The high utility value of soybean results from their nutritional value, in particular: the high content of well-digestible and balanced proteins that are rich in exogenous amino acids, the abundance of oil with a significant share of polyunsaturated fatty acids (PUFAs), and the vitamins and minerals they contain [1,2].
The development of optimal technology for converting soybeans into food or fodder continues to drive much research [3-9]. However, the use of soybean and soybean products as components of food or fodder involves various forms and methods of their heat treatment, which is applied to reduce the activity of numerous thermolabile antinutritional factors [10, 11], and to develop rheological and organoleptic properties typical of soybean-based products, while maintaining the digestibility of nutrients . In the context of modern agro-food soybean processing, characterized by a high rate of dynamism and large scale of production, combining these mutually opposed objectives, viz. preserving the highest possible nutritional value, while, at the same time, reducing the activity of the antinutritional factors, determines the application of adequate methods of assessing heat treatment efficiency, which are characterized by high sensitivity and precision combined with low price and simplicity of implementation.
The quality of soybean products obtained in the heating process can serve as a criterion of the form, method, temperature, and time of raw material processing. Assessment of the effectiveness of heating soybean or other soybean products, as well as other food or feed products, can be based on any of their properties that can change as a result of heating. The most common assessment methods can be methodically divided into five basic groups:
- nutritional tests,
- methods based on determination of the content of products of thermal transformations,
- assessment of non-protein properties and thermolabile quality traits,
- microbiological assays,
- analytical methods based on alterations of the properties of product proteins .
1.1 Nutritional tests
These are one of the simplest natural methods for evaluation of raw materials or food or feed products. A controlled factor is the impact of analysed mixtures or their components on livestock health via such indicators as the protein efficiency ratio (PER), biological value (BV) of proteins, net protein utilisation (NPU), and true digestibility (TD) .
The quality of food and feed components depends on the process of heating thereof, hence thermal treatment has a significant effect on the results of nutritional tests. Yet, their results may be distorted due to the high complexity of metabolic processes occurring in experimental animals and the interpretation may be difficult. Therefore, agro-food processing aims to minimise the extent of nutritional experiments in favour of other less time-consuming methods with a possibly a high correlation with their results .
1.2 Determination of the heating efficiency using non-protein components
The efficiency of heating of soybean seeds can be assessed by investigation of the activity of thermolabile vitamins, e.g. thiamine, riboflavin, tocopherols, and tocotrienols .
Thermal treatment, especially in the presence of oxygen, may induce many transformations in food or feed components, which result in an increase in the content of characteristic products of these transformations (furosine – , e-pyrrolysine – , or lysinoalanine – ).
A disadvantage of these determinations affecting their usefulness in the current assessment of the effectiveness of thermal treatment is the substantial length of time and labour consumption as well as the high complication of the analytical procedure .
1.3 Microbiological assay
Heating is a method for reduction of microbial contamination, whose level can be a measure of the heating length of a product at a given lethal temperature and results in a certain limitation of the product shelf life . A drawback of this type of determinations associated with the microbiological assays is the prolonged time of the determinations and susceptibility to the shortcomings of the analytical procedure .
1.4 Methods based on alterations in the properties of product proteins
Three categories are most frequently distinguished in this group:
- methods based on denaturing changes in the solubility or other functional properties of proteins (water absorption, emulsion stability),
- methods based on measurement of the activity of enzymatic proteins or some anti-nutritional factors,
- determinations carried out with the use of indicator compounds characterised by specific reactions with certain protein groups .
1.5 Determination of changes in the solubility or differences in the functional properties of proteins
One of the effects of protein heating is the change in their solubility resulting in coagulation. Hence, the degree of protein heating can be determined from solubility in water (NSI) or water solutions [18-19].
Thermal denaturation of proteins, including soybean proteins, can be carried out while analysing their properties in oil or water determined as the water absorption coefficient -WHC, emulsifying capacity - FAC, emulsifying activity index - EAI, and emulsion stability index - ESI
A shortcoming of these methods is the high labour intensity and time consumption as well as the complexity and multistage character of the analytical procedure .
1.6 Analysis of changes in enzymatic activity
The activity of enzymes contained in soybean seeds depends on the thermal denaturation of enzymatic proteins. In practice, such assays are based on the activity of urease (UA) or lipoxygenases [20-24].
