4), anti-espumante,
Indicador misto de vermelho de metila (C15H15N3O2) e verde de bromocresol(C21H14Br4O5S),
álcool etílico a 70 %, sulfato de potássio (K2SO4), sulfato de sódio anidro (Na2SO4), sulfato
de cobre pentahidratado (CuSO4.5H2O), ácido bórico (H3BO3), hidróxido de sódio (NaOH)
eZinco metálico granulado.
1.3.5 Cinzas
2. AÇAÍ
mm e o endocarpo é duro, revestido por fibras e contem uma única semente. Sendo assim, a
proporção de polpa para o fruto é baixa, variando de 5 a 15% (Nascimento, 2008; Rogez,
2000).
Açaí é uma parte importante da dieta na Amazônia, onde geralmente é consumido
como uma sopa grossa, no entanto também pode ser utilizado na produção de sorvetes,
chocolates, geleias e produtos liofilizados (Schauss, 2009). A cor violeta do açaí é uma
consequência da alta concentração de antocianinas cianidina-3-glucosídeo e cianidina-3-
rutinosídeo, flavonóides que exibem uma ampla gama de benefícios para a saúde como
propriedades antioxidante e anti-inflamatória (Bichara & Rogez, 2011; Velioglu et al., 1998;
Wycoff et al., 2015). Devido às suas propriedades funcionais, o consumo de polpa de açaí
vem aumentando(Yamaguchi et al., 2015). Esta bebida, nos últimos anos, tornou-se conhecida
em outras regiões do país, principalmente na região sudeste, e vem tendo grande e crescente
aceitação (Suframa, 2003). O açaí também contem compostos fenólicos não flavonoides
(protocatecuico, p-hidroxibenzóico, ácido vanílico, siríngico e ferúlico), lipídio saturados e
insaturados (ácido oléico, ácido linoleico, ácido linolênico e ácido palmitoléico), carboidratos,
cinzas, proteínas, fibras dietéticas, vitaminas (C, tiamina, riboflavina e niacina) e minerais
(Na, K, Ca, Mg, Fe e P) (Gordon et al., 2012; Pacheco-Palencia et al., 2009; Rogez, 2000).
O açaizeiro é explorado principalmente na forma de extrativismo, dadas as
grandes populações da espécie na floresta, com predominância para os Estados do Amapá e
Pará. Devido a sua exploração predatória, que chegou a representar um risco considerável
para a espécie até o início da década de 90, foram tomadas providências pelas instituições
competentes no sentido de racionalizar a exploração do açaí, tanto através de sistemas de
extrativismo manejado e sustentável quanto de seu cultivo (Suframa, 2003).
O açaí apresenta duas safras, a primeira de março a junho e a segunda de agosto a
dezembro; e duas entre safras nos períodos de janeiro a fevereiro e de junho a julho. Nos
períodos de entressafra o produto é encontrado em menor quantidade e apresenta uma menor
qualidade, visto que são encontrados frutos em estágios diferentes de maturação no mesmo
cacho. Enquanto que a safra de agosto a dezembro (estação mais seca) é mais expressiva e
apresenta uma maior quantidade de frutos, os quais se encontram em estágios de maturação
mais homogêneos (Rogez, 2000). Como consequência, o negócio açaí é diretamente afetado
pela sazonalidade, o que implica em escassez e escalação de preços nos períodos entre as
culturas (Binois, 2012). A polpa de açaí pode ser comercializada fresca, mas para garantir a
oferta ao longo do ano, toneladas de polpas são congeladas e liofilizadas (Schauss, 2009).
32
para formar um modo interactância, onde a radiação incidente é detectada em pontos distintos
da superfície de uma amostra(Manley et al., 2008).
Os avanços mais significativos na espectroscopia no infravermelho surgiram com
a introdução de espectrômetros com transformada de Fourier (FT). Este tipo de instrumento
emprega um interferômetro e explora o processo matemático da FT, o que melhora a
qualidade dos espectros de infravermelho e minimiza o tempo necessário para obtenção dos
dados(Stuart, 2005a). Quando a FT é aplicada na soma dos interferogramas acumulados
(dados de intensidade versus tempo), pode ser obtido um espectro com uma melhor relação
sinal/ruído, ou seja, maior sensibilidade do que um instrumento convencional de
dispersão(Pavia et al., 2009). A espectroscopia no infravermelho distante requer o uso de
fontes e materiais óticos específicos(Rouessac & Rouessac, 2007). A região do infravermelho
distante é mais limitada que a região no infravermelho médio por conter menos informações
acerca da estrutura das moléculas. Entretanto, pode fornecer informações de moléculas que
contém átomos pesados, moléculas que apresentam vibrações moleculares de cadeia, torsões
moleculares e vibrações de cristais em modo de rede (Lattice)(Stuart, 2005b).
A espectroscopia no infravermelho médio baseia-se na absorção de radiação na
região de 4000 a 400 cm-1 do espectro eletromagnético (Fagan & O'Donnell, 2010).A
espectroscopia no infravermelho médio (MIR) pode fornecer mais informações acerca da
estrutura dos compostos e, por isso, tem sido bastante utilizada na identificação de compostos
orgânicos (Stuart, 2005b).Existem no mercado vários analisadores automáticos de leite por
infravermelho médio, que tem a capacidade de analisar centenas de amostras por hora. Nestes
equipamentos, podem ser determinados simultaneamente os teores de gorduras, proteínas,
lactose, baseando-se nos comprimentos de onda de absorção dos principais grupos funcionais
de cada um desses compostos(Wehling, 1998). Devido ao sucesso da análise de leite por
FTIR, esta tecnologia tem sido comercializada em produtos como o MilkoScan™ FT120
(Foss, Hillerød, Dinamarca) que emprega este princípio em conformidade com os padrões
estabelecidos pelo International Dairy Federation (IDF) e Association of Official Analytical
Chemists (AOAC)(Fagan & O'Donnell, 2010).
A espectroscopia no infravermelho próximo (NIR) é considerada uma técnica
importante em análises quantitativas de controle de qualidade, embora seja caracterizada
como pobre em termos de absorções específicas por conter bandas largas e de baixa
intensidade geradas a partir de combinações(Stuart, 2005b). A sobreposição de várias bandas
individuais de absorção da radiação eletromagnética resulta em bandas de absorção largas,
que são características dos espectros de NIR. Estes espectros contém uma quantidade enorme
36
4. QUIMIOMETRIA
Com o avanço das tecnologias, tem se tornado cada vez mais comuma utilização
de instrumentos sofisticados (normalmente sob o controle de computadores) em métodos
analíticos aplicados em química. Isto tem gerado um campo rico tem termos de quantidade de
dados, abrindo assim a possibilidade de pesquisa movida pelos dados (Lavine & Davidson,
2006).Dentre os diversos métodos analíticos existentes, podem ser citadas as análises por
espectroscopia, que fornecem através de uma análise relativamente simples e rápida muitas
informações a partir de uma única amostra(Oliveri & Forina, 2012). Entretanto, a
disponibilidade de grandes conjuntos de dados não significa que a interpretação dos mesmos é
instantânea e simples. Na verdade, vários procedimentos são necessários para “extrair” e
interpretar adequadamente as informações contidas nos dados(Oliveri & Forina, 2012).
Dentro desse contexto se aplica a Quimiometria, que consiste na aplicação de métodos
estatísticos e matemáticos em análises de dados experimentais (Stuart, 2005b). Na
quimiometria são utilizados métodos matemáticos para a conversão de dados numéricos
(obtidos através dos instrumentos) em informação de caráter químico(Oliveri & Forina,
2012).
Em áreas como espectroscopia no infravermelho, onde os espectros obtidos
contêm uma quantidade enorme de informações sobre propriedades químicas e físicas das
amostras analisadas, os métodos quimiométricos são de extrema importância, visto que, por
exemplo, nos espectros obtidos na região do infravermelho próximo, ocorrem,
geralmente,sobreposições de bandas individuais de absorção da radiação eletromagnética,
resultando em bandas de absorção largas. Essas sobreposições dificultam a interpretação dos
resultados (Manley et al., 2008).Além de facilitar ou possibilitar a interpretação dos resultados
obtidos, a quimiometria pode mostrar relações “escondidas” entre os dados (Lavine &
Davidson, 2006).
Após aquisição dos espectros, cromatogramas, espectros de massas, etc, os dados
são organizados em vetores. Cada vetor (amostra) é ordenado, de modo a formar uma matriz
X constituída de um arranjo ordenado de linhas e colunas. Esses dados são, geralmente, pré-
tratados antes da análise quimiométrica através de diferentes tipos de tratamentos, sendo,
escolhido o que possibilita melhor interpretação ou resultado. O pré-tratamento tem como
objetivo principal de reduzir variações indesejáveis que não foram removidas durante a
obtenção dos dados e que podem comprometer a análise quimiométrica. Os pré-tratamentos
podem ser divididos em dois tipos: os que são aplicados às amostras são denominados de
38
X=CST Equação 5
Ĉ = XS/STS Equação 6
42
X=TPT + EX (Equação 8)
Y = Ŷ + EY = TC + EY (Equação 9)
C = T + Y = (TTT)-1TTY (Equação 10)
43
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52
CAPÍTULO II
Kleidson Brito de Sousa Lobatoa, Lucas Oliveira Montesinob, Rafael Leonardo Zampronib,
Ronei Jesus Poppic, Juliana Azevedo Lima Pallonea*
a
Department of Food Science, School of Food Engineering, University of Campinas,
Monteiro Lobato Street, 80, 13083-862, Campinas, São Paulo, Brazil
b
Laboratory of Physicochemical analyses of foods of animal origin and water, National
Agricultural Laboratory (Lanagro), Raul Ferrari street, 13100-105, Campinas, São Paulo,
Brazil
c
Institute of Chemistry, University of Campinas, 13083-970, Campinas, São Paulo, Brazil
*
Corresponding author. Department of Food Science, School of Food Engineering, University
of Campinas, Monteiro Lobato Street, 80, 13083-862, Campinas, São Paulo, Brazil
ABSTRACT
The majority of the studies involving the application of near- and mid-infrared spectroscopy
for the determination of quality parameters in milk are related to the analysis of raw or
pasteurized milk. To our knowledge, this is the first work with the objective to investigate the
performances of NIR and MIR spectroscopy in the determination of total solids, lactose, fats,
proteins and ash in UHT whole milk. During oneyear, 102 samples were analyzed by the
reference methods and by NIR (transflectance) and MIR (Transmittance by Attenuated total
reflectance- ATR),being used in the development ofPartial Least Squares (PLS) models. The
models developed with near-infrared spectral data (NIR models) were constructed with 2- 9
latent variables, resulting in low values ofRoot Mean Square Standard Error of calibration
(RMSEC) (0.0065- 0.155%), Root Mean Square Standard Error of Prediction (RMSEP)
(0.011- 0.1049%), Limit of detection (LD) (0.01- 0.57), Limit of quantification (LQ) (0.03-
1.74%), Relative standard deviation (RSD) (0.9-3.2%) and bias(-0.0106- 0.00476%). The
models developed with mid-infrared spectral data (MIR models) were constructed with 6-8
latent variables, resulting in values of RMSEC (0.0061- 0.0969%), RMSEP (0.0099-
0.0836%), LD (0.06- 0.9), LQ (0.17- 2.72%), RSD (0.7-2.7%) and bias (-0.0086-
0.000289%). The model developed for the quantification of total solids by NIRpresented
lower limits of detection and quantification, and higher R2pred, requiring fewer latent variables,
indicating a superior robustness compared to the MIR model. The models developed for the
quantification of lactose, fats, proteins and ash contents had similar values of RMSEC and
RMSEP, but different numbers of latent variables, bias, R2cal, R2pred, sensitivity, selectivity,
limits of detection and quantification. Taking into consideration all those figures of merit, the
best models for the quantification of quality parameters in UHT whole milk were developed
using the near-infrared spectroscopy data.
1. INTRODUCTION
Milk is the 4th most produced agricultural product in Brazil (33.62 billion
kg/year) (Faostat, 2016) per capita consumption of 169 kg/ year(Ifcn, 2012), which is
considered high and it is estimated that by the year of 2022 the consumption will be twice the
current(Ibge, 2014). In Brazil, about 20% of all milk production is used in the production of
UHT milk, corresponding to 81% of the liquid milk segment. It has been observed an increase
in the consumption of UHT milk, which is present in 88 percent of Brazilian households and,
on the other hand, it was observed a decrease in the consumption of pasteurized milk and milk
55
informal(Nogueira, 2014). The UHT milk is defined as the homogenized milk subjected to
heating during 2 to 4 seconds at 130-150° C in continuous flow, being immediately cooled to
a temperature below 32° C and packaged in sterile packages and hermetically sealed in
aseptic conditions(Mapa, 1997).
Usually, the reference procedures of analysis of dairy products applied in the
determination of proximate composition are time consuming and generate a lot of waste due
to the use of large amounts of reagents per sample analyzed (Mapa, 2006). Consequently,
there is a need for analytical methods that can provide quick results and low waste generation,
such as near- (NIR) and mid-infrared (MIR) spectroscopy.Infrared spectroscopy is a
technique based on vibration of atoms of a molecule. The infrared region is commonly
divided into 3 regions: Far infrared (< 400 cm-1), mid-infrared (4000-400 cm-1) and near-
Infrared (13000- 4000 cm-1). The mid-infrared spectrum can be divided into four regions: the
X-H stretching region (4000- 2500 cm-1), the triple-bond region (2500- 2000 cm-1), the
double-bond region (2000- 1500 cm-1) and the fingerprint region (1500- 600 cm-1). The
absorptions observed in the near-Infrared region are overtones or combinations of the
fundamental stretching bands which occur in the mid-infrared region. The bands in the near-
Infrared are often overlapped, making them less useful than the mid-infrared region for
qualitative analysis. However, there are important differences between the near-Infrared
positions related to the functional groups which can be exploited for quantitative analyses
(Stuart, 2005).
