Physicochemical and microbiological changes of bovine blood due to high hydrostatic pressure treatment

Bovine blood samples were treated with high hydrostatic pressure (HHP) to examine the changes that may occur in the blood related to its colour, microbiological characteristics, protein denaturation, and dynamic viscosity. Pressure treatments were carried out from 100 to 600 MPa in 100 MPa scale up, with 5 min holding time. The blood samples were treated with anticoagulant (EDTA) to eliminate the possible measurement distorting effects. We found that 2 log reduction in the microbial load could be achieved with a pressure treatment above 400 MPa. According to the protein denaturation measurements (DSC), blood proteins were resistant to pressure treatment, even at 300 – 400 MPa a substantial part of proteins remained in native state. The colour of the samples got darker with the rising pressure, however, visible colour change was observed only above 400 MPa. It can be established, that the HHP treatment was suitable to increase the microbiological stability of blood, without significantly changing its techno-functional properties.


INTRODUCTION
Blood is a specific connective tissue in liquid state, built up from plasma with suspended elements in it, a rich source of iron and protein (Ofori and Hsieh, 2011). Blood as a resource is currently scarcely used, despite the fact that the protein content of whole blood is approx. 18%, the protein content of the plasma is 6-8 g dL À1 in pigs and 7-9 g dL À1 in cattle Lynch et al., 2017). Apart from it being a protein source, knowing the proportion of anaemia caused by iron deficiency in the population -primarily among youngsters and women -the other most important nutrient in blood is iron. Pig blood contains 1,490.14 mg kg À1 of iron in terms of dry matter content, which is outstanding among food products (Sorapukdee and Narunatsopanon, 2017).
Last year's estimated "waste" of this rich protein source only from pigs was 6,465 tonnes in Hungary, 625,352 tonnes in Europe, and 435,524 tonnes in the USA according to Eurostat and USDA slaughter statistics from 2018 (Eurostat, 2020;USDA, 2020). It is estimated that China produces 1,500,000 tonnes of pig blood per year with a protein content equivalent to 2,000,000 tonnes of meat or 2,500,000 eggs (Wang et al., 2007). If the blood is not collected for further processing, it has to be treated as hazardous waste, so the industry is not simply wasting this opportunity, but paying for treatment and neutralisation. Some of the blood and blood proteins are used by the meat industry as a food ingredient as natural colourant, emulsifier, fat substitute, or texture modifying agent. Only about 30% of the blood from slaughterhouses is used by the food industry worldwide (Ofori and Hsieh, 2012). The blood reduces the amount of cooking loss released during heat treatment by binding water and fat, also forming a kind of matrix that can bind nutrients and flavourings (Chen and Lin, 2002;Toldr a et al., 2008). Blood, containing all its fractions, can only be added into food products in limited quantities. The characteristic smell, colour, and metallic taste of blood is given by the heme part (Duarte et al., 1999). Haemoglobin in the blood could be suitable for preventing the development of iron deficiency due to its higher bioavailability ratio (Liu et al., 1996). However, without compromising the organoleptic properties of the final product, blood can be added to only about 0.5-2.0% of the product (Ofori and Hsieh, 2012). According to this, blood must be divided into fractions for further use (even for food industry), or innovation of blood-based food products should be encouraged. Consumer preferences are significantly influenced by the colour of meat and meat products (Claus and Du, 2013). Therefore, it is essential that the colour of the finished products should meet the consumer's acceptance, and if possible and technologically feasible, colourants with natural origin should be used. Application of blood as natural colourant is one of the most popular way of using blood. However, the suitability of a natural colourant in a given product is greatly influenced by the form, quantity, and condition in which that specific colourant is available. The microbiological quality of blood from slaughterhouses must be improved before it is used as a food ingredient (Toldr a et al., 2004(Toldr a et al., , 2008. High hydrostatic pressure treatment (HHP) can be suitable for this. HHP is a gentle food preservation process, during treatments the microorganisms in food are partially or completely inactivated by treating the product at a pressure of 100-800 MPa, proteins in microbes are denatured (covalent bonds are broken down) and cell death occurs (Morales et al., 2008), meanwhile organoleptic properties of the product only slightly change (Campus, 2010). In case of the vegetative pathogens, 2-4 log reduction has been observed in various meat products, resulting increased food safety, microbiological stability, and shelf life (Bajovic et al., 2012). HHP treatment of blood and its use is a less researched topic.
Toldr a et al. (2004) found that HHP treatment of blood at 400 MPa for 15 min caused significant improvement in microbiological quality and did not adversely affect colour characteristics or protein solubility. However, it is essential to examine what kind of physical and chemical changes may occur in the blood as a result of the pressure treatment and how these changes can subsequently affect the properties of the final product.

Samples
Bovine blood was obtained from a cattle herd of a rural farm, breed of Hungarian Simmental, age of 6 years. For comparability and to analyse the effects of the pressure treatment, EDTA (ethylene-diamine-tetra-acetic acid) was used as anticoagulant. The samples were vacuum packed airtightly in polyethylene (Cryovac©, BB4L) package with an average weight of 50 g.

HHP treatments
High-pressure experiments were performed in a Resato FPU-100-2000 (laboratory scale, Resato International B.V, The Netherlands) high-pressure equipment, that contained a pressurising (1,600 mm 3 2,200 mm 3 830 mm) and a control (1,300 mm 3 950 mm 3 1,400 mm) unit. The samples were subjected to pressure treatments from 100 MPa to 600 MPa with 100 MPa scale up and 5 min holding time, uniformly. The pressure medium was propylene glycol (Resato PG fluid) in the vessel. The initial temperature of the samples was 1-3 8C (cooled in icy water), and due to the adiabatic heating, sample temperature increased by approximately 3 8C/100 MPa during the treatments. The pressure profiles in the sample holder during the treatments were logged.

