DOI: 10.3303/CET23102013 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Paper Received: 17 March 2023; Revised: 14 June 2023; Accepted: 16 July 2023 
Please cite this article as: Fasse S., Gottschalk A., 2023, In Vitro Analysis of the Anti-inflammatory Activity in Dairy Products Containing 
Salicornia Europaea, Chemical Engineering Transactions, 102, 73-78  DOI:10.3303/CET23102013 
  

 CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL. 102, 2023 

A publication of 

 
The Italian Association 

of Chemical Engineering 
Online at www.cetjournal.it 

Guest Editors: Laura Piazza, Mauro Moresi, Francesco Donsì 
Copyright © 2023, AIDIC Servizi S.r.l. 
ISBN 979-12-81206-01-4; ISSN 2283-9216 

In Vitro Analysis of the Anti-Inflammatory Activity in Dairy 
Products Containing Salicornia Europaea 

Sylvia Fasse*, Axel Gottschalk 
Institute of Process Engineering, Bremerhaven University of Applied Sciences, An der Karlstadt 8, 27568 Bremerhaven, 
Germany  
sfasse@hs-bremerhaven.de  

Novel solutions for ensuring food security have to be established in the context of a growing world population, 
and rising climate restrictions on agriculture and agriculturally usable lands. Simultaneously, there is a rising 
demand for healthy functional foods in today’s world population. Edible halophytes could be part of the solution, 
as they are capable to grow in saline environments and contain several bioactive compounds. The exploitation 
of these salt-tolerant plants in the formulation of novel functional dairy products is demonstrated. Therefore, the 
focus of this research is on the development of suited dairy products with the addition of different concentrations 
of Salicornia europaea biomass, while measuring the in vitro anti-inflammatory activity with the egg albumin 
denaturation method. The inhibition of protein denaturation observed is equivalent to the anti-inflammatory 
capabilities of the steroid reference drug hydrocortisone. Therefore, these initial results indicate that the 
formulation of cream cheese products with the addition of S. europaea biomass can be achieved. 

1. Introduction 
Global demand for food is rising. In addition to their caloric value, functional foods lead to health benefits. 
Therefore, they experience a huge compound annual growth rate (CAGR). The global nutraceutical market size 
is expected to grow at a CAGR of 9.4% from 2023 to 2030 (Grand View Research, 2023).  
Halophytes are salt-tolerant plants and have traditionally been used as medical plants worldwide. Today they 
are regaining more interest, as they have been reported to contain several bioactive compounds (Qasim et al., 
2017). As part of their defense mechanism against oxidative stress, induced by high salt concentrations, they 
synthesize bioactive secondary metabolites. These include phenolic acids, flavonoids, and tannins, which are 
considered health beneficial in foods (Jdey et al., 2017). A few halophytes, e.g. Aster tripolium and Salicornia 
europaea, were already used as a food or food ingredient before 15th May 1997, thus they do not need to be 
approved according to the Novel Food Regulation (CE) No 2015/2283 (Novel food catalogue, 2023). Therefore, 
this study examines the addition of S. europaea, as it is already widely available as a vegetable in the European 
market and should be considered for the formulation of innovative food products (Custódio et al., 2021). 
The combination of dairy products with halophytes seems reasonable, as plant additives are already added to 
several products and their bioactivity has been analysed (Wajs et al., 2023). As the addition of halophyte 
biomass also increases the salinity in dairy products, cream cheese was chosen in this research. Weragama et 
al. (2021) reported the sensory acceptability of cream cheese products with 2 % (w/w) salt. With the addition of 
health-beneficial plant sterols, as they have been reported in Salicornia spp., infused dairy products such as 
cream cheeses can be recognized as functional foods (Choi et al., 2014; Jędrusek-Golińska et al., 2020). Further 
the sensory properties of cream cheeses with addition of herbs are already widely known. Thus, a green color 
and alteration of the texture through addition of ground plant materials is already accepted by numerous 
consumers, e.g. herb cream cheese with parsley or chives are on the market. 
Nonetheless, to evaluate the functional properties of the formulated dairy products, the anti-inflammatory and 
anti-arthritic properties are investigated. Inflammation is a complex biological defense mechanism that enables 
the body to protect itself against pathogens and damaged cells, e.g. due to external stress factors (Giordano et 
al., 2021).  

