The effect of various spices on functional properties of plant-based cheese analogue

This research purpose was to study the effects of various spices on functional properties of plant-based cheese analogue (PBCA). PBCA was made from Geotrichum candidum (Gc: 3%), glucono-δ-lactone (GDL: 0.25%), and bromelain: 8 × 105 U/g. The sample was commercial dairy cheese (control), PBCA 1 (Gc + bromelain), PBCA 2 (GDL + bromelain), PBCA 3 (Gc + GDL), and PBCA 4 (Gc + GDL + bromelain). The results of observations processed using ANOVA and DMRT. In this study, different spices affect the chemical and sensory features of PBCA (p<0.05). The chemical features results were ash content (1.92%; 1.51%; 1.57%; 1.74%; 1.39%), fat content (29.7%; 25.0%; 24.9%; 26.7%; 24.7%), moisture content (55.4%; 49.6%; 50.1%; 47.3%; 50.3%), protein content (6.39%; 17.5%; 17.2%; 19.5%; 17.0%), and salt content (0.69%; 0.29%; 0.31%; 0.32%; 0.28%), respectively. The sensory features results were appearance (7.52; 7.18; 7.27; 7.48; 6.85), flavor (8.05; 7.68; 7.33; 7.67; 7.71), creaminess (7.73; 7.35; 7.33; 7.67; 7.25), firmness (6.40; 7.04; 6.37; 6.66; 6.58), spread ability (7.82; 7.48; 7.40; 7.78; 7.25), and overall (8.44; 7.91; 7.80; 7.48; 8.03), respectively. The effectiveness index test result was 1.00; 0.45; 0.53; 0.00; and 1.00, respectively. In conclusion, PBCA 4 was the best treatment.


