Key findings:

The key findings of this study on plant-based cheese fortified with herbs (tofu) include: significant sensory differences in flavor, texture, cohesiveness, and color between treatments with nigella sativa, mint, and control; microbial tests showed reduced bacterial, mold, and yeast levels in herb-treated samples after two weeks; chemical analysis revealed variations in moisture, fat, protein, carbohydrates, and mineral content among treatments.

What is known and what is new?

The known aspect in this abstract is the use of tofu as a plant-based cheese alternative. The new contribution is the fortification of tofu with herbs to extend its storage period, leading to significant sensory, microbial, and chemical differences, highlighting the potential for enhancing nutritional value and shelf life in plant-based products.

What is the implication, and what should change now?

The implication of this study is that fortifying plant-based cheese with herbs can improve sensory attributes, microbial quality, and nutritional content. Changes needed include promoting the use of herb-fortified tofu cheese as a nutritious and extended-shelf-life alternative, emphasizing the importance of incorporating natural ingredients for enhanced product quality and consumer appeal

Vegetarian and other dairy-free diets are so popular. There are now many different options available, such as vegan cheese, which comes in a range of flavors and types. Tofu is a commonly used vegetable protein and is an unfermented soy product, a type of vegan cheese made from soy milk, as it is a food of plant origin with nutritional and health benefits. It was first discovered by a Chinese cook about 2,000 years ago in Japan, and tofu was introduced in the eighteenth century. It is a curd soybean curd as it is manufactured in a process very similar to the processes of manufacturing soft cheese from cow’s milk where soybeans are ground in water, then subjected to heating to be coagulated with minerals such as calcium or magnesium salts, and pressed into plates [1].

It should be noted that tofu is a versatile food as it has a very mild flavor, and can be enhanced by roasting it. The texture and flavor of tofu varies according to the use in coagulants. Tofu should be smooth, firm and firm while hard texture and rubber are unacceptable.

Its consumption is widespread across the globe, encompassing regions such as the Americas, Australia, China, Europe, India, Indonesia, Japan, Korea, Malaysia, New Zealand, the Philippines, Singapore, Thailand, and Vietnam, with a particular prevalence in East Asia [2].

Tofu is hailed as an exemplary source of protein, especially for adherents of plant-based diets, as it furnishes all the essential amino acids that the human body cannot synthesize endogenously, including isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine – nutrients crucial for daily dietary inclusion. Moreover, it constitutes a prominent source of calcium, iron, manganese, selenium, phosphorus, and a viable source of vitamin B1, copper, zinc, and magnesium. Notably, tofu has been recognized for its capacity to mitigate the risk of cardiovascular diseases [3] by reducing levels of deleterious low-density lipoprotein cholesterol, thereby promoting heart and vascular health. 

Furthermore, it safeguards against colon cancer and diminishes the likelihood of prostate cancer [4], while women who consume substantial quantities of tofu exhibit a reduced susceptibility to breast cancer.

Soy milk is recognized as a nutritionally valuable food product, owing to its richness in proteins and unsaturated fatty acids. It represents a suitable dietary option for individuals afflicted with lactose intolerance or exhibiting sensitivity to dairy products, as well as for those adhering to plant-based diets. Furthermore, soy milk and its fermented derivatives constitute robust dietary supplements, replete with highly efficacious antioxidant compounds [5]. Taxonomically, soybeans belong to the legume family and are classified as oilseeds, having been utilized as a food and medicinal source for over five millennia. They are regarded as one of the world’s most crucial food and industrial crops, owing to their chemical composition, comprising 40% protein, 22% fat, and 34% carbohydrates [6].

Soy milk offers several health benefits, serving as an excellent alternative to cow’s milk, particularly for individuals afflicted with lactose intolerance or sensitivity, as the consumption of lactose can trigger symptoms such as diarrhea, vomiting, flatulence, and cramping. Besides, soy milk is distinguished by its abundance of fiber and manganese, which is crucial for enzymatic functions within the body, as well as thiamine, which contributes to enhancing muscle and nerve functions. Soy protein has been demonstrated to lower blood cholesterol levels, thereby diminishing the risk of cardiovascular disease, while also conferring protection against osteoporosis and proving beneficial for individuals with hypertension due to its low sodium content [7].

