Abstract: Background and Objective: The adulterated milk could be processed into fluid or other dairy products such as yoghurt and cheese. Milk is not cooled after the milking process or stored for a long period before processing so, the acidity increased and the milk became unfit for heat treatments. So, some raw milk suppliers may neutralize the milk acidity by neutralizing agents to be suitable for processing. So this study aimed to validate a suitable method for detecting the neutralizing substances in milk, yoghurt and soft cheese. Materials and Methods: The spectrophotometric method using FeCl3 was used to determine the lactate in sour milk and neutralized milk as well as yoghurt and soft cheese made from neutralized milk. Results: Results confirm that the spectrophotometric method could be used for the determination of the lactate in milk even after neutralizing by ammonia, sodium hydroxide or tri-sodium phosphate as there was no significant difference between sour milk (pH 6.2) before neutralization or after neutralization in lactate levels. The yoghurt and soft cheese physiochemical properties did not seem to be impacted by milk neutralization. Conclusion: The spectrophotometric method is unable to detect the neutralizing substances in yoghurt made using neutralized milk.
INTRODUCTION
Milk is the main source of proteins, fats, carbohydrates, vitamins and minerals that are all important for bodybuilding and health. It is consumed as a fluid product or converted into a variety of dairy products. For safety, milk flavour, shelf life and consumer satisfaction, milk must be quickly cooled and held at low temperature after milking until pasteurized or processed to dairy products. Keeping the milk without cooling becomes critical especially in warm weather. One of the important criteria of raw milk acceptance in processing plants is its titratable acidity and pH. Acidification in milk is mainly due to lactic acid bacteria which rapidly grow in absence of efficient cooling after milking1,2. The anion lactate, which is the final product of lactose fermentation by lactic acid bacteria, is an indicator of milk quality3. Therefore, high lactate values in milk indicate inefficient cooling and/or poor farm and supply chain practices4. Sometimes, milk producers use a small number of alkaline solutions such as sodium hydroxide, sodium carbonate and sodium phosphate to neutralize acidity in milk so that raw milk does not exceed the acceptable acidity limit5,6.
The adding of neutralizer substances decrease milk quality and may lead to serious consumers’ health issues7. Caustic soda (NaOH) contains sodium that acts as a slow poison for hypertension and heart patients8,9. It also prevents the body from using an essential amino acid in milk (Lysine), which is needed by growing infants10. Adding carbonates in milk causes gastrointestinal problems including gastric ulcer, diarrhoea, colon ulcer and electrolytes disturbance11,12. Carbonates and bicarbonates also cause interruptions in growth hormone signaling13.
The amount of milk and dairy products consumed worldwide makes these products target for potential adulteration with financial benefits for producers14. For that reason, it is important to protect the consumer from adulterated milk and dairy products by accurate results using suitable methods for the detection of adulteration. The standard method of Titratable Acidity (TA) is expressed as % lactic acid15, which is also known as Dornic acidity16,17. Also, the Association of Official Analytical Chemists (AOAC) recommend this method for acidity determination in milk18. Since milk acidity is due not only to lactic acid but also, the presence of caseins, albumins, CO2, citrate and phosphates, the results inducted by the TA method always over-estimate acid development18. Furthermore, the illegal use of neutralizing substances makes TA a useless method for detecting acid developed after milking. Therefore, the application of lactate detection method that works in case of added neutralizing substances has become very important especially in developing countries.
There were several efficient methods to determine lactic acid as the final product of fermentation such as ion-pair, ion-exclusion and reversed-phase chromatography19,20. In addition, oxidation of lactic acid can be oxidized to acetic aldehyde that can be quantified. Also, it is possible to use Atomic absorption spectroscopy which proved to estimate the abnormal rise in minerals concentration due to application to neutralizers21. Recently, a spectrophotometric method for lactic acid determination has been developed for the detection of the end product of the reaction between lactate ions and FeCl322.
The objective of the present study was to validate the spectrophotometric method of lactate determination for the detection of milk adulteration by neutralizing substances. In addition, the effect of the neutralizing substances on the physicochemical and rheological properties as well as the yield of curd produced from acid and rennet coagulation were also assessed.