Both NIR and MIR spectroscopy can be employed as non-destructive and non-
invasive measurements, require minimal or no sample preparation, and only need a few
seconds for spectral collection. We performed a search on Scopus and Web of Science
databases using the terms UHT milk and infrared and found two papers involving the analysis
of UHT milk by infrared. However, they aimed the analysis of physical and chemical changes
in UHT milk during storage(Grewal et al., 2017) and the construction of classification models
for the detection of adulterants(Luna et al., 2016). Most studies involving the analyses of milk
by infrared relates to the analysis of raw cow milk(Aernouts et al., 2011; Jankovská &
Sustová, 2003; Melfsen et al., 2012; Mlcek et al., 2016), dairy products(Barabássy, 2001),
ewe's milk (Mouazen et al., 2009) by NIR, and the analysis of raw cow milk (Bonfatti et al.,
2016; Etzion et al., 2004; Luinge et al., 1993) and commercial milk samples (type not
defined)by MIR(Iñón et al., 2004; Mazurek et al., 2015). Thus, taking into account the
importance of UHT milk and the need for tools that provide rapid analytical results, this
56
original work aims to develop calibration models for the quantification of total solids, lactose,
fats, proteins and ash in UHT milk.
2.1 Samples
UHT milk samples (29 brands) used in this study were acquired in supermarkets
in the city of Campinas-SP and are sourced from producers in the Brazilian States of Bahia
(5), Goiás (2), Minas Gerais (3), Rio Grande do Sul (12), Santa Catarina (10) and São Paulo
(70). During the period of 1 year (September 2015 to September 2016) 102 samples of 1 liter
were collected (different lots) and were immediately taken to Lanagro (National Agricultural
Laboratory-Campinas, São Paulo), where they were kept at 4° C for the reference analyses. At
the same time, 100 mL of each sample were stored in PET bottles for collection of near-
Infrared spectra in the Laboratory of Products of Animal Origin(Lanagro- SP), and the mid-
infrared spectra in the laboratory of Infrared spectroscopy and Raman of the Institute of
Chemistry of the State University of Campinas (Campinas, São Paulo).
The reference analyses for the quantification of the quality parameters of total
solids (%), proteins (%) (Kjeldahl principle), fats content (%) (Gerber Method) and ash (%)
were performed in triplicate according to the official methods of Ministry of Agriculture,
Livestock and Food Supply(Mapa, 2006) and results were expressed in wet basis. The
carbohydrates were determined in terms of lactose content (%)according to the method
ofGomis et al. (2001), using a High Performance Liquid Chromatography system (Series 200,
Perkin Elmer, Shelton, CT 06484, USA) equipped with a diode array detector, an online
degasser, a quaternary pump and an automatic injector and that was coupled to a C18 Zorbax
column (150 mm x 4.6 mm i.d.; 3.5µm particle size), adjusted at 30°C. An aliquot of 10 µL
was injected into the chromatographic system and separation was achieved using gradient
mobile phase of 0.1 % acetic acid: acetonitrile (96:4) during 3.5 minutes, 0.1 % acetic acid:
acetonitrile (65:35) during 6.5 minutes and 0.1 % acetic acid: acetonitrile (96:4) during 10
minutes, and chromatograms were processed at 303 nm. Data acquisition and processing were
performed using the Chrom Navigation (Version 6.3.2.0646). All of the solvents
(chromatographic grade)used in the HPLC separation were previously filtered through a
Millipore vacuum filtration system using a 0.22 µm membrane for organic solvents
57
(Millipore, Barueri, SP, Brazil). Samples were analyzed in triplicate. The averaged results
obtained in reference analyses were used for the development of calibration models.
Firstly, before the collection of NIR and MIR spectra, samples were stored during
1 hour to reach the room temperature. Near-Infrared (NIR) transflectance spectra were
collected at room temperature (24±1°C) using a FT-IR spectrometer (Perkin Elmer-Waltham,
USA, model Spectrum 100N) calibrated with a reference Spectralon®, equipped with an
integrating sphere and InGaAs detector. Then, each sample was homogenized and scanned in
triplicate (10000 to 4000 cm-1, with increment of 1 cm-1, resolution of 4 cm-1 and 32
scans/spectra) in a transflectance cell that consisted in a glass Petri dish combined with an
aluminum reflector with 0.5 mm path length. Mid-Infrared (MIR) transmittance spectra were
collected at room temperature (22±1°C) using a FT-IR spectrometer (model Cary 630,
Agilent Technologies, Santa Clara, USA) calibrated with air, equipped with a diamond
attenuated total reflectance (ATR) accessory. Each sample was homogenized and an aliquot
(around 300 µL) was scanned in triplicate (4000 to 400 cm-1, increment of 1.864 cm-1,
resolution of 4 cm-1 and 64 scans/spectra). Between the scanning of each sample, the cell was
cleaned with ethanol (99%). The averaged spectrum was used for multivariate analyses.
In order to obtain the best predictive ability, NIR and MIR spectral data were
treated with several pre-processingmethods and in some cases, with the combination of them,
like smoothing, mean centering, Standard Normal Variate, Multiplicative scatter correction,
first Savitzky-Golay derivative, second Savitzky-Golay derivative, etc. The pre-processing
was manually chosen through the analysis of the performance of the models (number of latent
variables, Root Mean Square Standard Error of calibration, Root Mean Square Standard Error
of cross-validation, Root Mean Square Standard Error of Prediction, bias, relative standard
deviation). Therefore, only the best models will be shown.
From the total of 102 samples, around 70% were used in the development of the
calibration models (calibration set) and 30% for the external validation (prediction set). Partial
least squares (PLS) regression models were developed using the SIMPLS algorithm with the
spectral data and the reference analyses results of the calibration set. Outliers were identified
by Q and T2 plot with a confidence limit of 0.95. The number of latent variables was chosen
by cross-validation (venetian blinds method with 8 data splits). The preprocessing and the
development of PLS models were performed by PLS-toolbox (version 7.3.1) for Matlab
58
(2014a).The quality of calibration models was assessed via some statistical parameters: The
Root Mean Square Standard Error of calibration (RMSEC) and Prediction (RMSEP) describe
the degree of agreement between the values of concentration estimated by the model
calibration and the values obtained by the reference analysis in calibration and prediction sets,
respectively. The lower the RMSEC and RMSEP values, the better the prediction ability of a
model. The magnitudes of the parameters R2cal and R2pred providethe proportion of total
variation in the response variable (Y matrix) explained by the calibration model for the sets of
calibration and prediction, respectively(Kalivas & Gemperline, 2006).The R2(R2cal or R2pred)
should be high to indicate a good prediction capability, while with a low value it is not
possible to obtain consistently high accuracy by infrared spectroscopy analyses. The bias
gives the average by which the results differ. As in the case of RMSEC and RMSEP, the bias
should be as low as possible(Manley et al., 2008).The figures of merit of sensitivity,
analytical sensitivity, selectivity, limit of detection, limit of quantification and relative
standard deviation were determined using Matlab(2014a) routines that were based on the
concept of net analyte signal (NAS)(Ferreira, 2015). All parameters related to the performance
of the models were evaluated together.
Figure 1 shows the variation of the contents of total solids, fats, proteins,
carbohydrates and ash of UHT whole milk samples during the collection period (September
2015 to September 2016). The results show that, in general, the average levels of total solids,
protein and ash are almost constant over the course of 1 year. The fat content is the main
parameter adopted on classification of UHT milk. However, in the present study, we found
that 5 samples showed mean levels of fats below 3%, a value described by (Mapa, 1997)as a
minimum for classification as whole milk, and therefore considered as semi-skimmed milks.
In addition, we verified that 5 samples of a same brand produced in the State of Bahia
presented fat levels above the global average (Fig. 1- A). This can be attributed to non-
submission of the raw material milk to the standardization step, where part of the fat is
removed, resulting in skimmed milk and cream (high value product). The standardized milk is
obtained by the addition of cream at a known concentration to the skimmed milk(Gunsing et
al., 2009).
59
Figure 1. Variation of the levels of total solids, fats (a), proteins, lactose and ash (b) of UHT
whole milk samples during 12 months of collection. Each symbol indicates the mean value
and the bars indicate standard deviation.
Figure 2.NIR (A) and MIR (B) spectra of UHT whole milk samples.
Figure 2- A shows the NIR spectra of 102 UHT whole milk samples.
Carbohydrates in general may have a free OH stretch absorption near 6940 cm-1 in the NIR
region. The bands associated with proteins are found in the NIR region at 10277 to 9804 cm-
61
1
(N-H stretch second overtone), 6667 to 6536 cm-1 (N-H stretching first overtone), 4878 to
4854 cm-1 (N-H stretching combinations), 4613 to 4587 cm-1 (N-H bend second overtone and
C=O stretch-N-H in-plane bending-C-N stretch combination bands)(Workman & Weyer,
2012). The bands associated with milk fats are found between 5780 and 5624 cm-1(assigned
to first overtone CH2 stretching vibrations) and 4325 cm-1 and 4248 cm-1 (assigned to the
combination stretching and bending ofCH2 and CH3) (McClure & Stanfield, 2006).
Theoretically, there are no absorption bands for mineral species in the NIR region. Although,
salts can be detected in high-moisture samples due to changes in hydrogen bonding are
resultant band shifts(Shenk et al., 2008).
Milk is made up of more than 80% water and has a NIR spectrum very similar to
water. Although, milk spectra have higher absorbances at all wavelengths(McClure &
Stanfield, 2006). In the NIR region, the spectrum of water exhibits two strong bands. The first
is located at 6934 cm-1, which is typical of the first harmonic of νOH vibration. The second is
located at 5176 cm-1 which is related to the combination of νOH + δOH (overtone of O-H
bending)(Dufour, 2009). The strength of the NIR absorption bands, the unique water
combination band at 5154 cm-1, and the sensitivity of the absorption bands to the
environment of the water molecules have all contributed to the success of NIR to study and
measure water(Workman & Weyer, 2012).
Figure 2- B shows the MIR spectra of 102 UHT whole milk samples.
Carbohydrates and proteins resulted from the polymerization of monosaccharides and amino
acids, respectively. In general, the MIR spectra of carbohydrates show four main zones of
absorbance. The main bands observed in the MIR region for lactose are 1167, 1139, 1116,
1093, 1083, 1070, 1058, 1031, 1018, 1005, 989 cm-1. Peptidic bond C-NH (Amide I, II and
III) is mainly responsible for the absorption occurring between 1700 and 1500cm-1. The
triacylglycerols ester linkage C-O (1175 cm-1), CO (1750 cm-1) group, and acyl chain C-H
(3000- 2800cm-1) stretch wavenumbers are commonly used to determine fat. In the MIR
range, water exhibits a broad and intense band at 3300 cm-1 corresponding to νOH. The δOH
bending band, less intense, is observed at 1638 cm-1(Dufour, 2009).
The NIR Spectra showed an offset in coordinate axis and an inclination of the
baseline as decreases the wavenumber (Fig. 2- A). The MIR spectra are almost overlapped,
with little offset and slope of the baseline (Fig. 2- B). Therefore, different ranges and pre-
processing were used in the development of models in order to minimize the effects of noise,
scattering, offset and slope of the baseline (Table S1). In the literature other studies show the
application of different treatments in spectral data in the NIR and MIR regions (Table S2).
62
Table 2 shows the statistical data of the samples used in the calibration and
prediction sets of NIR and MIR models. These results show that the concentration of samples
from the calibration sets are representative when compared with the prediction sets, which is
extremely important, since unknown samples are predicted by interpolation within the limits
of the model, because it is not guaranteed a good accuracy by extrapolation(Blanco &
Bernárdez, 2009).
Table 2: Quality parameters of the samples used in calibration and prediction sets.
Calibration set Prediction set
Component
nc Range Meanc SDc np Range Meanp SDp
Near-Infrared
Total Solids (%) 68 11.25- 13.39 11.74 0.41 27 11.79- 13.21 11.73 0.36
Lactose (%) 65 3.65- 5.17 4.47 0.39 30 3.85- 4.96 4.47 0.34
Fats (%) 61 2.80- 4.30 3.21 0.28 30 2.95- 4.25 3.22 0.26
Proteins (%) 68 2.83- 3.69 3.14 0.15 32 2.90- 3.68 3.16 0.16
Ash (%) 68 0.71- 0.85 0.77 0.029 30 0.72- 0.80 0.76 0.021
Mid-Infrared
Total Solids (%) 70 11.14- 12.34 11.67 0.27 25 11.40- 11.97 11.65 0.15
Lactose (%) 69 3.65- 5.35 4.46 0.42 30 3.95- 4.92 4.40 0.33
Fats (%) 69 2.80- 4.30 3.21 0.31 30 3.00- 3.75 3.20 0.17
Proteins (%) 71 2.83- 3.69 3.18 0.18 27 2.97- 3.30 3.14 0.10
Ash (%) 73 0.70- 0.85 0.76 0.032 27 0.72- 0.81 0.76 0.021
nc: number of samples in calibration set; Meanc: Mean value obtained in reference methods
for the calibration set; SDc: standard deviation of results obtained in reference methods for the
calibration set; np: number of samples in prediction set; Meanp: Mean value obtained in
reference methods for the prediction set; SDp: standard deviation of results obtained in
reference methods for the prediction set.
Table 3 shows the figures of merit obtained from the NIR and MIR models forthe
determination of quality parameters of UHT milk. For the quantification of total solids, the
NIR model was developed using the spectral data preprocessed by Smoothing, Orthogonal
signal correction and Mean-centering. The MIR model was developed with spectral data
preprocessed by standard normal variate (Table S1). The comparison of the values of RMSEC
and RMSEP obtained for the NIR model (Table 3) with the standard deviations obtained in
the analysis of reference for calibration and prediction samples (SDc and SDp) (Table 2)
shows that the errors associated with the prediction of the total solids content by NIR are low.