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To quantify the anti-inflammatory properties of plant materials and extracts, several methods have been 
described. There are albumin denaturation assays either using bovine serum albumin or egg albumin.  
The anti-inflammatory activity is compared to the activity measured in medicinal nonsteroidal anti-inflammatory 
drugs (NSAIDs) or steroid drugs on the market (Rahman et al., 2015; Dharmadeva et al., 2018). Further, there 
are also cell culture experiments analyzing the immune response, e.g. in murine macrophages, and then the 
suppressed synthesis of tumor necrosis factor in induced cells is quantified. Salicornia herbacea L. extracts 
have been described to exhibit anti-inflammatory activities. It was investigated to measure the suppression of 
the inflammatory response in lipopolysaccharide-induced RAW 264.7 macrophage cells (Kim et al., 2009; 
Giordano et al., 2021). Therefore, it is likely that also S. europaea included in food is health beneficial.  
In the scope of this research, an in vitro albumin denaturation assay using egg albumin is performed with the 
formulated products, as described by Rahman et al. (2015). This assay has been described to evaluate the anti-
inflammatory activity of plant extracts against protein denaturation. In this study, the assay will be adapted to 
assess the properties of cream cheese samples. 

2. Material and Methods 
On the one hand, the scope of this research was to assess the feasibility of cream cheese formulation with 
different levels of S. europaea and on the other hand to perform the laboratory anti-inflammatory analysis of the 
products. 

2.1 Fresh material and preparation of S. europaea biomass 

The fresh, food-grade S. europaea biomass was produced in hydroponic systems and obtained from Alpha-
Aqua in Denmark (DK). This fresh biomass was stored frozen until the biomass was dried at 50°C for 20 h in a 
convection dryer. Afterward, the biomass was ground. The dry matter content was measured in a KERN Dab 
100-3, in which 5 g of biomass were spread on the tray and heated to 120°C to evaporate the residual moisture. 
Measurement was performed in triplicate 

2.2 Dairy product formulation  

The cream cheese used in the experiments was commercially available double cream cheese with the following 
macronutrient composition: 25 g/100g fats, 3 g/100g carbohydrates, and 4.5 g/100g proteins. In a reactor, the 
ground S. europaea was mixed into cream cheese in concentrations of 0.5 %, 1.0 %, and 1.5 %. Thereafter the 
mixture was heated to 70°C and kept at the temperature for 30 s (see figure 1), as this is a standard procedure 
for the heat treatment of dairy products in the industry. This heat treatment was performed to eliminate the 
potential contamination through the S. europaea biomass, as majority of the bioactive compounds are phenolics 
the bioactivity is presumably not impacted (Friedman and Jürgens, 2000). A further measurement performed 
was the detection of the pH value over time (after preparation, after 5 days, and after 8 days) and was measured 
in triplicate. Additionally, the water activity was measured in the Novasina LabMaster-aw, where the sample is 
heated to 25°C. Also, the dry matter content was measured as described above. 
 

 
 
Figure 1: Product formulation of cream cheese with the addition of dried and ground green S. europaea biomass. 
Heat treatment was performed by heating the cream cheese to 70°C for 30 s.  

2.3 Chemicals and reference drug  

The following chemicals were used in the analysis: Phosphate buffered saline (PBS, pH 6.7) consisting of 1.79 g 
di-sodium hydrogen phosphate (ROTH, Karlsruhe, Germany), 1.36 g potassium dihydrogen phosphate (ROTH, 
Karlsruhe, Germany) and 7.02 g sodium chloride (ROTH, Karlsruhe, Germany) in 1,000 mL of distilled water. 
The commonly used steroid anti-inflammatory drug hydrocortisone (Soventol®, Iserlohn, Germany) was chosen 
as a reference (positive control) to quantify the anti-inflammatory properties. The hydrocortisone was diluted 
with distilled water to yield solutions with 1,000 µg/mL, 100 µg/mL, 10 µg/mL, and 1 µg/mL of active ingredient. 