Since the taste of soy milk is not palatable to everyone, the idea of manufacturing tofu emerged. Despite the importance of this product, its shelf life does not exceed 10 days. Therefore, the researchers aimed to manufacture tofu cheese from soy milk and fortify it with mint and nigella sativa herbs to extend its storage period. In view of the above, we seek in our research to address the following aims:

1. We aim to determine the sensory properties of the cheese. 

2. We also aim to determine the microbial properties of the cheese.

3. Lastly , we aim to determine the chemical properties of the cheese

In this section, we cover the materials used in this study and the methods we adopted for making the tofu cheese and evaluating its sensory qualities.

Table 1 lists the materials used to produce the tofu cheese, including the amounts and sources.

In this method we did the following:-

1. Weighed and prepared (6 cups) of soybeans, i.e. approximately 1 kg (cleaning and washing).
2. Soy seeds (2 liters of water) were soaked for 24 hours 
3. Measured 1 cup of soybeans per 3 cups of water 
4. The mixture was mashed using an electric mixer until it became a homogeneous liquid and then filtered with a piece of cloth (fidgeting).
5. Cooked the mixture on the fire at a certain temperature and then waited for it to become lukewarm.
6. Placed half a cup of acid per 1 cup of soybean milk 
7. Placed a quarter teaspoon of salt per 1 cup of soybean milk 
8. Allowed 45 minutes for the milk to curdle and then we put it in a cloth (fidgeting)

9. Then add the substance to the flavors such as mint or nigella, drain it and then leave it for the whole day until it becomes a cohesive cheese.

    

Firstly, Sensory Evaluation:

The sensory evaluation was conducted at the University of Baghdad / College of Education for Girls / Department of Home Economics, where the sensory evaluation of the cheese manufactured by (10) assessors with specialization was conducted according to the evaluation form prepared by (Musa, 1995) [8] represented by (7) degrees.

Table 2 sensory Evaluation Form for Cheese Samples.

Sensory assessment score ranges from 1-7

The highest score awarded to each sensory characteristic is 7 and the lowest score awarded to each sensory characteristic is 1.

 7= excellent, 6=very good, 5=good, 4=medium, 3=fair, 2=poor, 1=very bad

Secondly: Statistical Analysis:

The statistical program Statistical Analysis System – SAS (2018) [9] was used to analyze the data to study the effect of different parameters on the studied traits according to a completely randomized design (CRD), and the significant differences between the means were compared with the Least Significant Difference (LSD) test.

Thirdly: Microbial tests to estimate microbial growth:

An amount of 0.1 g of the sample was weighed, 15 ml of Normal Saline was added, and 0.1 ml of the emulsion was incubated on a plate prepared from Nutrient Agar for 18 hours at a temperature of 37 °C, and then the colonies were counted on the plate.

Fourth: Chemical tests for cheese:

The components of the cheese samples were estimated:

Weigh 0.2 of the sample H2SO4 acid and concentrated hydrochloric acid 1-4 and leave it for 16 hours in order to complete the digestion process. Then the samples are transferred to a heat source to change the color. It will be dark brown tending to black. The color will be changed on a heat source until it reaches a clear color after that.  Distilled water will be added to the model up to 50 ml and transferred to plastic containers for reading nitrogen and elements.

Protein determination: We take an amount of the digestate and add it to glass tubes belonging to the Kjeldahl device. Sodium hydroxide is added to it as a basic environment. It is received by 2% boric acid and a red dye. Then the nitrogen is released and it is received by the boric to change the color from red to green. We collect 35 to 50 ml of the sample and flush it with acid. Dilute hydrochloric acid to a standard of 0.05, then the color will return to red again according to the amount of acid consumed, and it is entered into a law to extract the amount of nitrogen and multiplied by a fixed number for the total protein.