Introduction
Cheese is an excellent source of nutritional food for human health.Nutrients contained in cheese include protein and fat.Protein serves as a body building substance while fat acts as a backup energy source.In addition, the content of vitamins (A, Thiamine, Riboflavin, Niacin, Choline, Pantothenic acid, B6, Biotin, Folic acid, B12, C, D, and E) and minerals (Ca, Mg, K, Na, Fe, Zn, and P) in cheese is also beneficial for human health [1].According to [2], The source of milk affects the nutritional content of cheese significantly.Therefore, the selection of the right milk source can produce quality cheese and in accordance with society demand.
Recently, public demands for the industry to produce food to be able to produce food that has a positive impact on health and environmental sustainability have become a challenge for the food and beverage industry [3].This is because livestock activities cause a decrease in environmental quality.According to [4], livestock activities consume a lot of water to meet animal needs, animal feed plant needs, sanitation needs, and post-harvest product processing needs, as well as the potential for water pollution from fecal waste that is not treated properly.On the other hand, livestock activities are also still needed to meet the needs of the society for animal protein.According to [5], the need for animal products is still very necessary even though it causes negative impacts that will optimistically be minimized along with the development of livestock technology.Therefore, the selection of raw materials is important for the food and beverage industry to create a product in accordance with the demand of the society.One of the current trending demands is plant-based food.
Soymilk is one of the trending ingredients used to produce cheese.Soymilk has a role in maintaining human health.According to [6], soymilk is a good source of potassium to lower cholesterol levels in the blood and can maintain the body condition of people with low blood pressure.In addition, the combination of soymilk with fermentation technology produces a peptide bond that has a positive impact on human health.According to [7], soymilk fermentation using protease enzymes can increase the ability of ACE inhibitory to suppress the rate of pressure in the blood.The ability of ACE inhibitory can be increased even greater up to 48% if using a combination of lactic acid bacteria (LAB) and protease enzymes in the fermentation process [8].Moreover, soymilk is relatively more economical compared to dairy milk as a protein source [4].
PBCA is a cheese-like food product produced from milk, milk fat or milk protein either whole or partially add with emulsifiers, preservatives, flavor enhancers and so on derived from animals and plants.Therefore, the replacement of raw materials will certainly affect cheese production either nutritionally or economically.According to [9], the use of plant-based ingredients can reduce the cost of cheese production.Nevertheless, PBCAs have different sensory qualities than commercial dairy cheese.This is because PBCA can be distinguished by its type of fat content, the use of coagulating materials and fermentation ingredients.According to [10], appropriate formulations such as regulating the ratio of animal and vegetable fats, selection of certain enzymes as coagulating agents and the use of probiotics as fermentation ingredients have the potential to increase the various advantages of analogue cheese as one of the functional foods.Art in combination of various spices to produce cheese affects the quality of PBCA nutritionally and sensorially [11].
The World Health Organization (WHO) in 2001 has defined probiotics as living microorganisms such as LAB and yeast which are officially recorded to be able to provide health benefits to their hosts in certain amounts [12].According to [9], the use of probiotics can produce PBCA that have an aroma like commercial dairy cheese, but the texture and taste are significantly different.In addition, gluconoδ -lactone (GDL) was also used as a coagulant to produce cheese.According to [13], GDL and hydrolysis enzymes can be used to produce cheese because they are able to provide an acidic atmosphere without lowering the pH value of dairy milk based.A few years later, the trend of using plant-based ingredients as alternative raw materials to produce cheese continues to emerge such as the use of soy protein concentrate [10], milk from corn extract [14], and soymilk [15].
According to [15], the addition of Lactobacillus rhamnosus and L. plantarum apparently causes microstructural hardening of PBCAs, reducing their dispersion and making the product surface rougher than commercial dairy cheese.They also report that LAB as a fermentation agent is known to grow more slowly in soymilk than in dairy milk, therefore other spices are needed as an alternative.According to [16], cheese produced from soymilk turns out to have a distinctive taste, namely beany flavor which is generally not found in commercial dairy cheese.Alternatives that can be used to solve the problem is utillization of yeast [13], GDL, and bromelain enzyme [14] to create a plant-based cheese.
Yeast is one type of probiotic that can be used to produce functional food.According to [13], Geotrichum candidum is the one type of yeast that can remove beany flavor from soymilk because of its ability to produce lipase enzymes.The lipase enzyme can degrade fat into fatty acids and glycerol to remove the beany flavor from soymilk during the fermentation process [17].Moreover, the increase in soluble nitrogen and protein breakdown was desireable for the development of flavor by adding bromelain enzyme to enhance physicochemical and sensory features of PBCA [2].In addition, the use of bromelain enzyme can also produce bioactive peptides [18].
Thus, it is the interest of the authors to develop different technical processes for making PBCA in form of soy cheese spreads with good functional properties, flavor characteristics, and stable homogeneous structure.In this study, PBCA will be made from soymilk using various spices such as Geotrichum candidum as fermentation material, GDL as coagulating material and or without the addition of bromelain as a proteolytic enzyme.The objective of this work is to develop the soy cheese spreads production, by using Geotrichum candidum and GDL together with enzymatic hydrolysis process.Our focus was on the differences in the spreadability and sensory features of every PBCA.In addition, proximate assay was used to obtain the salt, fat, protein, moisture, and ash content.Finally, the comparation between commercial dairy cheese and PBCA were discussed

Materials and methods
Material in this study was PBCA made from soymilk.The method used in this study was experimental design with 5 treatments and 4 repetitions.

Preparation of Geotrichum candidum
Geotrichum candidum (Gc) were purchased from the Cheese Making Supply, Indonesia.A starter culture of Gc was prepared by inoculating the strains (0.06%, w/v) in sterilized skim milk (12% TS) under neutralizing conditions and incubated at 37 •C for 16-18h.The milk had been stored under 4 •C before use.Gc was prepared according to Figure 1.

Proximate assay
A commercial dairy cheese (Puck Cream Cheese) purchased from Indomaret (local supermarket, produced by PT.Tigaraksa Satria, Indonesia).PBCA and commercial dairy cheese were analyzed in three repetition, and mean results were used for proximate assay.Fat was determined by a modified Babcock method [19].Total nitrogen content was obtained from Kjeldahl method [20] and a factor of 5.71 was used to convert nitrogen to protein content.Ash content was obatined from a muffle furnace at 550 •C and moisture were determined according to AOAC [13].Salt content was obtained from Mohr method [21].

Sensory analyses
Sensory analyses were carried out based on [22] with slight modification.The PBCA samples were organoleptically examined by untrained 30 panelist which consists of 15 males and 15 females at the Animal Product Laboratory, Animal Science Faculty of Universitas Brawijaya.In this test, the panelist presented personal responses written using a hedonic scale.The hedonic scale was then transformed into a numerical scale

Statistical analyses
Microsoft Excel with One Way Analysis of Variance (ANOVA) were used to analyze the data.If there was a significant difference between treatments followed by the Duncan Multiple Range Test (DMRT) analysis with a significance level of 0.05.