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2.4 In vitro anti-inflammatory activity 

Several methods have been described to estimate the anti-inflammatory activity by in vitro measurements. This 
described method is used for in vitro determination of the anti-inflammatory activity of S. europaea infused cream 
cheese products using the albumin denaturation method based on Rahman et al. (2015) with minor adjustments. 
For the cream cheese, samples are prepared by diluting the cream cheese with distilled water (1:10) and 
keeping it in a thermoshaker (Cell Media Thermoshaker Pro) at 50°C for 15 min at 500 rpm. Afterwards, the 
liquid was separated via centrifugation at 3,500 rpm for 15 min. The supernatant was diluted with distilled water 
to yield a concentration of 10 mg/mL. Further, cream cheese without the addition of S. europaea biomass was 
taken as control, following the same procedure. Also, a control with distilled water instead of the above-described 
solutions, was prepared for the reference drug measurements. 
Reaction mixtures were prepared by adding 2.5 mL of PBS (pH=6.7), 0.5 mL of albumin from fresh hens’ egg, 
and 2 mL of sample (reference drug, control, cream cheese control, and cream cheese sample). After mixing, 
the mixture was kept at 37°C for 15 min and shaken at 400 rpm. Then the mixture was heated to 70°C and kept 
for 5 min at 400 rpm. Before the absorbance measurement, the reaction mixture was stored at room temperature 
for 20 min. All measurements were taken in triplicates.  
The absorbance was measured in triplicates at 680 nm (VWR UV-3100PC). The calculation of the denaturation 
inhibition is based on Rahman et al. (2015): 

𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 (%) =  �
𝐴𝐴𝐴𝐴𝐴𝐴𝐷𝐷𝐷𝐷𝐴𝐴𝐷𝐷𝐷𝐷𝐴𝐴𝐷𝐷 𝐷𝐷𝑜𝑜 𝐴𝐴𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝐷𝐷/𝐷𝐷𝐷𝐷𝑜𝑜𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐴𝐴𝐷𝐷

𝐴𝐴𝐴𝐴𝐴𝐴𝐷𝐷𝐷𝐷𝐴𝐴𝐷𝐷𝐷𝐷𝐴𝐴𝐷𝐷 𝐷𝐷𝑜𝑜 𝐴𝐴𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝑠𝑠
�  ∙ 100 (1) 

The anti-inflammatory activity was measured after the cooling of the product and 5 days after the product 
packaging, while the sample was stored at 4°C. 

2.5 Statistical analysis 

To evaluate the statistical significance a one way analysis of variance (ANOVA) in Excel was performed, as 
described by Quirk (2012), to evaluate the results. The p-value was considered significant with p < 0.05. 

3. Results and Discussion 
3.1 Analysis of cream cheese and S. europaea biomass 

The dry matter content in the cream cheese amounts to 67.7 % ± 0.5 %. Water activity in the cream cheese is 
determined to be 0.94. Meanwhile, the dry matter content of the fresh S. europaea biomass was determined to 
be 91.9 % ± 0.9 %. After drying the dry matter content increased to 93.9 % ± 0.4 %. 

3.2 Cream cheese formulation 

The cream cheese products have been prepared and the pH value was measured directly after cooling, after 5 
days, and after 8 days. The pH values are displayed in Figure 2. Therein a slight shift towards an alkaline pH 
value can be observed. The one-way ANOVA statistical analysis calculated a p = 0.0032, which is evaluated 
statistically significant.  

 

Figure 2: Development of pH value of S. europaea infused cream cheese samples on the day of preparation, 
after 5 days, and after 8 days of storage at 4°C 

 

75



A possible explanation is the entry of molds in the samples (Goldfarb and Herrmann, 1956), therefore the anti-
inflammatory activity was also measured after 5 days. The microbiological examination of the samples should 
be performed. Herein the possible contamination with molds or bacteria most likely happened due to the 
processing performed, as the cream cheese was commercially available. Also, the contamination could be due 
to the addition of the biomass, which is possibly contaminated by molds. 