For the MIR model, the RMSEC and RMSEP were even lower, indicating that the model fits
63
to the data and has higherprediction ability. The RMSEP values close to the standard
deviations of the reference methods (SDp) indicate that the prediction of the quality
parameters by calibration models is similar to the reference method. Therefore, for a
satisfactory model, the SDp/RMSEP relationship must be greater, with anoptimum value
around 10(Ferreira, 2015). On determination of total solids content, the values of the
SDp/RMSEP ratio for NIR and MIR were 3.4 and 1.8, respectively, indicating that the NIR
model has more ability to predict the total solids content.
The MIR model had twice the sensitivity of the NIR model. On the other hand,
the NIR model showed high analytical sensitivity, greater selectivity and low limits of
detection and quantification, where all predicted samples had total solids concentrations
above these values. The parameter SENa-1 shows that 0.0013% is the smallest difference of
concentration that can be distinguished among the samples by the model of NIR. Both models
showed low RSD values indicating little error compared to the average of the predicted
values, being smaller for the model of MIR.
Table 3: Figures of merit obtained for the NIR and MIR models.
Component LV RMSEC RMSEP SEN SENa SENa-1 SEL LD LQ RSD
Near-Infrared
Total solids (%) 5 0.1550 0.1049 0.8315 34.1 0.0293 0.0800 0.097 0.294 0.9
Lactose (%) 2 0.1167 0.0878 0.0099 56.5 0.0177 0.1527 0.07 0.20 2.0
Fats (%) 8 0.0859 0.1029 0.0158 5.7 0.1744 0.0114 0.57 1.74 3.2
Proteins (%) 9 0.0745 0.0726 0.0430 22.9 0.0436 0.0208 0.14 0.44 2.3
Ash (%) 7 0.0065 0.0110 2.0651 372.0 0.0027 0.0006 0.01 0.03 1.4
Mid-Infrared
Total solids (%) 6 0.0762 0.0832 0.0522 9.7 0.1026 0.0004 0.34 1.03 0.7
Lactose (%) 8 0.0965 0.0642 0.0035 3.7 0.2723 0.0001 0.90 2.72 1.5
Fats (%) 8 0.0969 0.0809 0.0043 3.8 0.2660 0.0199 0.87 2.66 2.5
Proteins (%) 8 0.0445 0.0836 0.0004 4.3 0.2315 0.0440 0.76 2.31 2.7
Ash (%) 6 0.0061 0.0099 0.0154 57.7 0.0173 0.0679 0.06 0.17 1.3
LV: Number of latent variables; RMSEC: The Root Mean Square Standard Error of
calibration (%); RMSEP: The Root Mean Square Standard Error of Prediction (%); SEN:
Method sensitivity(A or T x %-1); A: Absorbance units; T: Transmittance units; SENa:
Analytical sensitivity(%-1); SENa-1: inverse of Analytical sensitivity(%); SEL: Selectivity;
LD: Limit of detection (%); LQ: Limit of quantification (%); RSD: Relative standard
deviation (%).
Figure 3 shows the relationship between the predicted values by the models and
the measured values obtained through the reference methods for the calibration and prediction
sets used in NIR and MIR models. The R2cal and R2pred values indicate that the NIR model had
good fitting and prediction ability. Although, the MIR model had presented lower R2pred, as
64
mentioned before, the RMSEC and RMSEP values indicate that this model fits the data well
and has superior prediction ability (Fig. 3- A, B). The biasvalues obtained for both models
show that the average values predicted are closer to the reference values.The R2cal value
obtained in the present study by the NIR model is similar to the R2cal obtained by Jankovská
& Sustová (2003), who developed PLS models for the determination of total solidsin raw
milk. The model for total solids was developed with 6 latent variables, resulting in SEC
(standard error of calibration) of 0.46 (Table S3). Nevertheless, in the study ofJankovská &
Sustová (2003), some important parameters are missing, such as the samples origin, collection
period, number of producers, etc. Besides, it wasn't carried out external validation. Therefore,
those results indicate the model fitting and the potential of NIR for the determination of
parameters in raw milk.
Mazurek et al. (2015)developed a PLS model with FTIR-ATR (4000- 400 cm-1)
for the determination of dry matter in commercial milk samples, including evaporated milk
(type not described). Models had low relative standard errors of calibration (RSEPcal) and
prediction (RSEPval) of 2.36 and 2.47, but higher than the value found in the present study
(0.7%). The model developed by Mazurek et al. (2015) had superior values of R2cal(0.995)
and R2CV(0.990) in comparison with the present study 0.918 (R2cal) and 0.684 (R2pred), but
indicate that the model is overfitted (Table S3). This overfitting may have been caused by the
type of samples used in calibration and prediction sets, since the authors prepared 75 samples
by mixing various commercial milk containing different concentrations of fat, including
evaporated milk. Moreover, authors did not cite the number of factors used in the model, the
other important figures of merit like RMSEC, RMSEP, bias.
3.3.2 Lactose
Among all the calibration models developed in this study, the models for the
determination of lactose presented the best results.The NIR spectral data were preprocessed
by Mean-centering and 1st Savitzky-Golay derivative, and the MIR spectra data were
preprocessed by Multiplicative scatter correction (Table S1), resulting in low RMSEC and
RMSEP values of NIR and MIR models (Table 3) when comparing with the standard
deviations of the HPLC analysis for the calibration and prediction sets (SDc and SDp,
respectively) (Table 2). The MIR model presented lowest RMSEC and RMSEP and a superior
SDp/RMSEP (5.2) than the NIR model (3.9), indicating that the model of MIR has superior
prediction ability. However, the NIR model was considered more robust, because it was
required only 2 latent variables in development (Table S1). The NIR model presented higher
65
values of sensitivity analytical sensitivity, selectivity and lowest SEN-1, limits of detection
and quantification. This shows that the errors associated to the prediction of the lactose
content by the models are low. All samples had levels above the limits of detection and
quantification. Both models showed low RSD values indicating that the values predicted by
the model are closer to the average, being slightly lower for the MIR model.
The plots of predicted values by the modelsversus measured values obtained
through the reference analysis (Fig. 3- C, D) for the calibration and prediction sets show that
both models presented R2cal and R2pred values >0.90. The MIR model presented higher values
of R2cal and R2pred, indicating that this model fits to the data well and has a higher prediction
ability. The bias values indicate that both models had little difference between the average
values predicted by the models and the reference values.
Tsenkova et al. (1999)investigated the potential of near-infrared spectroscopy to
determine the concentration of lactose contents in unhomogenized raw milk. The authors
collected 258 milk samples at different stages of the milking process from three Holstein
cows (during 6 months). The reference analyses for determination of lactose were carried out
in infrared equipment Milko scan (Foss Electric a/s, Hillerød, Denmark). Transmittance
spectra were collected in the range of 14285- 9090 cm-1 in cuvettes with optical paths of 1, 4
and 10 mm, and in the range of 9090- 4166 cm-1 in cuvette with optical path of 1 mm to
evaluate the influence of the path length on the performance. In comparison to our study, the
best model developed by Tsenkova et al. (1999)was constructed with 10 latent variables
(collected in cuvette with 1 mm optical path) and had lower values of R2cal (0.729) and R2CV
(0.685) (Table S3).
In comparison with the present study,Jankovská & Sustová (2003)developed a
PLS model using a superior number of latent variables (11) for the determination of lactose
content in raw milk by NIR, resulting in lower R2cal and SEC (standard error of calibration)
values of 0.87 and 0.08, respectively (Table S3).Aernouts et al. (2011), developed calibration
models for the determination of lactose in raw milk (spectral data of transmittance and
reflectance in different NIR ranges) requiring from 2 to 14 latent variables,resulting in
RMSEP from 0.115 to 0.328, R2pred from 0.05 to 0.883 and bias from -0.049 to 0.095 (Table
S3). The model developed in the present study can be considered more robust, since the best
model (transmittance spectra data in the range of 2500 to 5882 cm-1 preprocessed by first
Savitzky-Golay derivative and OSC), developed by Aernouts et al. (2011)was obtained with a
superior number of latent variables (9), resulting in similar RMSEP (0.115%), lower
R2pred(0.883) and superior bias (-0.043), respectively.
66
Figure 3. Predicted versus measured values plots for calibration (●) and prediction sets (○) for
the parameters of total solids, lactose, fats, proteins and ash by NIR (A, C, E, G and I) and
MIR (B, D, F, H and J). Dashed and solid lines indicate calibration and prediction,
respectively.
67
3.3.3 Fats
For the quantification of fats, the NIR spectral data were preprocessed by
orthogonal signal correction and mean-centering, and the MIR spectral data were
68
preprocessed by Mean-centering (Table S1). In comparison to the mean values and standard
deviations obtained in the reference analyses (SDc and SDp) (Table 2), both models of NIR
and MIR for the determination of fats showed low values of RMSEC and RMSEP (Table 3).
The ratios of SDp/RMSEP of 2.5 and 2.2 for NIR and MIR, respectively, are similar and
indicate that the fat content is well predicted. The NIR model showed lower RMSEC value,
indicating that fits the data better. On the other hand, the MIR model showed lower RMSEP
value, indicating better prediction. The NIR model is more sensitive and has a superior
analytical sensitivity, being possible the distinction between samples with a difference in the
concentration of 0.1744% (SENa-1). In addition, the MIR model is more selective, although
the NIR model presented low limits of detection and quantification, with all samples analyzed
with concentration above LD and LQ.
Both NIR and MIR models were developed with 8 latent variables (Table 3) and
when comparing the plots of predicted values by the models versus measured values obtained
through the reference analysis (Fig. 3- E, F) for the calibration and prediction sets, we
observed the MIR model fits the data worse and its prediction ability is inferior to the NIR
model, which showed lower bias. The fats content in milk samples analyzed in this study is
the quality parameter that more varied in UHT milk samples (coefficient of variation of
8.7%), and, consequently, the parameter that influenced the values of total solids. In this way,
it is also recommended the introduction of new samples to the model, with fat content
between 3.5 and 4.2% to improve the performance.
The present study is similar to the work of Tsenkova et al. (1999)that showed the
potential of near-infrared spectroscopy for the quantification of fats, with a difference, since
those authors applied in unhomogenized raw milk. Although, in comparison with the model
obtained in the present study, the best model developed by Tsenkova et al. (1999) can be
considered as overfitted, since it was developed with 10 latent variables, obtaining high
R2cal(0.998) and R2cv (0.996) values closer to 1.0 (Table S3).
Jankovská & Sustová (2003)developed a PLS model (4 latent variables) using
NIR data for the determination of fats in raw milk and obtained R2cal and SEC (standard error
of calibration) of 0.92 and 0.30, respectively (Table S3). The calibration model developed
byMelfsen et al. (2012)for determination of the fat content (%) using an in-line spectrometer
to predict some quality parameters during the milking process on a single property presented
SEC, R2cal, SEP, R2val and bias values of 0.09, 0.998, 0.09, 0.998 and -0.0004, respectively
(Table S3).
69
Mazurek et al. (2015)developed a PLS model with FTIR-ATR (4000- 400 cm-1)
for the determination of fats in commercial milk samples, including evaporated milk (type not
described). Despite the low values of RSEPcal and RSEPval, and higher values of R2cal(0.995)
and R2CV(0.977) than those found in the present study (0.900 and 0.776, respectively), the
model developed by Mazurek et al. (2015)indicate that the model is over-fitted (Table S3) as
explained in section 3.3.1.Luinge et al. (1993)developed models with lower values of RMSEC
and bias(0.017- 0.028 and 0.001- 0.013, respectively) in comparison to our study (0.0168).
Although, the authors did not cite the period and the number of samples collected in each
location(Table S3).
3.3.4 Proteins
For the quantification of proteins in the present study, the NIR spectral data were
preprocessed by Multiplicative scatter correction, Smoothing and Mean-centering, and the
MIR spectral data were preprocessed by Multiplicative scatter correction, 2nd Savitzky-Golay
derivative and Mean-centering (Table S1). In the determination of protein content, both
models of NIR and MIR showed low values of RMSEC and RMSEP (Table 3) when
compared to the mean values and standard deviations obtained by reference analyses (SDc and
SDp) (Table 2), indicating that the errors associated with the prediction by NIR and MIR are
low. The NIR model presented best fitting and prediction, since it was obtained smaller
RMSEC and RMSEP values, and greater value of the SDp/RMSEP ratio (2.2) than the MIR
model (1.2). The NIR model is more sensible, has more analytical sensibility, being possible
the distinction between samples with a concentration difference of 0.0436% of proteins.
Although less selective, the NIR model presented lower limits of detection and quantification
than the MIR model. Both models showed similar values of RSD, being considered low.
The plots of predicted values by the models versus measured values obtained
through the reference methods for the calibration and prediction sets showed that the NIR
model had superior prediction ability than the MIR model (Fig. 3- G, H). Despite the low
value of R2pred, the NIR model presented low bias, indicating a small difference between
predicted and measured values.Tsenkova et al. (1999)developed PLS models for
determination of protein in unhomogenized raw milk by NIR using 7 to 10 factors, being
obtained SEC (standard error of calibration) from 0.085 to 0.184, R2cal from 0.305 to 0.785,
SECV (standard error of cross-validation) from 0.096 to 0.202, R2CV from 0.149 to 0.719
(Table S3). The best model was developed with 10 latent variables, with data (9090- 4166 cm-
71
1
) preprocessed by first Savitzky-Golay derivative (25 pts), where it was obtained SEC, R2cal,
SECV and R2cv values of 0.085, 0.785, 0.096 and 0.719, respectively.
Jankovská & Sustová (2003)developed a PLS model using the NIR region of
10000- 4000 cm-1 (reflectance mode) to quantify the content of proteins in unhomogenized
raw milk. Authors selected 14 latent variables and the model presented R2cal and SEC
(standard error of calibration) values of 0.97 and 0.05, respectively (Table S3).