Best treatment determination
The test for determining the best formulation was carried out by the method of De Garmo [23].
Determining the best treatment begins with grouping all observation parameters and scoring.Then calculate the weight value (WV) to determine the treatment value, the best value, and the worst value.
Then calculated the effectiveness value (EV) with Equation 1. Where: After that, the calculation of the product value for each observation parameter is carried out using Equation 2. Where: Finally, summing up all product values is carried out and the largest value is sought to conclude as the best treatment.The panelists used in this study were 30 untrained panelists (15 males and 15 females).

Chemical feature of PBCA
In the present study, the ash, fat, moisture until salt content of PBCA decreased in line with the utilization of soymilk.On the contrary, the protein content of PBCA increased in line with the utilization of soymilk (Table 1).The result of the ANOVA showed that utilization of Gc, GDL and bromelain had a significant difference (p<0.05) on chemical features of PBCA.The total ash content indicates the dry matter content present in the product.Ash content was 1.39 ± 0.16 a % -1.92 ± 0.06 c %.The highest ash content was obtained in control made from commercial dairy cheese and the lowest ash content was obtained in PBCA 4 made from soymilk, Gc, GDL, and bromelain.According to [24], milk casein is coagulated by fermenter agents so that the dry matter in milk becomes increased.Fat content was 24.7 ± 0.07 a % -29.7 ± 0.06 d %.The highest fat content was obtained in control and the lowest fat content was obtained in PBCA 4 made from soymilk, Gc, GDL, and bromelain.The fat content in the control had the highest value because dairy milk originally had a higher fat content than plant-based milk [25].The fat content of dairy milk ranges from 2.25% -4.08% [26]and 24.00% -25.50% when processed into cheese [27].While the fat content of soymilk is 1.8% [6] and 21% when processed into cream-cheese style [10].This shows that acid can increase fat levels.The combination of Gc and GDL can increase fat levels.This is because they are both factors causing the sour atmosphere.However, fat levels decreased when the enzyme bromelain was introduced into the formula.This is because the bromelain enzyme can maximize the separation of oil with water in the soymilk emulsion so that the fat and ash content produced will be less than PBCA 3.
Moisture content 47.3 ± 0.06 a % -55.4 ± 0.06 c %.The highest moisture content was obtained in control and the lowest moisture content was obtained in PBCA 3 made from soymilk, Gc, and GDL.This increase in water content is influenced by using the enzyme bromelain.The bromelain enzyme used can hydrolyze the proteins in soymilk during the fermentation process.Hydrolysis causes coaagulation in cheese proteins [21].In addition, the use of Gc and GDL can make acid so that the protein becomes coagulated [28].Protein content 6.39 ± 0.06 a % -19.5 ± 0.06 c %.The highest protein content was obtained in PBCA 3 made from soymilk, Gc, and GDL and the lowest protein content was obtained in control.Soymilk has a protein content between 30 -35% [29].The results showed that the use of bromelain enzyme was able to increase water content so that salt levels in cheese decreased.Salt content 0.28 ± 0.05 a % -0.69 ± 0.04 c %.The highest salt content was obtained in control and the lowest ash content was obtained in PBCA 4 made from soymilk, Gc, GDL, and bromelain.
Control is a commercial cheese made from dairy milk.In general, dairy milk contains casein protein that can be coagulated by the enzyme renin.The enzyme renin can coagulate milk casein to form coagulation.The coagulation formed will turn into cheese.Unfortunatley, casein protein was not found in PBCA 1, PBCA 2, PBCA 3, and PBCA 4. Therefore, coagulation was carried out with the help of Gc and GDL.Gc and GDL can decrease the pH of proteins inside PBCA to form coagulation [17].
Control has a different flavor than others PBCA.This occurred because of the influence from type of milk origin, animal feed, age, and health status.According to [30], the degradation of lipids and proteins by enzymes and the probiotic produce a volatile compounds abundance like alcohols, aldehydes, carboxylic acids, ketones, and lactones.According to [16], bromelain can break peptide bonds to produce distinctive flavor.Lipase from Gc was capable to convert fat into fatty acids and glycerol [31].The spread-ability of cheese was influenced by a set of material characteristics.PBCA with have relatively high moisture contents increases their spread-ability [21].In contrast, hard cheeses, which have relatively low moisture contents may be too gummy and dry, which results in shattering into smaller particles during the shredding process [32].
The proteins used in PBCA 1, PBCA 2, PBCA 3, and PBCA 4 were mainly derived from soymilk.These proteins need to fulfill different chemical and functional attributes with gelation and flavor precursor properties being the most important ones.According to [33], the enzymatic modifications like crosslinking and proteolysis could affect the flavor of cheese.Fat content plays an important role in determining the sensory features and nutritional benefit of PBCA.Triglycerides was the most common fats found in cheese in form of three fatty acids and glycerol via ester bonds [34].The most common fatty acids in milk were saturated (around 70%), mono-unsaturated (around 25%), and poly-unsaturated (around 2.3%), but short-chain fatty acids (such as butyric and caprylic acid) are also present, which make an important contribution to the flavor [28].