3.3 In vitro anti-inflammatory activity 

The egg albumin denaturation assays with the reference drug hydrocortisone show an increase in anti-
inflammatory activity with increasing concentration of the drug, as displayed in figure 3. Approx. 10-30 % of 
protein denaturation is inhibited in the measurements including the hydrocortisone solutions. This is analog to 
the results Chandra et al. (2012) reported for diclofenac sodium as a NSAID reference drug and the measured 
differences are considered statistically significant (p = 0.0015). 

 

Figure 3: Egg albumin denaturation with different concentrations of hydrocortisone (1 µg/mL – 1,000 µg/mL) in 
the reaction mixture  

The results of the protein denaturation experiments are displayed in figure 4. The egg albumin denaturation is 
increasingly inhibited in the samples with increasing S. europaea concentration in comparison to the control. 
After 5 days of storage, the inhibition is still detected in similar quantities. Both direct measurement and 
measurement after 5 days are statistically significant with p = 0.0014 and p = 0.0031, respectively. From this, it 
can be concluded that the anti-inflammatory properties of the formulated cream cheese products are stable 
during the storage period, although a shift in pH value was detected. If this is also valid for longer storage periods 
has to be examined further. 

 

Figure 4: Egg albumin denaturation in 10 mg/mL concentrations of cream cheese with the addition of different 
concentrations of S. europaea (0.5 %; 1.0 %; 1.5 %) in reference to the control of cream cheese without the 
addition of S. europaea. The first measurement was taken directly after cooling (■) and the second was taken 
after 5 days of storage at 4°C (■). 

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The denaturation inhibition is also summarized in table 1. Measured inhibition rates are comparable with those 
of plant extracts as reported by Dharmadeva et al. (2018) to be between 10-20 %. 

Table 1: Protein denaturation inhibition in cream cheese samples with 0.5 %, 1.0 %, and 1.5 % addition of 
dried S. europaea biomass in reference to cream cheese without the addition of biomass (control). 
Measurements were taken directly and after 5 days of storage. 

 Control 0.5 % 
S. europaea 

1.0 % 
S. europaea 

1.5 % 
S. europaea 

Denaturation inhibition [%] direct 
measurement ± SD - 8.16 ± 1.05 6.70 ± 1.28 16.17 ± 2.65 

Denaturation inhibition [%] after 5 
days of storage ± SD - 13.43 ± 1.25 1.06 ± 4.48 11.16 ± 0.92 

4. Conclusions 
The analysis of the different levels of halophyte concentration in dairy products showed great potential as 
additive for functional food development. The measured anti-inflammatory activity proved to be comparable to 
those of prescribed anti-inflammatory drugs. This is supported by the traditional use of halophytes as medical 
plants. Therefore, bringing these S. europaea infused dairy products to the market becomes inherent. Further 
research should focus on evaluating the additional nutraceutical properties of these products. These 
assessments should include the estimation of antioxidant activity, anti-microbial activity and the assessment of 
shelf-life. Also, a larger scale sensory assessment is recommended as the salt concentration, as well as other 
changed sensory properties in the dairy products might affect the consumer acceptance. 

Nomenclature

CAGR – compound annual growth rate 
PBS – phosphate buffered saline 
NSAID – nonsteroidal anti-inflammatory drug

Acknowledgments 

This work is part of the European Horizon 2020 project AQUACOMBINE, which aims to demonstrate a 
biorefinery that includes the production of food and additionally extraction of bioactive compounds, production 
of feed, cosmetics, biogas, and biochar in combination with the use of aquaponics for the co-production of fish. 
This project has received funding from the European Union’s Horizon 2020 research and innovation programme 
under Grant Agreement No 862834. Any results of this project reflect only this consortium’s view and the 
European Research Executive Agency is not responsible for any use that may be made of the information it 
contains. Further the authors thank Frederike Reimold and Imke Lang for supporting the experiments and giving 
access to the laboratory and Sanketkumar Raval and Tutku Taşçı Çilak for proofreading the manuscript. 

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	In Vitro Analysis of the Anti-Inflammatory Activity in Dairy Products Containing Salicornia Europaea