Possibility of Concentrating Milk Enriched with Lemongrass (Cymbopogon) as a Preservative using Ultrafiltration Technology

Membrane technology, which has been introduced in the dairy industry over the past thirty years, is one of the more advanced technologies (Agarwal et al., 2015). It is preferred for many industrial processes that require the separation or concentration of products without the use of heat, particularly in the cheese industry and the fractionation of milk components (Arunkumar and Etzel, 2015).
       
Ultrafiltration membranes are semi-permeable membranes that prevent the passage of substances bigger than 30 nanometers, which is the basis for this method of concentrating milk. This spectrum encompasses most proteins. The separated fine components have molecular weights between 1 and 1000 kdalton and molecular sizes between 0.01 and 0.02 µm. The filtered milk is often separated into two portions: permeate, which comprises water, lactose, dissolved salts, NPN and dissolved vitamins and retentat, which contains proteins, lipids and dissolved salts. The liquid is typically forced to the surface under low pressure (Arunkumar and Etzel, 2018). The majority of industrialized nations, even those that are adjacent, now employ ultrafiltration technology more frequently in the production of different dairy products (Agarwal et al., 2015). This may be attributed to the several benefits this technology offers, chief among them being enhanced production and improved nutritional value (Muehlhoff et al., 2013). Low molecular weight (MWCP) milk protein concentrate (MPC) with a range of 10 kDa to 30 kDa is produced using ultrafiltration membrane technology. The membranes hold the proteins in place while allowing lactose and extremely minute particles to pass through Arunkumar and Etzel, (2015).
       
About 8.6% of total solids are found in cow milk; 3.2% are proteins; 5.4% are ash, non-protein nitrogen (NPN) and other tiny molecules; and the remaining 91.4% is water. Casein and whey proteins, which range in molecular mass from 14.4 to 150 kDa, make up the majority of milk proteins (Arunkumar et al., 2016).
       
One issue with low-pressure membrane technology is fouling, which compromises the precision and efficiency of the filtration process (Salunke et al., 2021). The buildup of particles that need to be separated, including dirt or proteins, on the surface or within the pores causes the treatment process to take longer. This increases energy and cleaning costs, decreases separation efficiency, or even totally clogs the pores (Muhammad et al., 2018). On the other hand, the accumulation of particles that create a thin layer on the membrane’s surface over the first few minute’s leads to the steady, balanced production. Because of electrostatic interactions on the surface, the flux steadily decreases and remains constant throughout the filtration process until the membrane stops filtering owing to a phenomena concentration polarization, necessitating washing as (Judd and Carra, 2021).
       
In an attempt to solve this issue, producers hurried to develop membranes that are lower pollutant and more selective in their manufacturing process Prestes et al., (2022), as well as regular cleaning systems, transverse speed control, uniform membrane pressure, the use of ultrasonic waves, ceramic membranes and other potential future fixes (Marella et al., 2013).
       
Lemongrass (Cymbopogon citratus) is native to Asia and in all Asian regions but now grows all over the world Sharma et al., (2022), for its subtle flavor in its fresh and dried leaves common to Asian cuisine and use in curries, tea, soups and milk, it is also suitable for cooking with poultry, seafood and fish (Wifek et al., 2016). And it was also used in folk medicine (Singh et al., 2011), where it was mixed with tea as a sedative and to treat various fever-related diseases. Additionally, it served as an immune stimulant in India, Egypt and Iran Amirdivani and Baba, 2011; Tilaye et al., 2018). The antimicrobial activity of lemongrass is due to its high content of phytochemicals such as citrate and essential oils (Zulfa et al., 2016; Al-Hamdani, 2022). Currently, in our country, there are many cow breeders and an abundance of milk. We must investigate the possibility of filtering and concentrating the milk using internationally advanced techniques, such as ultrafiltration technology and its application with the addition of lemongrass to the milk as a preservative and its effect on the concentrated milk produced chemically and microbial.

Five kilograms of raw cow’s milk was brought from the College of Agriculture/University of Baghdad/Abu-Ghraib to the laboratory and placed in the refrigerator. Experimental testing was conducted after preparing the instruments in the laboratory of the Ministry of Science and Technology/Environment and Water Department, using flat sheet ultra-filter membrane with 10 kdalton (Snyder USA) made from polyethylene-sulphone (PES) and surface area of 266 cm² and two percentage of lemongrass extract (100 mg/ml, 200 mg/ml) .
 