MATERIALS AND METHODS
Study area: The study was carried out at the Laboratory of Dairy Microorganisms and Cheese Research, Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University, Egypt from March-October, 2019.
Validation of a spectrophotometric method to detect neutralizing substances
Milk samples preparation: Fresh full-fat cow's milk Table 1, obtained from the Farm of Faculty of Agriculture, Alexandria University was divided into three parts and received the following treatments: (a) control untreated milk (acidity of 0.17%, pH of 6.78), (b) milk was acidified with natural flora to pH 6.4 and (c) milk was acidified with natural flora to pH 6.2. Acidified milk was then divided into three parts and each part was neutralized by one of the following neutralizers: sodium hydroxide 10%, trisodium phosphate 10% or Ammonia 37% to ~ pH 6.7. Milk composition was determined by Miko Scan (FT2, FOSS, Denmark). The pH value of milk was measured using a laboratory pH meter (Milwaukee Mi 150). Titratable acidity was determined as % of lactic acid according to the AOAC18.
| Table 1: Analysis of raw milk | |
| Parameters | Values |
| Total solids (%) | 12.66 |
| pH | 6.78 |
| Titratable acidity as lactic acid (%) | 0.17 |
Standard curve of lactate: Serial dilutions of lactic acid were prepared from the stock solution (80 g L–1) using two-fold dilutions according to Borshchevskaya et al.22. A total of 50 μL of each dilution containing lactic acid were added to 2 mL of a 0.2% FeCl3 and stirred for 5 min and absorbance was then measured at 390 nm (UV/Visible spectrophotometer, Pg T80+, England), against the reference solution (2 mL of a 0.2% FeCl3 solution). Three replicates were carried out.
Determination of lactate in milk samples by the spectrophotometric method: All milk samples (as treated above) were warmed to 40°C and coagulated by rennet in 30 min. After enzymatic coagulation of each milk sample, whey was separated by centrifugation (6000 rpm). In the case of lactate determination in soft cheese (enzymatic coagulation) and yoghurt (acid coagulation), whey after centrifugation was diluted 10 folds.
A total of 50 μL of each sample was added to 2 mL of a 0.2% FeCl322. The concentration of lactate in each sample was calculated using the standard curve.
Studying the effect of neutralizers on the physicochemical properties of acid and rennet curds
Milk preparation: Fresh full fat cow's milk was divided into two batches, batch 1 as control with normal pH (6.78) and batch 2 fermented with yoghurt starter (Lofast 259A, SACCO, Italy) to a pH value of 6.3. Milk at pH 6.3 (batch 2) was then divided into three parts. In part A, the pH was adjusted to 6.8 using Sodium hydroxide (10%), in part B, the pH was adjusted to 6.8 using Trisodium phosphate (10%) and in part C, the pH was adjusted to 6.8 using ammonia solution. Milk from the three parts was used in making yoghurt and soft cheese.
Yoghurt making: All milk samples (control and parts A, B, C) were heated at 90°C for 10 min. After heat treatment, the milk was cooled to the (42°C) followed by thermophilic starter culture (Lofast 259A, SACCO, Italy) addition. When the pH of yoghurt reached 4.7, samples were cooled to 4±2°C23.
Soft cheese making: Soft cheese was processed according to Awad et al.24 Milk samples were pasteurized at 63˚C for 30 min. Milk was transferred to cheese vats and a commercial starter culture (DVS Lofast 259A, SACCO, Italy) and CaCl2 (0.012%) were added to milk at 40°C. The inoculated milk was held for 1 hand then NaCl was added at a level of 5% (w/w). A suitable amount of chymosin preparation (2% Chymax, Chr. Hansen, Hørsholm, Denmark) was added to coagulate the milk in 90 min. The crud was then transferred to moulds, which were lined with cheesecloth. After 2-3 hrs, a plate and weights were placed to compact the curd. The weights were removed after 4-6 hrs and the cheese mass was divided with a knife into blocks and placed in storage cans.