Melfsen et al. (2012)developed a calibration model (NIR region of 11750- 6064
cm in diffuse reflection mode) with SEC, R2cal, SEP, R2val and bias values of 0.04, 0.99,
-1
3.3.5 Ash
For the quantification of ash, the NIR model was developed with data
preprocessed by Multiplicative scatter correction and orthogonal signal correction, and the
MIR model was developed with data preprocessed by Mean-centering, 1st Savitzky-Golay
derivative. These models showed similar values of RMSEC and RMSEP (Table 3), being
considered low when compared to the mean values and standard deviations obtained through
the reference analyses (SDc and SDp) (Table 2). The SDp/RMSEP ratio of 1.9 and 2.1 for the
model of NIR and MIR, respectively, shows that the prediction abilities of both are very
similar. The NIR model is more sensible and has more analytical sensibility, being possible
the distinction between samples with a concentration difference of 0.0027% ash. Despite the
lower selectivity, the NIR model showed lower limits of detection and quantification. Both
models showed low values of RSD, demonstrating that the predicted values are close to the
mean values determined by the reference analysis.
The plots of predicted values by the models versus measured values obtained
through the reference methods for the calibration and prediction sets showed both models
(Fig. 3-I, J) had good fitting, but low prediction ability. Although, the bias values show that
for both models the average difference between the predicted and measured values is small. In
order to demonstrate the application of infrared spectroscopy in this field, we show the
performances of models developed for the determination of ash and minerals in dairy products
and milks from other species.
Barabássy (2001)compared the performances of models developed for the
determination of ash in mixed powder milks (mixture of whole milk, skimmed milk, whey
protein concentrate and lactose intolerance), with data obtained from 2 different NIR
spectrometers. Models presented values of standard errors of calibration (0.179- 0.257) and
good R2cal values (0.990- 0.994) and (Table S3).This last parameter can indicate
overfitting.Mouazen et al. (2009)investigated the performances of calibration models
developed with spectral data obtained by 2 different NIR spectrometers for the determination
of ash in ewe milk.When comparing with the present study, the best model developed by
Mouazen et al. (2009) was developed with less number of latent variables, but showed higher
values of RMSEC and RMSEP (0.04% and 0.04%, respectively), and lower values of R2cal
and R2pred (0.83 and 0.80, respectively).These results indicate that the ash content can be
determined by near-infrared spectroscopy (Table S3).
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4. CONCLUSION
ACKNOWLEDGEMENTS
The authors acknowledge the Brazilian funding agency CAPES for the financial
support (PROEX/CAPES #3300301702P1) and National Agricultural Laboratory at
Campinas, São Paulo, for the assistance.
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infrared spectroscopy (2 ed). CRC Press, Boca Raton, Florida, USA.
78
SUPPLEMENTARY MATERIAL
Table S1: Spectral bands and pretreatment used in calibration models for determination of
UHT whole milk components by NIR and MIR.
Component Spectral region (cm-1) Pre-processing
Near-Infrared
Smoothing (order: 2, window: 15 pts),
Total solids (%) 10000- 4000 Orthogonal signal correction, Mean-
centering
9000-8500.5, 7500-7000.5, 5000- Mean-centering, 1st Savitzky-Golay
Lactose (%)
4500.5 derivative
Orthogonal signal correction, Mean-
Fats (%) 6335.5-5375.5
centering
Multiplicative scatter correction,
8600-8400.5, 6400-5800.5, 5600-
Proteins (%) Smoothing (2nd order, 63 pts), Mean-
5400.5, 4800-4400.5
centering
Multiplicative scatter correction,
Ash (%) 10000- 4000
Orthogonal signal correction
Mid-Infrared
3448.63-2952.77, 2250-1903.27,
Total solids (%) Standard normal variate
1498.76-1001.04
3441.17-3349.83, 2975.14-2883.8,
2695.52-2604.18, 2322.7-2231.36,
Lactose (%) Multiplicative scatter correction
1856.67-1765.33, 1390.64-1206.09,
1017.81-833.263, 738.193-646.851
3347.97-3070.21, 2975.14-2883.8,
Fats (%) 1856.67-1765.33, 1577.05-1485.71, Man-centering
1390.64-1019.68
Multiplicative scatter correction, 2nd
2425.22-1920.05, 1780.24-1526.72,
Proteins (%) Savitzky-Golay derivative, Mean-
1196.77-803.437
centering
4000.41-3815.86, 3627.59-3443.04, Mean-centering, 1st Savitzky-Golay
Ash (%)
3068.35-2697.39, 1763.46-646.851 derivative
79
Table S2: Spectral range, pre-processing used in calibration models for determination of milk
components by NIR and MIR found in the literature.
Spectral range (cm-1) Pre-processing Reference
Near-Infrared
Total solids (%)
10000- 4000 nd Jankovská & Sustová, 2003
Lactose (%)
10000- 4000 nd Jankovská & Sustová, 2003
st nd
14285- 9090 Smoothing, 1 and 2 derivative Tsenkova et al., 1999
2nd Savitzky-Golay derivative
25000- 5882 (51pts), Orthogonal signal Aernouts et al., 2011
correction
Kubelka-Munk, 1st Savitzky-Golay
25000- 10000 derivative (51pts), Orthogonal signal
correction
1st Savitzky-Golay derivative(9pts),
10000- 5882
Orthogonal signal correction
1st Savitzky-Golay derivative (39
25000- 4000
pts), Orthogonal signal correction
1st Savitzky-Golay derivative (27
25000- 5882
pts), Orthogonal signal correction
2nd Savitzky-Golay derivative (45
25000- 10000
pts), Orthogonal signal correction
1st Savitzky-Golay derivative (21
10000- 4000
pts), Orthogonal signal correction
1st Savitzky-Golay derivative( 33
10000- 5882
pts), Orthogonal signal correction
1st Savitzky-Golay derivative (51
5882- 4000
pts), Orthogonal signal correction
11750-6064 Normalization Melfsen et al., 2012
Fats (%)
10000- 4000 nd Jankovská & Sustová, 2003
st nd
14285- 9090 Smoothing, 1 and 2 derivative Tsenkova et al., 1999
2nd Savitzky-Golay derivative (47
25000- 5882 Aernouts et al., 2011
pts), Orthogonal signal correction
1st Savitzky-Golay derivative (15
25000- 10000
pts)
1st Savitzky-Golay derivative (27
10000- 5882
pts), Orthogonal signal correction
1st Savitzky-Golay derivative (39
25000- 4000
pts), Orthogonal signal correction
1st Savitzky-Golay derivative (27
25000- 5882
pts), Orthogonal signal correction
25000- 10000 Orthogonal signal correction
10000- 4000 Standard normal variate
st
1 Savitzky-Golay derivative (21
10000- 5882
pts)
nd: not described
80
Carbohydrates (%)
Subtraction of region between
3000- 900 Iñón et al., 2004
2000- 2200 cm-1
4000- 400 Mean-centering, Normalization Mazurek et al., 2015
Subtraction of region between
3000- 900 Iñón et al., 2004
2000- 2200 cm-1
2877- 2827, 1782-1705
4000- 400 Mean-centering, Normalization Mazurek et al., 2015
Subtraction of region between
3000- 1000 Luinge et al., 1993
1700- 1600 cm-1
1480- 1613, 1000-1300 Baseline correction Etzion et al., 2004
1480- 1613, 1200- 1280,
1060- 1100 Etzion et al., 2004
1480- 1613, 1000-1300
1480- 1613, 1000-1300
1480- 1613, 1200- 1280,
1060- 1100
1480- 1613, 1060-1100
Subtraction of region between
3000- 900 Iñón et al., 2004
2000- 2200 cm-1
1701- 1507
4000- 400 Mean-centering, Normalization Mazurek et al., 2015
Subtraction of region between
3000- 1000 Luinge et al., 1993
1700- 1600 cm-1
Calcium (mg/ 100 mL)
Subtraction of region between
3000- 900 Iñón et al., 2004
2000- 2200 cm-1
2877- 2827, 1782-1705,
1701- 1507
Calcium (mg/ kg)
Subtraction of region between
nd Bonfatti et al., 2016
1628- 1658, 3105- 3444 cm-1
nd nd Soyeurt et al., 2009
nd: not described
82
Table S3: Number of samples (n), number of latent variables (LV) and figures of calibration
models for determination of milk components by NIR and MIR found in the literature.
Near-infrared Calibration Prediction Reference
2 2
n LV RMSEC R cal n RMSEP bias R pred
Total solids (%)
Jankovská
50 6 0.46 0.86 nf 0.55* nf 0.80* &Sustová,
2003
Lactose (%)
Tsenkova,
258 7 0.086 0.66 nf 0.092* nf 0.605
et al., 1999
5 0.087 0.65 nf 0.092* nf 0.607
6 0.085 0.67 nf 0.092* nf 0.64
10 0.082 0.69 nf 0.089* nf 0.615
8 0.083 0.68 nf 0.09* nf 0.602
9 0.084 0.67 nf 0.089* nf 0.61
6 0.09 0.71 nf 0.096* nf 0.654
6 0.091 0.7 nf 0.094* nf 0.672
6 0.091 0.7 nf 0.099* nf 0.632
10 0.077 0.73 nf 0.086* nf 0.598
10 0.076 0.73 nf 0.082* nf 0.686
10 0.081 0.71 nf 0.091* nf 0.613
Jankovská
56 11 0.08 0.87 nf 0.15 nf 0.56* &Sustová,
2003
10 Aernouts,
200 2 nf 0.72 0.182 0.059 0.708
0 et al., 2011
3 nf 0.465 0.224 0.02 0.557
2 nf 0.681 0.282 -0.011 0.3
14 nf 0.893 0.164 0.095 0.761
9 nf 0.916 0.115 -0.043 0.883
3 nf 0.453 0.317 0.055 0.111
8 nf 0.886 0.162 -0.011 0.768
3 nf 0.875 0.128 -0.049 0.856
2 nf 0.297 0.328 0.005 0.05
26 Melfsen, et
523 nf 0.05 0.96 0.06 -0.007 0.92
2 al., 2012
Fats (%)
Tsenkova,
258 10 0.31 0.972 nf 0.376 nf 0.996
et al., 1999
9 0.284 0.976 nf 0.347 nf 0.962
10 0.236 0.984 nf 0.262 nf 0.978
10 0.172 0.992 nf 0.213 nf 0.988
10 0.178 0.992 nf 0.203 nf 0.988
nf: data not found; *indicates values obtained by cross validation
83
CAPÍTULO III
Kleidson Brito de Sousa Lobatoa, Lucas Oliveira Montesinob, Rafael Leonardo Zampronib,
Ronei Jesus Poppic, Juliana Azevedo Lima Pallonea*
a
Department of Food Science, School of Food Engineering, University of Campinas,
Monteiro Lobato Street, 80, 13083-862, Campinas, São Paulo, Brazil
b
Laboratory of Physicochemical analyses of foods of animal origin and water, National
Agricultural Laboratory (Lanagro), Raul Ferrari street, 13100-105, Campinas, São Paulo,
Brazil
c
Institute of Chemistry, University of Campinas, 13083-970, Campinas, São Paulo, Brazil
*Corresponding author. Department of Food Science, School of Food Engineering, University
of Campinas, Monteiro Lobato Street, 80, 13083-862, Campinas, São Paulo, Brazil
ABSTRACT
The UHT milk is heavily consumed by Brazilians, it has different properties compared to raw
milk and to our knowledge this is the first study showing the detection and identification of
adulterants by near-infrared (NIR) and mid-infrared (MIR) spectroscopy in UHT whole milk.
A total of 102 samples (29 brands) collected during 1 year were analyzed by reference
methods. The authentic samples were adulterated with water, water + urea, water + starch,
water + sucrose, water + urea + starch, water + urea + sucrose and water + urea + starch +
sucrose in different levels (1, 5, 10, 15 and 20%), resulting in 210 adulterated samples. The
NIR transflectance spectra and the MIR transmittance spectra of authentic and adulterated
samples were used for the development of Principal Component Analysis (PCA) and Partial
Least Squares Discriminant Analysis (PLS-DA). In General, the PCA has shown good
distinction between the authentic and adulterated samples. However, some authentic samples
presented similar scores to the adulterated samples. For the detection of adulteration, the PLS-
DA model using the NIR data (8 latent variables explaining 95.86% of total variance) and
MIR (9 latent variables explaining 89.56% of total variance) distinguished correctly all
authentic and adulterated samples. For the identification of adulterants, after considering the
classes of water, water + urea and water + starch as a single class, all the samples were
correctly classified using NIR, with specificity values (ROC plots) ranging from 84.95 to
100% and sensitivities of 100%. On the other hand, in MIR spectroscopy, with the exception
of authentic samples class, all classes presented misclassification, being obtained specificity
values ranging from 94.15 and 99.37% and sensitivity between 12.35 and 100%. This study
shows that both NIR and MIR feature potential to detect the presence of adulterants in UHT
whole milk. On the other hand, only the NIR spectroscopy has enabled the correct
identification of adulterants in UHT whole milk at levels used in this study.
1. INTRODUCTION
Milk is one of the most produced agricultural product in Brazil(Faostat, 2016) with a highper
capita consumption of 169 kg/year(Ifcn, 2012). The main by-product obtained from milk is
UHT milk, which corresponds to more than 80% of the liquid milk segment(Nogueira,
2014).For being a fairly marketed food, milk is one of the most adulterated ones, and the 2nd
food with a high number of search results about food fraud (143 results) found in research
databases (Web of Science, Pubmed and Google Scholar) (Moore et al., 2012).
Several adulterants can be applied in milk, being water considered as the main,
which is characterized by a fraud with the main purpose to increase the volume of the final
product and consequently the profits. The addition of water causes the dilution of other milk
components. Thus, other measures are taken in order to mask the dilution, like the addition of
urea, starch and sucrose.urea naturally occurs in cow milk (84-280 mg/kg milk), but it is
added to increase the content of Non-Protein Nitrogen (NPN) fraction (Walstra et al., 1999).