Equipment
 
To complete the experiment, the milk concentration process was carried out using Membrane testing device manufactured by the American company as shown in (Fig 1).
 

 
Stages of operating the system
 
The process was carried out to achieve a concentration approximately 2.7 times calculated based on the fat concentration, with an estimated retention rate of almost 99% and it took about a 3 hour and a half. For this study, five kilograms of fresh milk were used. It underwent pasteurization at 73°C for 15 minutes, followed by cooling to a temperature of 5°C. Subsequently, the milk was heated at a temperature of 35±2°C and a press by pumping it into the separation chamber (sepa), as illustrated in (Fig 1) the pressure and flow rate are monitored by recording the readings of the pressure gages and set the stopwatch to read the gradients of cylinder in which the permeate filter is collected  respectively., Samples of 50 ml of milk were taken in sterile glass containers upon receipt, from the center and the filtrate during the concentration process every 15 minutes. Subsequently, the samples taken were subjected to chemical tests to determine the changes in composition between the milk and the concentrate, comparing them with the filtrate.
 
Analytical methods
 
Analysis was performed on milk, UF concentrated samples. The Bradford method was chosen to measure the percentage of protein concentration in milk, which is a spectrophotometric method based on the principle of shifting the absorbance of the blue dye under acidic conditions from brown to blue. The spectrophotometer has a wavelength of 595 nm and the standard curve was made, then the readings were taken for the different models and the protein concentration was measured compared to the standard solution according to Bradford (1976); Schiano et al., (2017).
       
The percentage of lactose in whey was determined using the colorimetric method, based on its sugar reduction ability in the presence of substances that aid reduction (copper sulfate solution, sodium tungsten solution, sulfuric acid, phenol and lactose standard solution). The absorbance was then measured at a wavelength of 490 nm, following the procedure outlined by (Hieda et al., 2018).
       
The resultant concentrated milk were microbiologically examined for total bacterial count, mold and yeasts count and coliform group according to American Public Health Association (Vinderola and Reinheimer, 2000).

Ultra-filtration measuring
 
Fig 2 showed the scheme of working efficiency of the Ultra-filtration measuring on 10 kdalton to isolate milk protein in the form of concentrated milk and and exudes the lactose at pressure 2 bar.
 

 
Flow rate effect
 
Fig 3 represents the effect of changing the flow rate with constant  transmembrane pressure at 2 bar and the values of the flow rates are (0.15,0.25 L/min). The graph shows the flow decreasing with time. This rapid decrease occurs at the beginning of filtration and slows down when continuing, for both values. This is due to the decrease in the protein concentration at the boundary layer of the membrane, which leads to a decrease in the concentration polarization and with Continuing filtration, the protein is better preserved after approximately 25 minutes (Porter, 1972). The viscosity of the solution gradually increases due to the total solids it contains. This accumulation of dissolved materials spreads the protein molecules in the other direction, away from the membrane, because they spread from the higher concentration to the lower concentration. Fortunately, the protein concentration decreases from the surface of the membrane to the top of the boundary layer, its concentration is the same as the concentration of the bulk current through the membrane (Lipnizki, 2010).
 

 
Pressure effect
 
Fig 4, showed the filtration mechanism which can be followed by changing the pressure values (0.5, 1 and 2 bar) while maintaining a constant flow rate of filtration (0.15 L/min). The flux can be observed over time for the first 20 minutes and for all values   due to the natural decrease in hydraulic pressure on the surface of the membrane, which leads to the precipitation of dissolved substances in the milk, forming a layer of cake, which causes an increase in Polarizes protein and total solids (Li et al., 2017). The small filter pores that do not allow the passage of protein molecules larger than 10 kDa such as α-lactalbumin and its molecular weight is 14 kDa β-lactoglobulin with a molecular weight of 18 kDa. This could be a result of the gradual reducing flux over the 90-minute filtration period  (Ferrer et al., 2011).
 

 
Change in protein concentration over time
 
Fig 5 showed the change in protein concentration with time when the filtration conditions were fixed at the pressure and flow rate. Samples were taken every 15 minutes. From the graph, it can be seen that the percentage of protein coding in the milk increased from 3.2 mg/l to 8.1 mg/l and this increase can be monitored after 20 minutes of filtration (Meyer et al., 2015).
 