Their practical application is problematic due to the time-consuming and labour-intensive character of the analyses and the possibility of using varieties with genetically lowered or reduced activity of these enzymes .
1.7 Biochemical assays
This group of methods is based on determination of the activity of thermolabile anti-nutritional compounds present in soybean seeds, primarily the antitrypsin inhibitory activity (TIA) and inhibition of the activity of hemagglutinins, called lectins.
The basis for TIA determinations is the reaction of trypsin with substrates (casein or the synthetic substrate BAPA N-a-benzoyl-DL-arginine-p-nitroanilide) [24,25].
The results of these determinations are calculated in TUI units per mg dry weight of the sample or per unit of protein mass in dry matter. Raw soybean seeds of traditional varieties are usually characterised by an anti-trypsin activity level of ca. 50 TUI/mgd.w. . Thermal denaturation of inhibitory proteins may result in reduction of the TIA to a residual level; at the same time, 10% of the activity of raw seeds is regarded as a safe level of trypsin inhibitor activity that permits their application in feeds.
Proteins with hemagglutinating properties undergo thermal denaturation similarly to trypsin inhibitors. The hemagglutination activity is most frequently determined using the immunosorption method with the use of monoclonal [29,30] or polyclonal antibodies [31,35]. Immunosorption methods can be applied for determination of other soy proteins (b-conglycinin) as well .
1.8 Use of indicator substances
These types of methods are based on diagnostic reactions carried out with selected chemical reagents that specifically bind to certain groups of the heated product, facilitating assessment of the heating degree. Such reagents as orange-G, safranin , and coomassie blue  have long been used. The action of coomassie blue as a diagnostic reagent is based on the colour reaction with proteins dissolved in the test solution. Thermal denaturation of proteins reduces their solubility in the solution, which is reflected in the intensity of the colour of the blue-protein complex and can be a measure of the product heating intensity [34-36]. A disadvantage of the application of coomassie blue is the temporal instability of the staining intensity in the protein-blue complex, which may yield errors in the determinations [13,36].
The cresol red indicator (CRI) is one of the most important and often used methods for investigating the efficiency of heating soybean or its products - soybean meal. The application of cresol red in studies of the intensity of heating food and feed products was proposed already by Frolich , and the investigations were continued by Olomucki and Bornstein . With time, a standard was established based on these studies  to unify the procedure of application of cresol red as a diagnostic compound in assessment of the intensity of soybean product heating.
The CRI value increases along the thermal treatment of soybean seeds and soybean products, thus facilitating the assessment of the heating intensity .
The index of lysine absorption (LA), which is determined based on the reaction of the sample with the FDNB (1-fluoro-2,4-dinitrobenzene) reagent, in accordance with the methodology proposed by Booth , can be adopted as an approach for assessment of the efficiency of heating food or feed, including soybean seeds and their products.
A difficulty in the implementation of the FDNB-based determinations of CRI and LA is the necessity to defat samples before the determination process, which lengthens and complicates the analytical procedure .
Significant progress in simplification and shortening of analytical procedures in CRI- and FDNB-based studies on the efficiency of heating soybeans and soybean products was provided by the use of bromocresol purple as an active compound, which eliminated the necessity of the laborious process of sample defatting and significantly enhanced the sensitivity of the designed method referred to as the bromocresol purple index (BCPI).
Methods were developed to test the efficiency of heating of selected pulses and oilseeds, such as: soybean, chickpea. The studies were preceded by an initial selection of the possible indicator substances, of which acidic solutions of bromocresol purple (5’, 5’’- dibromo - 3’, 3’’ - dimethyl phenolsulfonphthalein) turned out to be the most effective ones, while in the analysis of soybean and soybean products also bromocresol green solutions (3,3’, 5, 5’ — tetrabromo-m — cresolsulfonphthalein) were effective. The threshold of concentration value for the two indicator substances in their solutions resulted from the linear association between their absorbency and their concentration, which was verified experimentally. It was also the condition for the correctness of the studies carried out, the results of which were calculated according to formula 1 .