The starch has been added to increase the density of milk that has been adulterated with
water.The addition of sucrose is a common practice of adulteration in the dairy industry,
which aims to increase the density of milk and thereby masking the addition of water, such as
in the case of addition of starch(Sharma, 2006).
Analytical detection methods are an important first line of defense for both
detecting and deterring food ingredient fraud(Moore et al., 2012). The development of rapid
analytical methods for food products relies mainly upon two approaches: the use of physical
properties of substrates as an information supply and the automation of chemical methods.
Most rapid analytical methods based on the physical properties of food products are
spectroscopic methods (Dufour, 2009). Those methods can substitute the traditional
techniques for the analysis of adulterants in foods, like near- and mid-infrared Spectroscopy
(NIR), fast analyses with minimum sample preparation that has been successfully applied in
the food industry for many purposes, including the search of adulterants(Moore et al., 2012).
The near-infrared (NIR) region (800–2500 nm or 12 500–4000 cm-1) is the first
spectral region exhibiting absorption bands related to molecule vibrations. This region is
characterized by harmonics and combination bands and is widely used for composition
analyses of food products. The mid-infrared (MIR) region (2500–25 000 nm or 4000–400 cm-
1
) is the main region of vibrational spectroscopy. This region retains information, allowing
organic molecules to be identified and the structure and conformation of molecules such as
proteins, polysaccharides, and lipids to be characterized. In general, the absorption of an
infrared radiation corresponds to an energy change ranging between 2 and 10kcal mol-
1
(Dufour, 2009).
89
We performed a search on Scopus and Web of Science databases using the terms
UHT milk and infrared, resulting in 16 articles by Scopus and 20 articles by Web of
Science. Among them, only 2 were related to the detection of adulterants in UHT milk. In the
first, the authors showed a method to detect the presence of melamine (>100 ppm) spiked in
different levels (100 ppm- 10,000 ppm) in UHT milk by MIR and the technique of local
Moving Window PCA(Fernández Pierna et al., 2016). In the second study, the authors
investigated the detection of water (5, 10, 20, 30 and 40%), urea (1, 2, 3, 4 and 5 g/L) and
formaldehyde (1, 2, 3, 4 and 5 g/L) in UHT milk by NIR(Luna et al., 2016). To date no work
involving the detection of adulterants in milk in Brazil by NIR or MIR was based on the
proportions adopted by fraudsters (Bertão, 2014; Mprs, 2013, 2017). Thus, taking into
account the importance of UHT milk and the need analytical methods for the detection of
adulterants, this study aims to develop classification models for the detection of adulteration
with common adulterants found in Brazilian UHT whole milk.
2.1 Materials
urea (>99%) was purchased from Sigma Aldrich (Saint Louis, Missouri, USA).
Cornstarch (Maizena®, Unilever Bestfoods, Mogi Guaçu, São Paulo, Brazil) and sucrose
(Caravelas, Usina Colombo S/A Açúcar e Álcool, Ariranha, São Paulo, Brazil) were obtained
on the local Market of Campinas, São Paulo.
2.2 Samples
A total of 102 commercial UHT milk samples (29 brands) of different lots were
periodically (around 8 samples/month) acquired in the supermarkets in the city of Campinas-
SP during 1 year (September 2015 to September 2016). Samples are sourced from producers
in the Brazilian States of Bahia (5), Goiás (2), Minas Gerais (3), Rio Grande do Sul (12),
Santa Catarina (10) and São Paulo (70). Samples were immediately taken to the Laboratory of
Products of Animal Origin(LANAGRO- National Agricultural Laboratory- Campinas, São
Paulo) for the investigation of the presence of adulterants and the collection of NIR spectra.
Then, 100 mL of each sample were stored in PET bottles for the collection of the MIR spectra
in the laboratory of Infrared spectroscopy and Raman of the Institute of Chemistry of the
University of Campinas (Campinas, São Paulo).
90
‘batch) of that brand were analyzed and presented mean levels of total solids, lactose, proteins
and fats of 12.83 ± 0.05 %, 4.50 ± 0.07 %, 3.25 ± 0.002 %, 3.95 ± 0.07 %, respectively.
The proportion of water used for the adulteration was based in the investigations
during the phases of operation called Leite Compen$ado organized by the Public Ministry of
Rio Grande do Sul, where it was verified that in relation to the quantity of milk, the water was
the main adulterant applied, in the proportions of water:milk of 1:12.9 (Mprs, 2013), 1:10
(Bertão, 2014) and 1:9 (Mprs, 2017). In the present study we added urea to compensate the
reduction of proteins, and cornstarch and sucrose to compensate de reduction of lactose.
In the present study, samples were adulterated through the addition ofa “mix” to
100 ml of authentic milk (Table 1). Each mix was added separately in different volumes(1, 5,
10, 15 and 20 ml).For example, in the preparation of the samples containing 1% of mix 1, we
added 1 mL of the mix 1 to 100 mL of authentic milk, totaling 101 mLof adulterated milk.
Each sample was prepared in sextuplicate, totaling 210 adulterated samples.
The concentration of urea to be added in the mixes 2, 5, 6 and 7 was calculated
taking into account the nitrogen content in authentic milk (0.51%) that was determined by the
Kjeldahl method. We considered that urea contains 46.67% of nitrogen. Mixtures containing
cornstarch (3, 5 and 7) were produced using a solution of cornstarch in water. This solution
was heated to promote starch gelatinization, in order to simulate the heating step where milk
is submitted during the UHT milk production. The mixes containing starch (3 and 5) and
sucrose (4 and 6) were prepared in the same concentration of lactose in authentic milk (4.5%).
The 7th mix was prepared using starch and sucrose at concentrations of 2.25% and 2.25%,
respectively. This approach was taken in order to produce an adulterated milk containing a
sum of starch and sucrose in a final concentration of 4.5%.
(ATR) accessory. Each sample was homogenized and scanned in triplicate (4000 to 400 cm-1,
increment of 1.864 cm-1, resolution of 4 cm-1 and 64 scans/spectra). Between the scanning of
each sample, the cell was cleaned with ethanol (99%). The averaged spectrum was used for
the development of multivariate analyses.
The exploratory analysis of the NIR and MIR spectra of all samples was
performed through the Principal Component Analysisusing the Singular Value
Decomposition (SVD) algorithm with a confidence level of 0.95 for Q and T2. The number of
principal components for each model was chosen by cross-validation (venetian blinds method
with 10 data splits) through the analysis of the evaluation of cumulative variance (%), Root
Mean Squared Error of Calibration (RMSEC) and Root Mean Squared Error of Cross-
Validation (RMSECV).
This analysis was carried out by the method of Partial Least Squares Discriminant
Analysis (PLS-DA) using the SIMPLS algorithm and the NIR and MIR spectra data
separately. Models were validated using an external group (prediction set)and the number of
latent variables was chosen through the evaluation of sensitivity, specificity, classification
error of calibration and prediction. The samples that were considered as outliers were
identified and removed when necessary by Hotelling’s T2 x Qresidual plot (confidence level
of 95%).Thus, different numbers of authentic samples were used in the development of the
models. For the NIR model, 77 authentic samples were used in the calibration set and 21
authentic samples were used in the prediction set. For the MIR model, 79 authentic samples
were used in the calibration set and 22 authentic samples in the prediction set, respectively.
For both NIR and MIR models, 20 samples of each adulterant mixturewere used in the
calibration set (4 samples at each adulteration level: 1, 5, 10, 15 and 20%), totaling 140
94
adulterated samples. In the prediction set, 10 samples of each mixture of adulterants were
used (2 samples at each adulteration level), totaling 70 adulterated samples.
For the identification of adulterants, the PLS-DA was also applied in the same
conditions demonstrated in the section 2.6.2. Although, it was established 1 class for authentic
samples, and 7 classes for adulterated samples based in the type of adulterant: water,
water+urea, water+starch, water+sucrose, water+urea+starch, water+urea+sucrose,
water+urea+starch+sucrose. Similarly to the section 2.6.2, samples were divided into
calibration set, which was composed of 80 authentic samples and 140 adulterated samples,
being 4 samples in each level of adulteration (1, 5, 10, 15 and 20%). The prediction setwas
composed of 22 authentic samples and 70 adulterated samples (2 samples in each level of
adulteration: 1, 5, 10, 15 and 20%).
According toBlanco & Bernárdez (2009)the samples in the calibration set must
contain all sources of variation from samples that will be predicted in the future. In the present
study, we collected 102 samples in order to have samples with all the sources of variation
inherent to this type of product. The raw NIR and MIR spectra (without pre-processing) of
102 UHT whole milk samples are shown in Figure 1. The NIR spectra showed an offset in
coordinate axis and an inclination of the baseline as decreases the wavenumber (Fig. 1- A).
The MIR spectra are almost overlapped, with little offset and slope of the baseline (Fig. 1- B).
The absorption bands observed in the NIR range (Fig. 1- A) occur due to the
presence of overtones, which tend to be weak in intensity compared to the same effect in the
MIR region. Although, this can be considered as an advantage, since absorption bands that
have sufficient intensity to be observed in the NIR region arise primarily from functional
groups that have the hydrogen atom attached to carbon, nitrogen or oxygen, which are
common groups in the major constituents of water, proteins, lipids, and
carbohydrates(Wehling, 1998).
95
Figure 1.Raw (without pre-processing) NIR (A) and MIR (B) spectra of authentic and
adulterated samples.
The milk NIR spectrum is very similar to the spectrum of water, since milk is
made up of more than 80% water (McClure & Stanfield, 2006). The main bands exhibited by
water in the NIR region can be found at 6934 cm-1, which is typical of the first harmonic of
νOH vibration, and at 5176 cm-1 which is related to the combination of νOH + δOH (overtone
of O-H bending)(Dufour, 2009). These bands feature a great contribution to the success of
NIR to study and measure water in foods (Workman & Weyer, 2012).
Other major compounds present in milk also feature specific bands in the near-
infrared spectrum. For example, carbohydrates, in general, may have an absorption band near
6940 cm-1caused by a free OH stretch. Proteins bands can be at 10277 to 9804 cm-1which is a
result of aN-H stretch second overtone. Moreover, proteins bands can be found in different
ranges, like the range from 6667 to 6536 cm-1, which is a result of N-H stretching first
overtone, 4878 to 4854 cm-1caused by N-H stretching combinations, 4613 to 4587 cm-1
caused by N-H bend second overtone and C=O stretch-N-H in-plane bending-C-N stretch
combination bands(Workman & Weyer, 2012).
Milk fats bands can be found in different ranges, like: from 5780 to 5624 cm-
1
(assigned to first overtone CH2 stretching vibrations) and 4325 cm-1 and 4248 cm-1 (assigned
to the combination stretching and bending of CH2 and CH3)(McClure & Stanfield, 2006). As
well as in the near-infrared region, in the mid-infrared region, water also displays intense and
broad bands that can be characteristic of high moisture samples, such as milk. In the MIR
region, the water features two main absorption bands, being the first located at 3300 cm-1,
corresponding to νOH, and the second located at 1638 cm-1, corresponding to a less intense
band caused by bending δOH (Fig. 1- B). Carbohydrates exhibit four main zones of
absorbance, butspecifically in relation to lactose, the main bands observed in the MIR region
96
are located in the range from 1167 to 989 cm-1. Proteins are made of amino acids joined by
peptide bonds C-NH (Amide I, II and III), considered as the main responsible for the
absorption occurring between 1700 and 1500cm-1. Those bands are known to be sensitive to
the conformation of protein backbone. The acyl chain of fatty acids is mainly responsible for
the absorption observed between 3000 and 2800cm-1. Moreover, the triacylglycerols ester
linkage C-O (1175 cm-1), CO (1750 cm-1) group, and acyl chain C-H (3000- 2800cm-1)
wavenumbers are commonly used to determine fat(Dufour, 2009).
3.2.1 Near-Infrared
Figure 2. Pre-processed NIR spectra of authentic and adulterated samples (A), RMSEC,
RMSECV, Cumulative variance (%) (B), Scores plot (C) and Loadings plot (D) obtained
during the Principal Components Analysis.
3.2.2 Mid-Infrared
principal component, being highlighted the ranges between 3500 and 3000 cm-1, and 1750
and 750 cm-1.
Figure 3. Pre-processed MIR spectra of authentic and adulterated samples (A), RMSEC,
RMSECV, Cumulative variance (%) (B), Scores plot (C) and Loadings plots (D) obtained
during the Principal Components Analysis.
Although the band in the region of 400 cm-1 contributed enough to the formation
of the component 2, it must have been caused by noise, as can be seen in Figure 1-B. In
addition, in previous experiments (data not shown) we verified that by increasing the number
of principal components from 2 to 6, increased the contribution in the region of 4000 cm-1, a
common behavior, since the greater the number of principal components, the greater the
amount of residue added to the model. Another fact that corroborates our assertion about the
noise is in high residue presented in the region of 400 cm-1(Fig.S1 in the Supplementary
Materials). In order to improve the separation of the samples in the scores plot, we tested the
exclusion of some spectrum bands including the region around 400 cm-1(data not shown),
considering the pre-processed data and the loadings, but it was not obtained better results,
99
because again some authentic samples were located in the same quadrants of adulterated
samples. Thus, we kept all the range from 4000 to 400 cm-1 for the Principal Component
Analysis.