Physicochemical properties of dairy products (yoghurt and soft cheese): The pH of yoghurt and cheese was measured using a laboratory pH meter model (Milwaukee Mi 150). Moisture was analyzed by the oven method18, total protein by macro-Kjeldahl25 and fat content by the Gerber method18. While total solid, protein, fat, lactose and freezing point in whey were measured using Miko Scan (FT2, FOSS, Denmark).
Cheese yield calculation: The actual yield of cheese was determined as the quantity of cheese obtained from a given quantity of milk26.
Measuring the syneresis in yoghurt: Syneresis of yoghurt samples was determined during storage periods using the centrifugation method as described by Saffon et al.27.
Texture analysis: The textural characterization of yoghurt was evaluated by back extrusion (Stable Micro Systems Ltd., Godalming, UK) with a 35 mm disc28. The speed was 1 mm s–1, the distance target was 30 mm and the trigger was 10 g. In cheese and curd, the speed was 1 mm s–1, the distance target was 10 mm and the trigger was 5 g. The results were reported as the mean of three measurements.
Statistical analysis: Data reported are the average of 3 replicates and 3 measurements for each trial. The SAS software package29 was used for one-way ANOVA. Differences were considered significant at p<0.05.
RESULTS AND DISCUSSION
Validation of the spectrophotometric method to detect neutralizing substances: The result of Fig. 1 shows the standard curve of the absorbance of iron-lactate solution (A) versus the concentration of lactic acid (c). The obtained statistical assurance is approximation 0.9989 as calculated from the standard curve.
Data in Table 2 show that the spectrophotometer method of Borshchevskaya et al.22 using FeCl3 was suitable to determine if milk was neutralized by determining the concentration of lactate. The lactate was significantly increased after fermentation of milk and dropping the pH from 6.78-6.2.
| Fig. 1: | Standard curve of lactate concentration |
| Table 2: Lactate concentration (g L-1) in artificial adulterated milk with some neutralizer agents at different pH | ||
| Samples | pH value |
Concentration of lactate (g L–1) |
| Control 1 | 6.78 |
1.2022±0.01B |
| Control 2 | 6.2 |
1.5744±0.00A |
| Treatment with NaOH | 6.71 |
1.5488±0.00A |
| Treatment with Na3PO4 | 6.74 |
1.5578±0.00A |
| Treatment with NH3 | 6.79 |
1.5330±0.03A |
| Control 1 | 6.78 |
1.2022±0.01B |
| Control 3 | 6.4 |
1.5248±0.01A |
| Treatment with NaOH | 6.77 |
1.5248±0.01A |
| Treatment with Na3PO4 | 6.79 |
1.5330±0.00A |
| Treatment with NH3 | 6.77 |
1.5661±0.00A |
| Control 1: Refer to natural milk without any additives, Control 2: Milk after reducing, pH to 6.2, Control 3: Milk after reducing pH to 6.4, *Same letters indicate no significant differences at the 95% confidence level. Different letters refer to differences among samples. Mean±standard error | ||
The lactate concentration was not affected by the neutralizer addition to milk as there were no significant differences between acidified milk before and after neutralization. Detection of milk adulteration by neutralizing agents would provide useful information to dairy processing plants. The addition of neutralizing agents in milk did not lead to significant changes in physicochemical characteristics of milk, such as freezing point, density, alcohol stability, acidity and pH (Data not shown). Therefore, specific qualitative tests for the detection of these substances are required in the dairy industry to test the quality of receiving milk. Silva et al.30 couldn’t detect sodium hydroxide used to neutralize acidity in the milk. The neutralizing agent is a challenge for food safety and human health as they can cause serious health problems31.
Milagres et al.4 developed and validated the HPLC method to detect lactic acid in sour milk and after neutralization. However, such methods are expensive and HPLC may not be available especially in countries where adulteration is a common practice. Therefore, the spectrophotometric method would be a simple and easy method to detect adulteration and protect consumers with a relatively low running cost.