 
Change in total solids percentage with time
 
Fig 6 showed the change of percentage of total solids with time in minutes for milk concentration using ultrafiltration technique which was used in this study. Through the results, an increase in the percentage of total dry matter can be observed during the filtration process with a significant difference. The average rate at the beginning of the filtration process for raw milk reached 11%, while the average rate at the end of the filtration reached 24%. The reason is attributed to the presence of some dissolved components in the water such as mineral salts, lactose and others, which are included in the dry ingredients, which the membrane cannot seize. These results are similar to what was founded by (Moreno-Montoro et al., 2015).
 

 
Microbial assay
 
The contamination of membranes with microbes is a significant concern in the dairy industry, posing limitations on the use of membrane filtration. This includes issues such as pore blockage and the formation of a layer of various sediments and bacteria present in milk, commonly referred to as a ‘cake.’ These challenges can reduce efficiency and hinder the preservation of milk after concentration for an extended period. To address these concerns, an extract of lemongrass was prepared with two concentrations: the first at 100 mg/L and the second at 200 mg/L and added to the milk concentrate. Both concentrations were cultured on standard media for the purpose of conducting a biological examination, The results of the current study on the total bacterial counts (T.C.) of concentrated and fortified milk at a concentration of 100,200 mg/L milk from lemongrass extract are represented in (Table 1).
 

       
The results of the study showed a highly significant decrease (p≤0.05) in the total bacterial counts for both concentrations, especially after 24 hours of storage in the refrigerator, while increased significantly and by a small percentage, the total number of bacteria for both concentrations of 100 and 200 mg/L became (62 × 10³) and (50 × 10²) respectively after 48 hours of storage (Knight-Jones et al., 2016), but this increase is much less than what was stipulated in the Iraqi standard (1/693, 1988).  to the acceptable microbial limits in concentrated milk. The reason for the high decrease in bacterial numbers is attributed to several factors, including passing the concentrated milk through many heat treatments and the filtration used ultrafiltration for this study, which leads to the absence of microorganisms. In addition, the use of the lemongrass, which contains many active substances such as poly phenols, flavonoid, polysaccharides, also contributed to preserving the microbial load of the laboratory-produced concentrated milk (Tilaye et al., 2018). The presence of a high percentage of solids in the concentrated milk, represented by sugar (lactose) and the concentration of the milk generated another pressure on the contaminated microorganisms, which increases the effect of thermal treatments on them, as most anaerobic microbes, if they exist, can survive and have the ability to produce high sugar concentrations if they are packaged and stored well. These results were identical to what was found by (Knight-Jones et al., 2016; Muhammad et al., 2018)

Milk filtration methods rely on the membrane’s selectivity for proteins in particular, as well as on the membrane’s general selectivity for proteins and flow rate, which are governed by the membrane’s pressure and flow rate laws. Based on the findings, the proper operating parameters were established, which included a flow rate of 0.15 L/min and 2 bar, as well as the percentage of particulates and lactose isolation, in order to concentrate the milk to 2.7 times. Taking into account the flow rate entering the system, the size of the pores, the molecular weight of the protein molecules and the total number of dissolved components in the raw milk, we can deduce that the flow rate is all connected. The results of the study also showed that adding 200 mg/L of lemongrass extract to milk significantly reduced the total bacterial count, which confirms to us the effectiveness of lemongrass as a preservative in terms of microbial growth. It was found the change of the total solid and protein were excellent percentages with time of filtration and also more acceptable to the consumer in terms of flavor, color and overall appearance, making it more attractive.

I would like to extend my sincere thanks to the Market Research and Consumer Protection Center at the University of Baghdad in Iraq and the Ministry of Science and Technology, specifically the Directorate of Environment and Water in Baghdad. Their significant assistance and support were instrumental in completing this research.

We, authors of this research project collectively declare that we do not have any conflicts of interest related to this research. We have no financial interests, personal relationships, or any other factors that could potentially influence the impartiality and objectivity of this research. We affirm our commitment to conducting this research in accordance with the highest ethical standards and in compliance with all relevant institutional or organizational policies. We acknowledge that the information provided in this statement is true and complete to the best of our knowledge.