3.3.1 Near-Infrared
The PLS-DA model was constructed with data pre-processedby 2nd Savitzky-
Golay derivative (2nd order, 25 points) followed by mean centering, with 8 latent variables
selected by cross-validation (venetian blinds with 10 splits), that explained a total variance of
63.79% of the spectral data (matrix X) and 95.86% of the class data (matrix Y) and showed an
average rating error of 0 for cross-validation and calibration (Fig. 4-A).Figure 4-B shows the
classification of samples (represented by triangles) in authentic class (class 1) and adulterated
(class 2). All authentic samples (black triangles) and adulterated samples (red triangles) used
in the construction of the model and on external validation were correctly classified without
the occurrence of false positive or negative results in both classes. Figure 4 C shows the
ranges of spectra that most contributed to the formation of 8 latent variables, being
highlighted the regions of 7500- 4500 cm-1. The bands near 10000 cm-1 and 4000 cm-1 can be
a result of the pre-processing by 2nd Savitzky-Golay derivative.
Figure 4. Spectral cumulative Variance (%), Calibration Classification Error Average, Cross-
Validation Classification Error Average (A), Most Probable Classification for calibration and
prediction phases (B), Loadings (C) obtained by NIR and PLS-DA.
Positives + False Negatives and TNR= True Negatives/ True Negatives+ False Positives,
which are commonly known as sensitivity and specificity, respectively.It is possible to plot
sensitivity versus specificity, making it possible to accommodate all possible thresholds.
Although, the plot of sensitivity versus one minus specificity is more appealing, called as
Receiver Operating Characteristic Curve (ROC), which is a statistical tool that summarizes
the accuracy of prediction method(Gönen, 2007). In statistical terms, a ROC curve shows the
relationship between the alpha error (probability of obtaining a result false positive) and the
beta error (probability of obtaining a false negative result), allowing the estimation of the
performance of a test in a wide range of decision thresholds(Massart et al., 1997). In this
study we found that the authentic and adulterated samples in the prediction set were correctly
classified with high specificity and sensitivity of 100% and 97.28%, respectively (Fig. S2 in
the Supplementary Materials). These results show that it is possible to detect samples
containing the adulterants applied in this study using NIR spectroscopy and PLS-DA.
The Selectivity Ratio (Fig. S3- A in the Supplementary Materials) is defined as
the relationship between the portion of the total variance explained by the bilinear model
predictors and their corresponding residual variance. The VIP Scores plot (Fig. S3- B in the
Supplementary Materials) shows how a variable is relevant to explain the variation in the X
matrix (spectra) and its correlation with the Y matrix (classes, in our case). The variables with
VIP scores values near or greater than 1 can be considered as important for the model.
Normally, the limit of significance chosen is 1, because the mean of the squared VIP scores
over the variables is 1 (Westad et al., 2013). The Table S1 shows the bands in the NIR region
associated with the main compounds present in UHT milk samples analyzed in this study
emphasized by PLS-DA through the Selectivity ratio and VIP Scores plots (Figure 3-A, B).
In the same way as in the loadings obtained through the PCA analysis, the
loadings from the PLS-DA in this phase of the work also presented high-intensity at the
boundaries of the spectrum (10000 and 4000 cm-1), which can also be caused by treatment of
second derivative (Fig. 4- C), as mentioned before in the section 3.2.1 . The selectivity Ratio
plot (Fig. S3- A in the Supplementary Materials) and the Qresiduals plot (Fig. S3- C in the
Supplementary Materials) show that the bands from 10000- 9988 cm -1 and 4050- 4000 have
little relevance to the model, despite having high value in VIP Scores plot (Fig. S3- B in the
Supplementary Materials).We kept these bands, since the exclusion (data not shown) did not
improve the performance of the model.
101
3.3.2 Mid-Infrared
Figure 5. Spectral cumulative Variance (%), Calibration Classification Error Average, Cross-
Validation Classification Error Average (A), Most Probable Classification for calibration and
prediction phases (B) obtained by MIR and PLS-DA.
All authentic samples (class 1) and adulterated samples (class 2) from the
calibration and prediction sets were correctly classified (Fig. 5- B). The authentic and
adulterated samples in the prediction set were classified with high specificity and sensitivity
of 99.72% and 100%, respectively (Fig. S4 in the Supplementary Materials). These results
show that it is possible to detect an adulterated sample containing the adulterants used in this
study by MIR and PLS-DA.
The Supplementary Table 2 shows the main bands in the MIR associated to the
main compounds present in UHT milk samples analyzed in this study emphasized by the
PLS-DA through the plot of Selectivity ratio VIP Scores (Figure S5- A, B). Although the VIP
scores above 1, the range between 420 and 400 cm-1 can also be considered as a result of
noise, as shown by the high values of Qresiduals values in Figure S5- C in the Supplementary
Materials. This occurred due to the number of latent variables. We tested the model with the
low number of latent variables (data not shown) and the VIP scores of 420-400 cm-
102
1
decreased. The VIP scores of the bands of 3430-2832 cm-1 and 1760-910 cm -1 practically did
not change with the decrease in the number of latent variables (data not shown). As explained
in the section 3.2.2, since the greater the number of latent variables, the greater the amount of
residue added to the model.
The identification of the type of adulterant present in the samples was carried out
through the development of a PLS-DA model. Therefore, each group of samples was
considered as a class, resulting in 8 classes: authentic, water, water + urea, water + starch,
water + sucrose, water + urea + starch, water + urea + sucrose, water + urea + starch +
sucrose.
3.4.1 Near-Infrared
Among the pre-processing that we tested, the best model was developed using
data preprocessed by the treatment of mean centering and 1st Savitzky-Golay derivative (2nd
order, 11pts). The model was constructed with 7 latent variables that explained a total
variance of 50.41 % of the spectral data (matrix X) and 47.66 % of the class data (matrix Y),
being obtained average errors of classification of 0.0412 and 0.0479 for calibration and cross-
validation, respectively (Fig. 6- A). None authentic samples were classified as adulterated,
and no adulterated samples were classified as authentic samples. However, some samples of
the classes of water, water + urea and water + starch were incorrectly classified. The other
classes were also correctly classified (Figure 6-B).
Some samples from the calibration set were incorrectly classified. 7 samples
adulterated with water (5%, 5%, 10%, 15%, 15%, 15% and 20%) were classified as water +
urea, and 5 samples (1%, 1%, 1%, 1% and 10%) were classified as water + starch. From the
20 samples adulterated with water + urea, 1 sample (15%) was classified as water, and
1sample (10%) was classified as water + starch. Of the 20 samples adulterated with water +
starch, 1 sample (20%) was classified as water, and 1 sample (20%) was classified as water +
urea. Several samples from the prediction set belonging to the classes of water, water + urea
and water + starch were incorrectly classified(Fig. 6-B).In order to improve the model's
performance, other pre-processing (described in section 2.6) and the combination of them
were tested along with the selection of variables. However, they were not obtained better
results than those that were presented. These results show that it is difficult to distinguish the
samples adulterated with water, water + urea and water + starch, which can be verified on the
103
similarity of the plots of Selectivity Ratio (Fig. S6- A in the Supplementary Materials) and
VIP Scores (Fig.S6- B in the Supplementary Materials).
Figure 6. Spectral cumulative Variance (%), Calibration Classification Error Average, Cross-
Validation Classification Error Average (A), Most Probable Classification for calibration and
prediction phases (B) obtained by NIR and PLS-DA.
Taking into account the results, a new model was developed with a difference: the
classes of water, water + urea, water + starch were considered as a single class called as
water- water + urea- water + starch. Thus, 60 samples of this new class were used in the
calibration set and 30 samples in the prediction set. In that way, all samples were divided into
six classes: authentic, water-water+urea-water+starch, water + sucrose, water + urea + starch,
water + urea + sucrose, and water + urea + starch + sucrose. The data were pre-processed by
mean centering followed by 1st Savitzky-Golay derivative (2nd order, 11pts), being chosen 7
latent variables that explained a total variance of 50.35 % of the spectral data (matrix X) and
81.87% of the class data (matrix Y), resulting in average errors of classification of 0.0142 and
0.01612 for calibration and cross-validation, respectively (Figure 7- A).
Figure 7-B shows that all the samples were correctly classified. Despite not
having achieved the initial aim to classify properly all samples in 8 classes, it is possible to
affirm that the unknown samples which were classified in class water-water+urea-
water+starch contains at least water, or water and urea, or water and starch. If a sample is
classified as water-water+urea-water+starch, the presence of starch can be investigated by the
reference analysis. If the result is negative, the sample was adulterated at least with water or
water + urea. Anyway, being classified in this class, the sample is considered adulterated,
regardless the type of adulterant present.In addition, as previously mentioned, the adulteration
verified in Brazil occur by adding at least 3 adulterants that may bewater + urea+ starch
104
orwater + urea + sucrose, which would be the classes 4 (yellow) and 5 (cyan), respectively.
Thus, our work shows that it would be possible to identify the adulteration by NIR and PLS-
DA. Moreover, we verified that it is possible the identification of the other classes of
adulterated samples: water + sucrose, water + urea + starch and water + urea + starch +
sucrose. It is noteworthy that this last class had concentration of starch and sucrose adulterants
less than the other samples.
Figure 7. Spectral cumulative Variance (%), Calibration Classification Error Average, Cross-
Validation Classification Error Average (A), Most Probable Classification for calibration and
prediction phases (B) obtained by NIR and PLS-DA.
Samples were scanned in a FT-NIR spectrometer in the range of 10000- 4000 cm-1 (1100-
2500 nm) in reflectance mode. The authors verified that the discrimination was carried out for
all samples together independent of the level of adulteration. Although, the amount of urea
did not correspond to the amount required to compensate the dilution of proteins. Moreover,
the authors divided the samples into groups of training (120) and prediction (117) using the
algorithm ofKennard & Stone (1969), however, it is not clear if the classification models
(SIMCA, SVM-DA, PLS-DA) were developed with adequate number of authentic and
adulterated samples. The authors did not show the figures of merit for authentic samples,
which is a result of great importance, considering that if authentic samples are classified as
adulterated, it would consists as a false positive result. On the other hand, if adulterated
samples are classified as authentic, it consists of false negative results. Figures of merit show
that no classification model presented 100% of sensitivity (48- 100%) and specificity (43-
100%) for the classes of water, formaldehyde and urea.
Liu et al. (2011)investigated the use of classification models (SIMCA and PLS-
DA) developed with NIR data to detect adulterants in raw milk. The SIMCA models showed
69.2 69.2%, 64.7 of correct classification in the prediction for the samples adulterated with
urea (1- 2000 mg/dL), glucose (1- 2000 mg/dL) and melamine (0.8- 300 mg/dL), respectively.
The comparison was performed separately among the adulterated and authentic samples,
which showed correct classification ranging between 61.5 and 100%. The PLS models
developed comparing the pairs of adulterated and authentic samples showed correct
classification of 69.2, 47.1 and 18.4% for the class of adulterated samples with urea, glucose
and melamine, respectively.The authentic samples were classified with a correct classification
ranging from 18.4 to 92.3%. When authors analyzed all the samples together, correct
classification rates ranged from 75, 50, 80 and 93.3% for the classes of urea, glucose,
melamine and authentic, respectively. The authors concluded that NIR and PLS-DA can be
used to identify the presence of adulterants in milk. Although, since they did not add water to
increase the volume of milk, the models developed could be indicated in cases where milk
quality is low and the fraudsters commonly add adulterants to mask the low levels of milk
components. However, the authors did not analyze the nitrogen content of the samples to
verify if the amounts of added urea and melamine were sufficient to only correct the nitrogen
content or if they exceeded the normal levels of nitrogen in milk. In both SIMCA and PLS-
DA models, the class of authentic samples had a correct classification rate <100%, indicating
false positive results.
106
3.4.2 Mid-Infrared
Figure 8. Spectral cumulative Variance (%), Calibration Classification Error Average, Cross-
Validation Classification Error Average (A), Most Probable Classification obtained for
calibration and prediction phases (B) obtained by MIR and PLS-DA.
In this study we found that samples from the classes of Authentic, water,
water+urea, water + starch, water + sucrose, water + urea + starch, water + urea + sucrose e
water + urea + sucrose were classified with specificity of 99.37%, 99.04%, 94.15%, 98.78%,
97.56%, 98.78%, 96.09%, 98.38%, and sensitivity of 100%, 70.00%, 50.00, 80.85, 77.42%,
12.35%, 50.00% and 20.00%, respectively (Fig. S8 in the Supplementary Materials). These
107
results show that no class presented simultaneously specificity and sensitivity values ≥90%.
We give emphasis to low the sensitivity values obtained primarily for water + urea, water +
urea + starch, water + urea + sucrose e water + urea + starch + sucrose, which indicate high
probability of occurrence of the type II error (false negative), indicating the difficulty in
identifying adulterants in this study using MIR spectroscopy and PLS-DA.
Botelho et al. (2015)proposed a new screening method for the simultaneous
detection of five common adulterants in raw cow milk by using attenuated total reflectance
(ATR) mid-infrared spectroscopy and multivariate supervised classification (partial least
squares discrimination analysis – PLS-DA). The authors collected 15 samples of milk and
produced 71 adulterated samples containing from 2 to 5 adulterants (water, formaldehyde,
sodium citrate, starch and sucrose) in concentrations ranging from 0.5 to 10%. A multivariate
qualitative validation was performed, estimating specific figures of merit, such as false
positive and false negative rates, selectivity, specificity and efficiency rates, accordance and
concordance. The authors found that in the prediction set, only the samples adulterated with
sodium citrate presented 100% of sensitivity and specificity. The other classes showed
sensitivity and specificity ranging from 88.5-100% and 75.3-100%, respectively, indicating
the rate of false positive and negative results. Samples containing water, starch, formaldehyde
and sucrose were wrongly classified, which may have been caused due to the low
concentration of adulterants.
Santos et al. (2013)applied the attenuated total reflectance mid-infrared
microspectroscopy as a rapid method for detection and quantification of milk adulteration.
The authors collected 20 samples of 1 brand (10 different lots in duplicate) and produced 310
adulterated samples containing different levels of whey, urea, hydrogen peroxide, synthetic
urine and synthetic milk (vegetable oils emulsified with detergents and urea). By investigating
the detection of adulterants, it was possible to correctly classify authentic and adulterated
samples. However, when comparing all classes of samples, they obtained correct
classification percentages ranging from 90% (authentic samples) to 98% (samples containing
urea). Moreover, about 3% of the samples were not classified in any class.