The sensitivity of the method (c) was the basic criterion for comparing the test variants. It was calculated as the absolute value of the direction coefficient of linear regression formula (the result of sorption of the active substance – T, calculated in accordance with formula 1, as the function of the heating time of the seeds), taking as the basis of matching the results for raw seeds samples and seeds autoclaved at the temperature of 121oC for 120 minutes . The sensitivity of the method increases with the higher value of the index c.
For each of the tested variants of the working solution, i.e. bromocresol purple and bromocresol green, the sorption of the active substance – T was calculated, separately for raw and autoclaved (at 121oC for 120 minutes) seeds, as the difference in the solution content before and after the contact with the ground, for sieving through a 0.20 mm mesh, seed sample, measured using absorbance Eb and Eo (formula 1). For each of the tested variants of working solution, the sorption of active substance – T was measured for raw seeds (Ts) as well as autoclaved seeds (Ta) and calculated according to formula (1). For each of the tested variants of working solution, the sensitivity of the analytical method (c) was calculated on the basis of Ta and Ts values (for autoclaved and raw seeds respectively).
T = (Eo - Eb)*D*F*Eo-1m-1*(h/100)-1(z/100)-1 
For the variant of working solution with the greatest sensitivity in the seed testing, the name of bromocresol purple index (BCPI) and bromocresol green index (BCGI) was used, respectively. Additionally, the lower index in BCPIBSM and BCGIBSM indicates that the calculations were made per unit weight of protein in the seeds dry weight.
Both the bromocresol purple index (BCPI) and the bromocresol green index (BCGI) were characterized by high precision (π), sensitivity (χ) and high discernability (ρ) of the determinations , making it possible to accurately assess the characteristics of the samples, in the case of which enzyme analysis (UA) and biochemical assay (TIA) was difficult to perform (Table 1).
Moreover, it was also demonstrated that the results of the tests carried out using BCPI and BCGI methods were highly interdependent with the selected quality discriminants, i.e. urease activity - UA, trypsin inhibitor activity - TIA, cresol red indicator - CRI, lysine absorption – LA, activity of grass pea neurotoxin - BOAA, as well as acid value (AV) and peroxide value (PV) of oils and a tocopherol content — AE, of soybean, grass pea, bean, and rapeseed. The high and significant correlation factors enabled to formulate regression equations correlating the soybean, grass pea, chickpea, soybean oil, and rapeseed oil quality discriminants with the values of the developed BCPI and BCGI test as a dependent variable . The usefulness of some of the developed regression equations was confirmed in the studies on selected characteristics of soybean meal and chickpea meal carried out on a semi-technical scale .
By providing the possibility to convert the results obtained the use of these regression equations may replace some of the labour-intensive and time-consuming analytical procedures used so far, for testing quality discriminants by fast and effective BCPI and BCGI tests.
The practical use of the developed assessment methods (BCPI and BCGI) may include:
- complementing the existing methods for the evaluation of quality discriminants of seeds or products derived from them,
- replacement of the existing evaluation methods with the possibility of converting the results using the regression equations developed,
- using them as a replacement for the evaluation methods used so far in testing selected quality discriminants of soybean, grass pea, bean, chickpea, and rapeseed seed and their products.
So far, the most commonly used traditional methods of analysing soybean seeds and products, based on the activity of selected enzymes (urease activity – UA) or thermolabile antinutritional factors (trypsin inhibitor activity – TIA) [43-46], often supplemented by methods based on protein solubility [18,43,45,46], despite their labour intensity and time-consuming nature, as well as complicated analytical procedures and the necessity to use expensive, specialized laboratory equipment operated by highly qualified personnel, are still widely used in practice, even though thus obtained results are sometimes inaccurate and difficult to interpret. It should be noted that the UA and TIA test methods have been well verified in soybean meal studies and the obtained results of these parameters in this type of products were sufficiently correlated .
It appears that under the conditions of industrial soybean processing the bromocresol purple index (BCPI) method should meet the demands of practical implementation. This method is the answer to the approach postulating increased sensitivity of analytical methods with simultaneous shortening and simplification of the methodology , as it offers high sensitivity and precision results that allow for distinguishing wide range of samples with subtle differences in properties.
The study attempts to compare the usefulness of the BCPI method with the two evaluation methods, i.e. TIA and UA, which are currently most frequently used in the assessment of the effectiveness of soybean heating.