Colour measurements
The colour measurements were performed with a Minolta CR-400 (Minolta Co. Ltd., Osaka, Japan) tristimulus colorimeter, repeated five times. During reflection colour measurement, the three data provided by the instrument are L p , a p , and b p , which can be used to infer the colour and colour change of the samples. To calculate the total colour difference ðΔE * Þ between untreated and treated samples, average values of L * ; a * , and b * in the following equation were used: where ΔL * , Δa * , es Δb * are the differences between the values of L * , a * , and b * in the untreated and treated samples (Salamon et al., 2016).

Dynamic viscosity
For dynamic viscosity measurements Rheomat 115 (Contraves, Malaysia) rotary viscometer was used based on the modified method of Islam and Azemi (1997). For coaxial cylindrical (concentric) viscometers, the liquid is between the two measuring cylinders. Prior to measurement, all samples were set to the same temperature (5 8C). Measurements were carried out in triplicates. Dynamic viscosity (for non-Newtonian fluids) can be calculated from the reading (a), constant of the measuring system ðz ¼ 195:5Þ and the velocity gradient (D 5 57.20 s À1 ).

Statistical analysis
Statistical analyses were performed using SPSS 20.0 for Windows (Chicago, Illinois, USA). To determine the effect of treatments, one-way analysis of variance (ANOVA) was performed at a significance level of P < 0.05. Tukey's test was applied to compare the mean values when ANOVA showed significant differences.

Colour measurement
Due to the high pressure treatments, the L p colour parameter values decreased significantly (P < 0.05), highest level of decrease could be observed particularly in case of 300, 400, and 500 MPa treatments (Fig. 1). The samples lost from their original brightness and became darker. The a p red colour component results showed an intense decrease due to 300 MPa and above levels of pressure treatments, changes were significant compared to the control sample (P < 0.05). The b p yellow colour parameter also showed a significant change in the colour due to pressure treatments at each level (P < 0.05). The colour of the blood is mostly given by the haemoglobin in it, so the colour change after the pressure treatment is presumably related to haemoglobin. Bou et al. (2019) found that chicken haemoglobin remains relatively stable after high hydrostatic pressure treatment, however, methaemoglobin and insoluble forms of heme appear. Heme molecule part and its Fe 2þ -ion redox chemistry properties are presumably responsible for the colour change. Figure 2 illustrates the shear stress values obtained as a function of different shear rates. It can be seen from the flow curves that with increasing levels of pressure treatments, the shear stress values also increased. After 400 MPa treatment, the blood sample remained in liquid state, but particularly in case of the 500 and 600 MPa pressure treatments, there was a significant increase in the viscosity of bovine blood samples (h 400 MPa 5 23.95 Pas, h 500 MPa 5 119 Pas, h 600 MPa 5 62.54 Pas, at highest shear rate), the observed differences were even in the order of magnitude of the values. The proteins coagulate due to high hydrostatic pressure treatment, causing to the blood to flow more densely. Presumably the protein denaturation in the samples treated at 600 MPa caused a small amount of plasma release, which resulted in lower values than in case of the 500 MPa. The Ostwald-de-Waele model was fitted to the different flow curves so that the parameters of the flow curve could be quantified. The models proved to be adequate in all cases based on the correlation coefficients (r 2 5 0.79-0.99). The power exponent "n" was less than 1 in each case, which indicates a shear thinning rheological behaviour, therefore, by increasing the deformation rate, the slope of the flow curve decreases, ergo the viscosity also decreases. This probably happens because the damaged (denatured/aggregated) protein fragments are more easily aligned with the direction of shearing during the measurement.

Thermodynamic measurement
The enthalpy peaks in Fig. 3 indicate the aggregation and denaturation of the proteins. According to literature data, the detected peak (68 8C -70 8C) probably belongs to albumin, which provides the largest part of blood proteins (D avila et al., 2007). On the thermogram, a decrease both in the temperature peak and enthalpy values can be observed compared to the control sample due to protein denaturation. Particularly at higher levels of pressure, such as 500 and 600 MPa, the enthalpy values decreased with 50% and 68%, respectively (Csehi et al., 2017). This suggests that the amount of proteins that could be denatured by heat during the thermal analysis was greatly reduced.

Microbiological examinations
The results of the microbiological measurements are shown in Fig. 4. It is clearly visible from the initial values that blood is a great substance for microbial growth, 10 3 CFU g À1 was detected already in the control sample even as the samples were only minimally exposed to contamination. From the results it can be concluded that even 100 MPa pressure treatment resulted in an order of magnitude reduction of the number of microbes. As we increased the level of pressure, the TVC number showed a decreasing trend. 400 MPa treatment destroyed nearly all microbes in the blood sample, and above 500 MPa treatment their numbers were below detection limit. Consequently, high pressure treatment can be a good alternative for blood preservation.

CONCLUSIONS
Based on the above results, it can be established that high pressure treatment of 400 MPa is already effective to reach the sufficient microbiological stability of bovine blood, as it results in a 2 order of magnitude microbial count reduction, possibly providing more stable raw material for further processes from food safety perspective. According to the thermophysical measurements (DSC), the pressure of 400 MPa does not yet cause severe changes in the protein structure, so the blood remains in a liquid state, therefore, it can be used conveniently from a techno-functional point of view.