Effect of neutralizing agents on the properties of yoghurt and soft cheese: Data in Table 3 show that the titratable acidity % of yoghurt decreased by adding the neutralizing substances to the sour milk before yoghurt making when compared to yoghurt made using non-neutralized milk as control. The lactate concentration as measured by the spectrophotometric method, was significantly (p<0.05) higher in yoghurt samples made from neutralized milk than in the product made from normal milk. The concentration of lactate was very high in treatment 1 (neutralized with NaOH), followed by treatment 2 (neutralized with Na3PO4), while treatment 3 (neutralized with NH3) had the lowest concentration. While there were no significant differences in lactate concentration among sour milk samples before or after neutralization with NaOH, NH3 or Na3PO4 (Table 2), there were differences in lactate concentration in yoghurt and cheese samples made from milk neutralized by NaOH and NH3. This could be caused by differences in the impact of the neutralizing agents on the metabolic activity of starter cultures. Acidity was significantly higher in the control yoghurt than in the samples made from neutralized milk (Table 3). While the lactate concentration in yoghurt treatment-1(neutralized with NaOH) was three times that in control its acidity was only 10% higher. Whereas the lactate concentration in yoghurt treatment-3 (neutralized with NH3) was about 40% of that in yoghurt treatment-1, both samples had the same acidity. This may support our hypothesis that there are differences in the impact of the neutralizing agents on the metabolic activity of the starter culture.
| Table 3: Physicochemical properties of yoghurt and cheese, which produced from artificial adulterated milk with some neutralizer agents | |||||
| Samples | Concentration of lactate ( g L–1) |
Acidity (%) |
Total solids (%) |
pH |
Syneresis (%) |
| Yoghurt analysis | |||||
| Control | 2.9253±0.04D |
0.771±0.00A |
15.35±0.08AB |
4.88 |
44.13±0.03A |
| Treatment Y1 | 8.7143±0.14A |
0.698±0.00B |
15.44±0.20B |
4.8 |
41.33±0.16B |
| Treatment Y2 | 7.9700±0.00B |
0.664±0.01C |
16.23±0.51A |
4.89 |
37.28±0.32C |
| Treatment Y3 | 3.9177±0.04C |
0.702±0.01B |
16.14±0.36AB |
4.88 |
37.12±0.07C |
| Cheese analysis | |||||
| Control | 1.6848±0.00C |
0.380±0.01C |
35.68±60.13A |
6.46 |
ND |
| Treatment CH1 | 3.6696±0.19A |
0.615±0.02A |
35.39±0.63A |
5.99 |
ND |
| Treatment CH2 | 2.6772±0.16B |
0.52±0.03B |
35.08±0.56A |
6.24 |
ND |
| Treatment CH3 | 2.2637±0.14BC |
0.24±0.001D |
34.58±0.07A |
6.67 |
ND |
| Treatment Y1: Yogurt made from adulterated milk with NaOH, Treatment Y2: Yogurt made from adulterated milk with Na3PO4, Treatment Y3: Yogurt made from adulterated milk with NH3, Treatment CH1: Soft cheese made from adulterated milk with NaOH, Treatment CH2: Soft cheese made from adulterated milk with Na3PO4, Treatment CH3: Soft cheese made from adulterated milk with NH3. ND: Not determined. *Same letters indicate no significant differences at the 95% confidence level. Different letters refer to differences among samples. Mean±standard error | |||||
| Table 4: Yield of cheese produced from adulterated milk with some neutralizer agents | |
| Samples | Yield (%) |
| Control | 21.09±0.0B |
| Treatment CH1 | 20.49±0.0C |
| Treatment CH2 | 20.55±0.0C |
| Treatment CH3 | 23.18±0.0A |
| Treatment CH1: Soft cheese made from adulterated milk with NaOH, Treatment CH2: Soft cheese made from adulterated milk with Na3PO4, Treatment CH3: Soft cheese made from adulterated milk with NH3. *Same letters indicate no significant differences at the 95% confidence level. Different letters refer to differences among samples. Mean±standard error | |
The lactate concentration in soft cheese treatment-1 (neutralized with NaOH) was about two times higher than that in the control sample but the lactate concentration was only 1.6 and 1.3-times higher in treatments 2 and 3, respectively than in the control sample. This finding is similar to what we found in yoghurt but the titratable acidity in soft cheese is different from that in yoghurt. The acidity in treatment-1 (neutralized with NaOH) is significantly higher than that in control by about 1.6 times while the acidity in treatment-3 (neutralized with NH3) is significantly lower than in control. Some differences between yoghurt and soft cheese include salt addition at 5% and concentration of proteins and fat by drainage. The addition of salt to cheese milk could influence the metabolic activities of the starter in soft cheese. The obtained results confirmed that the determination of lactate by the spectrophotometric method is not suitable to detect the neutralization of milk used in yoghurt or cheese making. While the titratable acidity in yoghurt was not influenced by milk neutralization it increased in soft cheese made from milk neutralized by NaOH and decreased in cheese made from milk neutralized by NH3.