The exploratory analysis (PCA) using NIR data exhibited a good separation of the
samples. However, 6 authentic samples were located in the same quadrants of adulterated
samples. In the case of MIR data, it was also verified a good separation between the samples,
but 12 samples were located in the same quadrants of adulterated samples. On detection of
108
adulteration utilizing the model PLS-DA, both the NIR and MIR classified correctly all the
samples, and the latter were obtained higher values for specificity and sensitivity (99.72% and
100%, respectively) via the ROC plots. Thus, both NIR and MIR techniques, along with the
PLS-DA can be used in the detection of adulteration in UHT milk.
For the identification of adulterants in the first NIR data modeling, samples of
water, water+urea and water+starch have been erroneously classified among themselves.
When we considered these samples as a single class (water-water+urea-water+starch), all
samples were correctly classified, being obtained specificity rates ranging from 84.95% to
99.92% and sensitivity of 100% for all classes. In identifying adulterants using MIR data, the
samples from the calibration set were correctly classified. However, in the prediction set, with
the exception of authentic samples, all the other classes have been erroneously classified,
resulting in lower values when compared to the results obtained for the NIR, with specificity
ranging from 94.15% to 99.37% and sensitivity ranging from 12.35% to 100%. Then, we
concluded that the NIR spectroscopy coupled to PLS-DA can be applied in identification of
adulterants in UHT milk.
water + urea + starch + sucrose were correctly classified in both the calibration and prediction
set (section 3.4.1).
Figure 9.Investigation of starch in Adulterated Samples. BL: Blank; W+ST: water + starch;
W+U+ST: water + urea + starch; W+U+ST+SU: water + urea + starch + sucrose.
Figure 10- A shows the results obtained during the investigation of sucrose.
Samples containing sucrose showed a red color, which can be checked in the sample with
water + sucrose (second tube), water + urea + sucrose (third tube). During the analysis of the
sample with water + urea + starch + sucrose (fourth tube), it was not possible to detect the
presence of sucrose. This may be due to the low concentration of sucrose in the sample.
Furthermore, the method for detection of sucrose requires 1 mL of sample during analysis.
Thus, we increased the volume of added sample during analysis from 1 mL to 5, 10, 15 and
20 mL, being possible the detection of sucrose from the samples with 15 and 20 mL, i.e. it is
not possible to detect the presence of sucrose by the reference method using 1, 5 and 10 mLof
samples (Figure 10- B).
4. CONCLUSION
In this work we investigated the use of near- and mid-infrared spectroscopy, along
with PCA and PLS-DA for the detection and identification of water, water+urea,
water+starch, water+sucrose, water+urea + starch, water+urea+sucrose, and
water+urea+starch+sucrose at concentrations used in adulteration of UHT milk. It was
possible to distinguish authentic (without adulterants) and adulterated samples through the
PCA using NIR or MIR data.For the identification of the type of adulterant, using the NIR
data when considering the classes of water, water+urea e water+starch as a single class, all the
samples were correctly classified in the calibration and prediction sets. On the other hand,
when using MIR data even after the use of different ranges and different pre-processing, it
was not obtained a better performance. The best model was obtained with data pre-processed
by mean centering. However, only the calibration set samples were correctly classified. When
compared to reference methods, the NIR method presented similar performance in detecting
the presence of starch and higher in the detection of sucrose in the sample with water + urea +
starch + sucrose. These results show that both NIR and MIR can be applied in the detection of
adulteration. On the other hand, for the identification of the type of adulterant, it is
recommended to use the NIR spectroscopy. Our study suggests that the combination of NIR
spectroscopy and chemometrics is an effective, non-destructive and rapid method for
detection and identification of adulterants in UHT whole milk.
ACKNOWLEDGEMENTS
The authors acknowledge the Brazilian funding agency CAPES for the financial
support (PROEX/CAPES #3300301702P1) and National Agricultural Laboratory at
Campinas- São Paulo, for the assistance.
111
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Santos, P.M., Pereira-Filho, E.R., Rodriguez-Saona, L.E. (2013). Rapid detection and
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SUPPLEMENTARY MATERIAL
Figure S1. Qresiduals obtained during the Principal Components Analysis of MIR spectra of
authentic and adulterated samples.
Figure S2. Predicted ROC Curve (A) and Predicted Responses (B) for the obtained PLS-DA
model of the NIR Spectra for the classification of authentic and adulterated samples.
115
Figure S3. Selectivity ratio (A), VIP Scores (B) and Qresiduals (C) obtained by PLS-DA model
of the NIR spectra.
Figure S4. Predicted ROC Curve (A) and Predicted Responses (B) for the obtained PLS-DA
model of the MIR spectra for the classification of authentic and adulterated samples.
Figure S5. Selectivity ratio (A), VIP Scores (B) and Qresiduals (C) obtained by PLS-DA model
of the MIR Spectra.
116
Figure S6. Selectivity ratio (A), VIP Scores (B) obtained by PLS-DA model of the NIR
Spectra for the classes of water, water+urea and water+starch.
117
Figure S7. Predicted ROC Curves and Predicted Responses for the obtained PLS-DA model
of the NIR Spectra for the classification of authentic and adulterated samples.
118
Figure S8. Predicted ROC Curves and Predicted Responses for the obtained PLS-DA model
of the MIR Spectra for the classification of authentic and adulterated samples.
119
Table S1. Bands Associated with the authentic and adulterated UHT milk samples in the
present study by NIR. Source: Workman & Weyer (2012) and Workman Jr (2001).
Absorption bands of Band location (cm-1) Reported bands assignments
Lipids 7067 O-H stretch 1st overtone
Amide 5208 C=O (3ν), C=ONH
O-H assigned to molecular water O-H
O-H molecular water 5200 stretching and HOH deformation
combination
C-H (2νCH2 Asymmetric stretching
Hydrocarbons, aliphatic 4335
and δCH2) combination
C-H stretching and CH2 deformation
Glucose and Saccharide 4307
combination
C-H stretching and CH2 deformation
Polysaccharides(glucose) 4307
combination
C-H stretching and CH2 deformation
Glucose and Saccharide 4292
combination
starches and Sugars as C-H C-H stretching and CH2 deformation
4386-4286
and O-H related bands combination
Glucose and Saccharide 4252 C-H (3δ)
Polysaccharides 4252 C-H (3δ)
C-H (2νCH2 symmetric stretching and
Hydrocarbons, aliphatic 4232
δCH2) combination
Hydrocarbons, aliphatic 4068 C-H (3δ CH3)
Table S2. Bands associated with the authentic and adulterated UHT milk samples in the
present study by MIR. Source:Stuart (2005) and Workman & Weyer (2012).
Absorption bands of Band location (cm-1) Reported bands assignments
Peptide groups 2925 CH2 antisymmetric stretching
Peptide groups 2853 CH2 symmetric stretching
Lipids 2850 CH2 symmetric stretching
80% C=O stretching; 10% C–N stretching;
Proteins 1653
10% N–H bending
Proteins 1567 60% N–H bending; 40% C–N stretching
Peptide groups 1550 -NCHO- group vibration
Lipids 1468 CH2 scissoring
C-N stretch coupled with NH2 deformation
urea 1467
(amide III)
urea 1150 NH2 wagging
REFERENCES
Stuart, B., (2005). Infrared Spectroscopy: Fundamentals and Applications. John Wiley &
Sons, Hoboken, EUA.
Workman, J.J., Weyer, L., (2012). Practical guide and spectral atlas for interpretive near-
infrared spectroscopy (2 ed). CRC Press, Boca Raton, Florida, USA.
Workman Jr, J., (2001). The Handbook of Organic Compounds. Academic Press, Burlington.
120
CAPÍTULO IV
Kleidson Brito de Sousa Lobatoa, Priscila Domingues Alamara, Elem Tamirys dos Santos
Caramêsa, Juliana Azevedo Lima Pallonea*.
a
Department of Food Science, School of Food Engineering, University of Campinas,
Monteiro Lobato Street, 80, 13083-862, Campinas, São Paulo, Brazil
*Corresponding author. Department of Food Science, School of Food Engineering, University
of Campinas, Monteiro Lobato Street, 80, 13083-862, Campinas, São Paulo, Brazil
Kleidson Brito de Sousa Lobato, Priscila Domingues Alamar, Elem Tamirys dos Santos
Caramês, Juliana Azevedo Lima Pallone. Authenticity of freeze-dried açai pulp by near-
infrared spectroscopy,Journal of Food Engineering,Volume 224,2018, Pages 105-111.
ISSN 0260-8774,
https://doi.org/10.1016/j.jfoodeng.2017.12.019.
121
122
123
124
125
126
127
128
SUPPLEMENTARY MATERIAL
Supplementary Table 1: Statistics of the SIMCA models obtained for 2 and 6 classes.
Pre-processing Class PC CV (%) RMSEC RMSECV
2 Classes
MC Authentic 3 99.43 2.05x10-3 1.30x10-1
Adulterated 11 100.00 4.53x10-4 4.05x10-2
-2
SNV Authentic 2 99.99 1.13x10 6.85x10-1
-3
Adulterated 11 100.00 2.44x10 2.09x10-1
MSC Authentic 2 100.00 2.13x10-3 1.29x10-1
-3
Adulterated 4 100.00 3.19x10 2.04x10-1
2SGD Authentic 6 99.97 1.08x10-6 7.09x10-5
-7
Adulterated 12 99.96 9.75x10 8.27x10-5
6 Classes
MC Authentic 3 99.43 2.05x10-3 1.30x10-1
Corn starch 3 99.97 1.15x10-3 1.11x10-1
-3
Beet 2 99.42 2.14x10 1.77x10-1
-3
Cassava starch 3 99.97 1.40x10 9.83x10-2
Maltodextrin 3 99.87 1.93x10-3 2.10x10-1
-3
Grape Juice 3 99.88 2.97x10 2.89x10-1
SNV Authentic 3 99.99 7.81x10-3 4.81x10-1
-3
Corn starch 4 100.00 2.80x10 2.82x10-1
Beet 4 100.00 3.50x10-3 4.08x10-1
-3
Cassava starch 5 100.00 2.10x10 2.65 x10-1
Maltodextrin 3 99.99 7.50x10-3 6.37x10-1
-3
Grape Juice 5 100.00 3.40x10 4.05x10-1
MSC Authentic 2 100.00 2.13x10-3 1.29x10-1
-4
Corn starch 4 100.00 4.23x10 4.21x10-2
-4
Beet 4 100.00 6.01x10 7.05x10-2
Cassava starch 5 100.00 3.06x10-4 3.83x10-2
-4
Maltodextrin 4 100.00 7.43x10 7.33x10-2
Grape Juice 6 100.00 3.36x10-8 7.68x10-2
-6
2SGD Authentic 6 99.97 1.08x10 7.09x10-5
Corn starch 5 99.96 7.88x10-7 9.43x10-5
-6
Beet 4 99.97 1.02x10 1.11x10-4
Cassava starch 5 99.96 7.69x10-5 9.78x10-5
-7
Maltodextrin 6 99.96 9.60x10 1.42x10-4
-7
Grape Juice 7 99.98 8.77x10 1.79x10-4
PC: Number of principal components; CV: Cumulative variance.
129
Fig. S1. Loadings obtained from PCA models developed using the pre-processing of mean
centering (a), Standard normal variate (b), Multiplicative scatter correction (c) and second
Savitzky-Golay derivative (d). PC1: first principal component; PC2: second principal
component. Values within parentheses indicate the percentage of variance explained by the
principal component.
131
Fig. S3. Representation of samples incorrectly classified by the k-NN models for 6 classes,
obtained by the pre-processing of mean centering (a) and Second Savitzky-Golay derivative
(b). AUT: class of authentic samples; COS: class of samples adulterated with corn starch; BP:
class of samples adulterated with beet pulp; CAS: class of samples adulterated with cassava
starch; MD: class of samples adulterated with maltodextrin; GJ: class of samples adulterated
with grape juice.
Fig. S4. Models distances for 2 (AUT and ADU) and 6 classes (AUT, COS, BP, CAS, MD
and GJ) in relation to the models for authentic samples obtained by the pre-processing of
Mean centering, Standard normal variate, Multiplicative Scatter correction and 2nd Savitzky-
Golay derivative.
133
Fig. S5. Representation of samples incorrectly classified by the SIMCA models for 6 classes,
obtained by pre-processing of mean centering (a), Standard normal variate or multiplicative
scatter correction (b) and second Savitzky-Golay derivative (c). AUT: class of authentic
samples; COS: class of samples adulterated with corn starch; BP: class of samples adulterated
with beet pulp; CAS: class of samples adulterated with cassava starch; MD: class of samples
adulterated with maltodextrin; GJ: class of samples adulterated with grape juice.
Fig. S6. Representation of samples incorrectly classified by the PLS-DA models for 6 classes,
obtained by pre-processing of mean centering (a), Standard normal variate or multiplicative
scatter correction (b) and second Savitzky-Golay derivative (c). AUT: class of authentic
samples; COS: class of samples adulterated with corn starch; BP: class of samples adulterated
with beet pulp; CAS: class of samples adulterated with cassava starch; GJ: class of samples
adulterated with grape juice.
134
DISCUSSÃO GERAL
predição superior) e maior seletividade. Ao comparar os gráficos dos valores preditos pelos
modelos versus os valores medidos obtidos através do méto de Gerber para os conjuntos de
calibração e predição, observamos que o modelo MIR apresentou menor ajuste e habilidade
de predição que o modelo NIR, que apresentou menor bias, indicando pequena diferença entre
os valores previstos e os valores medidos.