As for the determination of mineral elements, an amount of the digestate is taken to read calcium in the optical flame device, and an amount of diluted acid is taken to read iron in the atomic absorption device.

As for phosphorus, reagents such as vitamin C and ammonium molybdate are added and read in a spectrophotometer.

Fat: We place the model in filter papers with a specific weight of 3-5 grams, tightly wrapped, and placed in an organic solvent for 16 hours. After that, the model is transferred to volumetric paper attached to the hydrometer from the top and a receiving flask from the bottom. Here, the thermal source will be used to estimate the fat again, but  With heat, and after an hour, the form is withdrawn and the solvent is recovered. Small amounts of the solvent are applied, placed in a 1.5°C convection oven, and we take the weight of the beaker again. Here the difference between the two weights will be the amount of fat, and the percentage of fat will be estimated using the Soxolite device.

Carbohydrates: We take a weight of 0.2 from the sample, add 1 molar hydrochloric acid to it, put it in a water bath at a temperature of 60 degrees Celsius, for 30 minutes, then filter the sample and add it, take a weight of 1 ml, add 5% phenol, and add H2SO4 to it.  The concentration is 5 ml, then the model is left for half an hour to read. We use a spectrometer at 490 nm.

3-1- Sensory Evaluation Results of Tofu Samples

Table (3): the sensory evaluation results of the tofu samples.

The results in Table (3) for the sensory evaluation of the manufactured cheese (tofu) showed no significant differences at the 0.05 P< level for the flavor attribute between the treatments with added nigella sativa and mint, which scored (5.4a) and (6.5a), respectively. However, significant differences were observed between these two treatments and the control treatment, which scored (4.0b). The mint sample had the highest flavor score. This can be attributed to the role of the active flavor compounds present in the mint and nigella sativa herbs. These findings are consistent with Mahgoubet al. (2013) [10], who found that adding 0.2% nigella sativa oil to cheese improved its sensory properties (flavor).

As for the texture characteristic, the results showed that there were no statistically significant differences at the level of (P<0.05) between the control and mint treatment, which amounted to (5.4 a) and (5.8 a), respectively, while significant differences were found between the previous two treatments and the added treatment.  It has Nigella sativa, which reached (4.1b).

Regarding the two characteristics: cohesion and sour taste, significant differences were found with statistical significance at the P<0.05 level) between the mint treatment, which amounted to (5.1a) (6.3a), and the control and nigella sativa treatments, which amounted to (4.1b), (5.0b), and (4.0).  b) (5.1b) respectively, while there are no significant differences between the previous two treatments.  The L.S.D value of the previous coefficients for the two studied characteristics (cohesion and sour taste) reached (0.983) and (1.19), respectively.  The researchers attribute the difference in the mint treatment from the previous two treatments in terms of the pungent taste to the mint herb containing active compounds such as menthol [11].

As for the color trait, the results showed that there were no statistically significant differences between the control and mint treatment, which amounted to (5.8a) and (5.5a), while significant differences were found between the previous two treatments and the nigella sativa treatment, which amounted to (4.1b), and the L.S.D value. The previous coefficients for the studied characteristic (color) are (1.26).

3-2-Analysis results and storage durations for soy milk cheese microbial growth.

Table (4): the microbial growth and storage periods for the cheese manufactured from soy milk

The results of the study appear in Table (4) of microbial growth and storage periods for cheese made from soybean milk. The average total number of bacteria in the cheese sample when treated with control on the first day was (27 x 10³). As for the total number of yeasts and molds, it was (25 × 10⁴) The percentage of the total number of bacteria, molds and yeasts increased after 7 days of storage and was (45 × 10³ and 47 × 10⁴), respectively. Other tests were conducted after 14 days of storage, and it was found that the percentage of microbes had exceeded that of the previous ones, and the percentage of bacteria had become (55 x 10³) while the percentage of yeasts and molds had increased to 57 x 10⁴).