The result of Table 3 shows that making yoghurt from neutralized milk has little effect on moisture and total solids contents. Syneresis (%) was lower decreased in yoghurt made from neutralized sour milk than normal milk. Using ammonia as a neutralizing agent produced the lowest syneresis (37.12%) in yoghurt among the other treatments. At the same conditions, data in Table 3 indicated that there were non-significant differences among treatments and control cheese in moisture content. Kamthania et al.32, Ahirwar33 reported that the physicochemical properties of milk did not change by adulteration of milk with neutralizing substances.
Effect of neutralizing substances on cheese yield and whey properties: There were significant differences in cheese yield among treatments and between them and the control Table 4. Using ammonia as a neutralizing agent caused increased cheese yield, while sodium hydroxide and tri-sodium phosphate decreased it as compared with control. This means that the milk adulterated with sodium hydroxide or tri-sodium phosphate produced a negative effect on cheese yield unlike adulteration with ammonia. There were no significant differences among the yield of cheeses made from milk neutralized by sodium hydroxide (treatment CH2) and tri-sodium phosphate. Whey of cheese made using milk neutralized by tri-sodium phosphate contained higher fat and protein than that of cheese made using milk neutralized by sodium hydroxide or ammonia (Table 5).
However, using sodium hydroxide or ammonia as neutralizing substances decreased the fat and protein in acid and sweet whey than those in control (cheese made from natural milk). The freezing point of acid and sweet whey was not affected by neutralizing substances as there were no differences between treatments and control (Table 5). The low cheese yield and high losses of fat and protein in whey of cheese made using milk neutralized by tri-sodium phosphate are related to the solubilization of colloidal calcium phosphate in casein. Neutralization with tri-sodium phosphate occurs more slowly than with sodium hydroxide.
| Fig. 2(a-d): | Rheological properties of curd producing from acid coagulation (yoghurt) and enzymatic coagulation (cheese) 1: Curd neutralized with sodium hydroxide, 2: Curd neutralized with trisodium phosphate and 3: Curd neutralized with ammonia |
| Table 5: Chemical analysis of yoghurt and cheese whey | ||||||
| Samples | Protein |
Fat |
TS |
SNF |
Freezing point |
Lactose |
| Yoghurt whey analysis | ||||||
| Control | 0.74±0.03B |
0.44±0.01A |
6.47±0.01A |
6.03±0.01AB |
-0.33±0.0A |
4.08±0.01A |
| Treatment wY1 | 0.68±0.01BC |
0.14±0.01C |
6.10±0.06B |
5.96±0.05B |
-0.33±0.0A |
4.05±0.03A |
| Treatment wY2 | 0.94±0.01A |
0.23±0.01B |
6.35±0.01A |
6.12±0.01A |
-0.32±0.0A |
3.30±0.01C |
| Treatment wY3 | 0.61±0.01C |
0.14±0.00C |
5.80±0.01C |
5.66±0.01C |
-0.31±0.0A |
3.79±0.01B |
| Cheese whey analysis | ||||||
| Control | 0.94±0.01B |
0.20±0.01B |
6.90±0.01A |
6.78±0.01A |
-0.40±0.01A |
4.60±0.01A |
| Treatment wCH1 | 0.77±0.01C |
0.10±0.00C |
6.45±0.01B |
6.35±0.01B |
-0.36±0.01A |
4.48±0.01B |
| Treatment wCH2 | 1.04±0.01A |
0.96±0.01A |
6.92±0.01A |
5.96±0.01C |
-0.36±0.01A |
4.39±0.01B |
| Treatment wCH3 | 0.76±0.01C |
0.10±0.00C |
6.10±0.01C |
6.03±0.03C |
-0.32±0.01A |
4.05±0.01C |
| Treatment wY1: Yogurt whey produced from adulterated milk with NaOH, Treatment wY2: Yogurt whey produced from adulterated milk with Na3PO4, Treatment wY3: Yogurt whey produced from adulterated milk with NH3, Treatment wCH1: Soft cheese whey made from adulterated milk with NaOH, Treatment wCH2: Soft cheese whey made from adulterated milk with Na3PO4, Treatment wCH3: Soft cheese whey made from adulterated milk with NH3. *Same letters indicate no significant differences at the 95% confidence level. Different letters refer to differences among samples. Mean±standard error | ||||||