Na determinação do teor de proteínas, ambos os modelos de NIR e MIR
apresentaram valores baixos de RMSEC e RMSEP quando comparados aos desvios-padrão
obtidos pelo método de Kjeldahl (referência), indicando que os erros associados à predição
pelos modelos são baixos. Ao comparar as figuras de mérito, foi verificado que ambos os
modelos, apresentaram baixos desvios padrão relativos. Por outro lado, apesar de menos
sensível, o modelo NIR apresentoumenores limites de detecção e quantificação, melhor
ajuste, habilidade de previsão (menores RMSEC e RMSEP), maior valor da relação
SDp/RMSEP, maior sensibilidade e sensibilidade analítica.Os gráficos de valores previstos
pelos modelos versus valores obtidos através do método de Kjeldahl para as amostras de
calibração e predição mostraram que o modelo NIR apresentou capacidade de predição
superior e baixo valor de bias, indicando uma pequena diferença entre os valores previstos e
os valores medidos.
Na quantificação do teor de cinzas, os modelos NIR e MIR apresentaram valores
semelhantes de RMSEC e RMSEP, sendo considerados baixos quando comparados aos
valores médios e desvios padrão obtidos através das análises de referência. A relação
SDP/RMSEP de 1,9 e 2,1 para os modelos NIR e MIR, respectivamente, mostra que as
habilidades de predição de ambo os modelos são semelhantes. O modelo NIR foi mais
sensível, apresentou mior sensibilidade analítica, sendo possível a distinção entre amostras
com uma diferença de concentração de 0,0027% de cinzas, teve menor seletividade, menores
limites de detecção e quantificação. Ambos os modelos apresentaram valores baixos de RSD,
demonstrando que os valores previstos estão mais próximos dos valores médios determinados
pela análise de referência. Os gráficos de valores previstos pelos modelos versus valores
obtidos através dos métodos de referência para os conjuntos de calibração e predição
mostraram que ambos os modelos se ajustam bem aos dados, mas baixa capacidade de
predição. Entretanto, os valores de biasmostram que, para ambos os modelos, a diferença
média entre os valores previstos e medidos é pequena.
A detecção e identificação de adulterante em leite UHT integral foi realizada
através das análises quimiométricas de Principal Components Analysis (PCA) e Partial Least
Squares discriminant Analysis (PLS-DA). Na detecção da presença de adulterantes a análise
137
de componentes principais (PCA) foi realizada para explorar os dados espectrais nas regiões
do infravermelho próximo (NIR) e médio(MIR). Os espectros das amostras autênticas e
adulteradas foram pré-processados pelo tratamento de segunda derivada de Savitzky-Golay
(2ª ordem, 25 pontos) seguido de centragem na média. O PCA foi construído com 8
componentes principais, sendo obtidos os seguintes valores de Root Mean Square Error of
calibration Standard (RMSEC), Root Mean Square Standard Error of Cross-Validation
(RMSECV) e variância acumulada de 9.22x10-6, 1.51x10-3 e 66,48%, respectivamente.
Atrav´pes da análise do gráfico de scores foi verificada uma separação entre a maioria das
amostras autênticas e adulteradas. No entanto, 6 amostras autênticas foram localizadas nos
mesmos quadrantes de amostras adulteradas, indicando scores similares. Das 6 amostras,
apenas uma é da mesma marca da amostra utilizada na produção das amostras adulteradas.
Estes resultados mostram que é possível a distinção entre as amostras autênticas e adulteradas
utilizando a espectroscopia NIR e PCA.
Os dados espectrais na região do infravermelho médio das amostras autênticas e
adulteradas foram tratados pelo pré-processamento de Multiplicativescattercorrectione
centragem na média. O PCA dos dados MIR foi desenvolvido com 6 componentes principais,
sendo obtidos os valores de Root Mean Square Standard Error of Calibration (RMSEC),
Root Mean Square Standard Error of Cross-Validation (RMSECV) e variância acumulada de
9.03x10-4, 6.16 x10-2 e 84,21%, respectivamente. Da mesma forma que na análise dos dados
NIR, os scores obtidos através do PCA dos dados MIR mostram a formação de dois grupos.
No entanto, 12 amostras autênticas estão localizadas nos mesmos quadrantes de amostras
adulteradas, indicando valores de scores semelhantes. Portanto, para melhorar a distinção
entre as amostras autênticas e adulteradas utilizando os dados de NIR e MIR, foram
desenvolvidos modelos de Partial Least Squares Discriminant Analysis, visto que o PLS
melhora as diferenças entre as classes de amostras, sendo usado quando as informações que
evidenciam as diferenças entre as classes não aparecem nas primeiras componentes principais.
Na detecção de amostras adulteradas utilizando os dados na região do
infravermelho próximo (NIR), o modelo PLS-DA foi construído com 8 variáveis. Todas as
amostras autênticas e amostras adulteradas utilizadas na construção do modelo e na validação
externa foram corretamente classificadas sem a ocorrência de resultados falsos positivos ou
negativos. As amostras autênticas e adulteradas no conjunto de predição foram corretamente
classificadas com alta especificidade e sensibilidade de 100% e 97,28%, respectivamente.
Estes resultados mostram que é possível detectar amostras contendo os adulterantes aplicados
neste estudo usando espectroscopia NIR e a análise quimiométrica de PLS-DA.
138
através do NIR e PLS-DA. Além disso, verificamos que é possível a identificação das outras
classes de amostras adulteradas: água + sacarose, água + uréia + amido e água + uréia +
amido + sacarose. Vale ressaltar que esta última classe teve concentração de
adulterantesamido e de sacarose menor do que nas outras amostras adulteradas. A classe de
água + Sacarose apresentou o menor valor de especificidade, indicando maior probabilidade
de ocorrência de erro de tipo I (falso positivo). Por outro lado, os resultados obtidos para as
outras classes são considerados elevados. Consequentemente, é possível identificar a maioria
dos adulterantes utilizados neste estudo com alta especificidade e sensibilidade através da
espectroscopia NIR e PLS-DA.
A identificação do tipo de adulterante presente nas amostras de leite UHT integral
através dos dados espectrais na região do infravermelho médio, os dados foram pré-
processados por Multiplicativescattercorrectionseguida de centragem na média, sendo
escolhidas 8 variáveis latentes. Nesse modelo, várias amostras do conjunto de calibração
foram incorretamente classificadas (dados não mostrados). As melhores taxas de classificação
foram obtidas após o aumento do número de variáveis latentes para 19. Este último modelo
foi desenvolvido usando os dados espectrais nas faixas de 3534,38-271,71 cm-1, 1903,27-
973,073 cm-1, 738,1993- 507,042 cm-1 pré-processadas pela centragem na média, sendo
possível a classificação correta apenas das amostras em o conjunto de calibração. Estes
resultados indicam que o modelo se ajusta às amostras de calibração, mas apresenta baixa
capacidade de predição.
Neste estudo, verificamos que as amostras das classes de autêntica, água, água +
uréia, água + amido, água + sucrose, água + uréia + amido, água + uréia + sucrose e água +
uréia + sacarose foram classificadas com especificidade de 99,37%, 99,04%, 94,15%,
98,78%, 97,56%, 98,78%, 96,09%, 98,38% e sensibilidade de 100%, 70,00%, 50,00, 80,85,
77,42%, 12,35%, 50,00% e 20,00% respectivamente. Estes resultados mostram que nenhuma
classe apresentou simultaneamente especificidade e sensibilidade, indicando a dificuldade em
identificar adulterantes neste estudo usando espectroscopia MIR e PLS-DA.
Após a detecção e identificação de adulterantes através dos modelos PLS-DA,
avaliamos as amostras adulteradas através dos métodos de detecção de amido e sacarose. As
amostras contendo amido (água + amido, água + uréia + amido e água + uréia + amido +
sacarose) e sacarose (água + sacarose, água + uréia + sacarose e água + uréia + amido +
sacarose) foram testadas no menor nível de concentração preparado neste estudo (1% da
mistura em relação à quantidade de leite integral UHT), juntamente com uma amostra de
controle sem adulterantes (branco).A análise de espectroscopia NIR acoplada à análise de
140
PLS-DA permitiram resultados semelhantes aos obtidos durante a análise de referência, uma
vez que todas as amostras contendo água + sacarose e água + uréia + sacarose foram
corretamente classificadas nos conjuntos de calibração e predição. Por outro lado, no caso da
amostra contendo água + uréia + amido + sacarose, o desempenho do modelo NIR foi
superior, uma vez que não foi possível detectar sacarose pelo método de referência, mas foi
possível determinar pelo método de NIR.
A detecção e a identificação de adulterantes em polpa de açaí liofilizada foram
realizadas utilizando diferentes análises quimiométricas. Na detecção da presença de
adulterantes foram utilizadas as Cartas controle de Qresiduals, modelos de k-NearestNeighbors
(k-NN), Soft Independent Modelling of Class Analogy (SIMCA) e Partial Least Squares
Discriminant Analysis (PLS-DA). Na identificação do tipo de adulterante foram utilizadas os
modelos de k-NearestNeighbors (k-NN), Soft Independent Modelling of Class Analogy
(SIMCA) e Partial Least Squares Discriminant Analysis (PLS-DA). As cartas de controle de
Qresiduals desenvolvidas através da Análise de Componentes Principais com dados tratados
pelos pré-processamentos de Multiplicative scatter correction(MSC), Standard normal
variate(SNV), segunda derivada de Savitzky-Golay (2SGD) e Centragem na média (MC)
foram adequados para identificar corretamente as amostras adulteradas, visto que algumas
amostras adulteradas no conjunto de predição foram identificadas como autênticas e algumas
amostras autênticas foram identificadas como adulteradas. Os loadings desses modelos
indicaram que o intervalo entre 7449 e 4000 cm-1 continha a informação mais importante para
a detecção de adulterantes.
Dentre os pré-processamentos avaliados, as cartas de controle desenvolvidas com
dados tratados pelos pré-processamentos de SNV e MSC apresentaram os melhores
resultados. No entanto, o pré-processamento de MSC foi escolhido como o melhor, pois
exibiu uma variância acumulada elevada (%), valores mais baixos de RMSEC e RMSECV em
comparação com o modelo SNV. Além disso, amostras adulteradas em todos os níveis de
adulteração foram distinguidas de amostras autênticas, uma vez que apresentaram Qresidual>
Qlimit.
Na detecção de amostras adulteradas utilizando o modelo de k-Nearest Neighbors
(k-NN), apenas os modelos desenvolvidos pelos tratamentos de SNV e MSC distinguiram
todas as amostras adulteradas de amostras autênticas no conjunto de predição. Na
identificação do tipo de adulterante presente nas amostras, os melhores modelos de k-NN
foram desenvolvidos com dados tratados pelos pré-processamentos de SNV e MSC, uma vez
que todas as amostras no conjunto de predição foram corretamente classificadas. Assim,
141
CONCLUSÕES GERAIS
detecção de 0,01 a 0,57%, limite de quantificação de 0,03 a 1,74%, desvio padrão relativo de
0,9 a 3,2%, R2cal 0,741 a 0,948, R2pred de 0,777 a 0,947 e biasde 4,76 x 10-3 a 3,63 x 10-2.
Os modelos MIR para determinação se sólidos totais, lactose, gorduras, proteínas
e cinzas foram desenvolvidos com 6, 8, 8, 8 e 6 variáveis latentes, sendo obtidos sendo
obtidos valores de RMSEC de 0,0061 a 0,0969%, RMSEP de 0,0099 a 0,0836, sensibilidade
de 0,0004 a 0,0522%, sensibilidade analítica de 9,7 a 57,7, seletividade de 0,0173 a 0,2660,
limite de detecção de 0,06 a 0,90%, limite de quantificação de 0,17 a 2,72% e desvio padrão
relativo de 0,7 a 2,7%, R2cal 0,900 a 0,962, R2pred de 0,571 a 0,967 e biasde 2,89 x 10-4 a -1,02
x 10-2.Estes resultados mostram que tanto para os modelos NIR quanto MIR foram obtidos
baixos valores de RMSEC, RMSEP, baixos limites de detecção e quantificação, baixos
desvios padrões relativos (RSD) e bias. Ao comparar as figuras de mérito de RMSEC,
RMSEP, R2cal, R2pred, sensibilidade, seletividade, limites de detecção e quantificação, os
melhores resultados para a quantificação de sólidos totais, lactose, gorduras, proteínas e
cinzas em leite integral UHT foram obtidos através do modelo NIR.
Todas as amostras de leite UHT integral foram avaliadas periodicamente através
da pesquisa de adulterantes, onde não foram observados resultados positivos para a adição de
água, presença de amido e presença de sacarose. Com base nisso, uma amostra foi escolhida
para simulação de adulteração pela adição de água, uréia, sacarose e amido de milho em
diferentes proporções (1, 5, 10, 15 e 20%).O gráfico de scores obtido através da análise de
componentes principais (PCA) mostrou, ao utilizar os espectros no NIR, uma separação entre
as amostras autênticas e adulteradas. Entretanto, algumas amostras adulteradas estavam
localizadas nos mesmos quadrantes das amostras autênticas, indicando similaridades, que não
estavam associadas à marca, visto que de 6 amostras (total), somente 1 era da mesma marca
da amostra autêntica utilizada na produção das amostras adulteradas. Ao aplicar a análise de
componentes principais aos dados espectrais no MIR, verificamos através do gráfico de scores
uma separação entre as amostras autênticas e adulteradas, sendo que também algumas
amostras adulteradas (12) também estavam localizadas nos quadrantes das amostras
autênticas, indicando similaridade. Apesar da similaridade de algumas amostras adulteradas
com algumas amostras autênticas, tanto a espectroscopia no NIR quanto no MIR associadas à
análise de componentes principais demonstraram potencial na distinção de amostras de leite
UHT integral autênticas de adulteradas.
Os modelos de classificação PLS-DA desenvolvidos tanto com os dador no NIR
quanto no MIR permitiram a detecção de amostras de leite UHT adulteradas nos conjuntos de
calibração e predição sem a ocorrência de resultados falso positivos e falso negativos. Isto
145
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