As for the cheese sample to which nigella sativa was added, the percentage of bacteria on the first day was (25 x 10³) as for yeasts and molds, the percentage was (22 x 10⁴). The percentage of microbes increased after a week of manufacturing, so it was (39 x 10³) As for yeasts and molds, their percentage was (40 x 10⁴), as the percentage of microbes exceeded that after two weeks of manufacturing, as the percentage of bacteria, yeasts, and molds became (51 x 10⁴) and (50 x 10³) respectively.

As for the cheese sample with mint added, the percentage of bacteria on the first day of manufacturing was (23 x 10³), while the percentage of yeasts and molds was (20 x 10⁴). After two weeks of manufacturing, the percentage of microbes increased slightly to 47 x 104 and 45 x 10³, respectively, for bacteria, yeasts, and molds. This is due to its high moisture content, which makes it more susceptible to microbial contamination and cases of food poisoning after consumption, especially when it is not followed.  Sanitary conditions for its preservation, and this is consistent with (Garnieret al., 2017) [12].

We note from the above that adding herbs to tofu helped reduce bacterial growth, yeasts, and molds, but it did not eliminate them completely.  Dry plants contribute more to the biological benefits and antioxidant properties.  These plant herbs and their extracts have the ability to act as natural preservatives and antioxidants [13], and this is what we observed in the cheese sample to which nigella sativa was added, as the results showed that the percentage of bacteria, molds and yeasts decreased clearly on the seventh day and also after. Two weeks compared to the control sample for the same sample. This is consistent with the findings of Ali et al. (2017) [14], where adding oil found the maximum possible antimicrobial effectiveness on pathogens and improved the chemical, sensory, and microbial properties of the cheese.  (Hassanienet al., 2014) [15] also reported an antimicrobial effect of black cumin seed oil to cheese particularly when it is stored in a cool place.

After a fortnight, we noticed that the cheese fortified with mint displayed lower levels of bacteria, yeasts, and molds than those in the control sample. We also noticed that the sample with added herb in it displayed the highest decrease rate of the sample with Nigella sativa added to it compared to the control sample. This actually helps in prolonging the storage time frame of herbal fortified cheese. 

We can ascribe the aforementioned effect to the containment of peppermint herb such as menthol and menthone, which are known for their inhibitory capability for microorganisms. This comes in addition to the antimicrobial phenolic compounds that herbs have. These in turn can be utilized as an good replacement to synthetic antimicrobial agents often used in producing food. These findings are similar to Mustafa, et al., 2018 and Paswan et al., 2021) [16, 17] found in this regard. 

Cheese is often considered as a conduit for many microorganisms to grow. This is because it boasts a good humidity level as well as some carbohydrate, nitrogenous compounds, vitamins and minerals. All surrender it a safe home for microorganisms that in turn are instrumental in nurturing the biological and chemical conditions that determine a number of aspects pertaining to cheese such as its quality, nutritional value, and its validity for human consumption. Al-Saadi ( 2003) [18] contends in this regard that this leads to consumers to encounter cases of food poisoning in addition to potential damage and putrescence of the product.

3-3-The chemical analysis of the nutrients of soy-milk cheese.

Table 5 displays the components of the aforementioned analysis.

A glance at table 5 tells us that there are no statistically significant differences that we can see across the factors assessed for protein and carbohydrates. However, there does appear to be such significant differences in the remainder of the parameters for both moisture and fat. The percentage of protein in the control treatment was (10.00), carbohydrates (6.3%), and fats (8.30%). The percentage increase in protein came after integrating nigella sativa and mint. The former had (10.61%) while the latter had (10.34%) for both treatments. The higher protein percentage in both treatments compared to the control treatment is due to the fact that the fortified cheese has a lower moisture level than the non-fortified soy cheese, which leads to a higher concentration.  The percentage of solids in soy cheese is fortified with herbs, including protein.  It was also observed that the percentage of carbohydrates increased after adding the nigella sativa and mint herbs to (6.25% and 6.18%), respectively, compared to the control treatment, which was (6.03%). The reason is attributed to the addition of medicinal herbs to the cheese, which led to a decrease in the moisture percentage of the fortified cheese and an increase in the percentage of solids, including carbohydrates. Awad (2003) [19] reported a similar finding to ours in that moisture is inversely proportional in percentage of solids to the increase in cheese.