Simultaneously to increasing the net negative charge with sodium hydroxide, calcium bridging would occur between individual proteins within the colloidal particles, keeping them intact and preventing further disintegration of the supramolecular structure34. Casein micelles in milk neutralized with ammonia would then be expected to have a more compact structure due to cross-linking by numerous calcium bridges predominantly occurring via phosphoserine in caseins.
Effect of neutralizing substances on the texture properties of yoghurt and soft cheese: Milk neutralization impacted the rheological properties of yoghurt and soft cheese (Fig. 2a-d). The firmness is lower in yoghurt made from treated milk than in control. Milk Neutralization with ammonia produced higher firmness than other neutralizing substances (Fig. 2a). In cheese curd, adding trisodium phosphate increased firmness compared to control. Adhesiveness was significantly lower in yoghurt made from neutralized milk than in control. In contrast, adhesiveness was higher in cheese curd made from milk neutralized by sodium hydroxide and tri-sodium phosphate (Fig. 2b). Adding neutralizing substances to sour milk reduced the consistency in yoghurt (acid coagulation). Cheese curd made using milk neutralized with tri-sodium phosphate has the highest consistency value (Fig. 2c). All neutralizing substances caused a decrease in the viscosity value of yoghurt (Fig. 2d).
Trisodium phosphate (food grade) is allowed as an acidity regulator in milk powder but not ammonia or sodium hydroxide. However, there is no guarantee that adulterants would use expensive food-grade substances to neutralize milk which can cause health problems. Yoghurt made from milk neutralized with ammonia has acceptable physicochemical properties. Moreover, yoghurt made from milk neutralized with ammonia had better syneresis and good texture properties than control. The soft cheese made from milk neutralized with ammonia had a better yield and textural properties than the control. Because it is not possible to detect neutralization in yoghurt or cheese milk by testing the finished product with spectrophotometric, raw milk should be tested before processing.
CONCLUSION
The spectrophotometric method using FeCl3 was able to detect the neutralizers in milk. This is a simple, reliable and cheap method that can be used by quality control to detect milk adulteration by chemicals. The texture and physiochemical properties of yoghurt and soft cheese were not impacted by milk neutralization. However, since this method could not detect adulteration in yoghurt and cheese, it would be recommended for only raw milk testing before processing.
SIGNIFICANCE STATEMENT
This study discovers a new reliable and fast method to detect the adulterated milk by neutralizing agents that can be beneficial for the dairy industry to reject the adulterated milk with neutralizing substances. This study will help the authorities in food safety foundations to uncover the critical areas of the adulterate milk with neutralizing agents that researchers were not able to detect this substance in a short time.
ACKNOWLEDGMENT
Authors thank the Academy of Scientific Research and Technology (ASRT) for funding the project (JESORnumber32) entitled “Microbial and Chemical Safety Criteria of Traditional Egyptian Cheeses”.