The table 5 also tells us that there are indeed statistically significant differences in humidity between the mint treatment on the one hand and the control and nigella sativa treatments on the other. The former reached (60.0 b) while the latter treatments amounted to (70.0 a) and (66.0 a), respectively. When it comes to the LSD value, moisture recorded a value of (5.037). Moisture, as it appeared, decreased in the herb-fortified treatments while it had a higher value in the control treatment at (70.0a). If we want to translate this finding we may ascribe it to the relative rate of solids that the added herbs displayed. This in turn facilitated the absorption of moisture. Al-Rawi (2020) [20] reported a similar finding to that.

Results from table 5 also tell us that both nigella sativa and mint treatments displayed statistically significant differences between them in terms of fat. The former stood at (8.80 a) while the latter was (8.16 b). Having said that, we did not find such differences between both these aforementioned treatments and the control treatment, which had (8.30 ab). We can also report an LSD value of (0.549) for fat. If we are to interpret the relatively high rate of fat in the Nigella sativa treatment we perhaps need to return to the fact that it is an oil seed, which is known for containing a high level of unsaturated oils. We can also report a decrease in the arithmetic mean of fat observed in the mint treatment. This particular finding may be interpreted as a result of the relatively low rate of fat in this herb. Al-Khalil (2020) and Al-Rawi (2020) [20, 21] both reported similar findings to ours in this regard.

3-4- The chemical analysis of nutrients in soy-milk cheese.

Table (6) displays the details of the aforementioned analysis.

It does appear from table 6 above that there are no significant differences in tofu’s elements. One exception that can be noted here is an increase in the arithmetic mean of calcium  in both: the Nigella sativa and mint treatments. The former amounted to (0.40%) while the latter amounted to (0.39%). The level of calcium mineral in the control treatment was (0.30%). Another element we can report here is iron, which displayed an increase of (0.66% and 0.65%), respectively, while that of the control treatment was (0.60%). Also, the Nigella sativa and mint treatments saw an increase in the arithmetic average of phosphorus amounting (0.31% and 0.29%), respectively, while the control treatment had (0.25%). If we are to interpret this increase, we may find reason in the high rate of minerals in soymilk as well as the herbs added to the mixture. Paswanet al. (2021) [17] note that these normally represent a rich source of vitamins, minerals and antioxidants.

• Herbs in dairy products can perform a dual function. Namely they serve as functional foods  as well as natural preservatives instead of synthetic ones, which have been reported to be of negative health effects.

• The sensory evaluation results we reported also showed that mint-fortified tofu cheese was superior to the other treatments.

• The adding of herbs to tofu cheese increased its shelf life.

• The addition of mint and Nigella sativa helped improve the texture and eliminate the undesirable vegetative taste of soybeans.

Recommendations:

• Conducting a study to compare the shelf life between regular cheese and cheese fortified with medicinal plants.

• Determining the effect of various medicinal plants fortified in soy cheese on the microbial content of the cheese.

• Studying the use of other medicinal plants and their fortification in food processing products to contribute to reducing cholesterol and triglyceride levels without leaving harmful side effects on human and animal health.

• Promoting health education towards increasing the consumption of plant-based cheeses for vegetarians and individuals with lactose intolerance.

• Increasing the consumption of medicinal plants due to their good flavor properties, antioxidant effects, and ability to improve liver and kidney tissues.

Funding: No funding sources.

Conflict of interest: None declared.

Ethical approval: The study was approved by the Institutional Ethics Committee of Indira Gandhi Medical College & Hospital.

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