Sensory features of PBCA
The result of sensory feature evaluation showed that the utilization of soymilk significantly effects (p<0.05) the appearance, flavor, creaminess, firmness, spread-ability, and overall acceptance of PBCA (Table 2).The mean score to each of the features evaluated: appearance, flavor, creaminess, firmness, spread ability, and overall acceptance of all sample is shown in Table 2.The hedonic scoring for appearance acceptance of control had a score 7.52 (like moderately); PBCA 1 made from soymilk, Gc, and bromelain had a score 7.18 (like moderately); PBCA 2 made from soymilk, GDL, and bromelain had a score 7.27 (like moderately), PBCA 3 made from soymilk, Gc, and GL had a score 7.48 (like moderately) and PBCA 4 made from soymilk, Gc, GDL, and bromelain had a score 6.85 (like slightly).This is due to the addition of bromelain which can break peptide bonds.The breakdown of peptide bonds.causes the appearance of PBCA to be less attractive.This is due to the breaking of protein bonds which causes a rough texture to appear and causes a griny taste [18].
The hedonic scoring for flavor acceptance of control had a score 8.05 (like very much); PBCA 1 made from soymilk, Gc, and bromelain had a score 7.68 (like moderately); PBCA 2 made from soymilk, GDL, and bromelain had a score 7.33 (like moderately), PBCA 3 made from soymilk, Gc, and GL had a score 7.67 (like moderately) and PBCA 4 made from soymilk, Gc, GDL, and bromelain had a score 7.71 (like moderately).The results showed that different spices affect flavor development (p<0.05).This flavor is a result of the composition of the milk used and the breakdown of the components within the cheese during the ripening phase.According to [10], the degradation of lipids, proteins, and carbohydrates by probiotic incombination with enzyme produces an abundance of volatile compounds including carboxylic acids, lactones, ketones, alcohols, and aldehydes.
The hedonic scoring for creaminess acceptance of control made from commercial dairy milk had a score 7.73 (like moderately); PBCA 1 made from soymilk, Gc, and bromelain had a score 7.35 (like moderately); PBCA 2 made from soymilk, GDL, and bromelain had a score 7.33 (like moderately), PBCA 3 made from soymilk, Gc, and GL had a score 7.67 (like moderately) and PBCA 4 made from soymilk, Gc, GDL, and bromelain had a score 7.25 (like moderately).
Firmness determines the texture that leads from soft to hard cheese.The hedonic scoring for firmness acceptance of control made from commercial dairy milk had a score 6.40 (like slighly); PBCA 1 made from soymilk, Gc, and bromelain had a score 7.04 (like moderately); PBCA 2 made from soymilk, GDL, and bromelain had a score 6.37 (like slightly), PBCA 3 made from soymilk, Gc, and GL had a score 6.66 (like slightly) and PBCA 4 made from soymilk, Gc, GDL, and bromelain had a score 6.58 (like slighty).The results showed that firmness is inversely proportional to spread ability.The lower the firmness value, the higher the spread-ability.The use of Gc and bromelain enzyme can increase the firmness value (p<0.05).This is because the combination of Gc and bromelain enzymes can change the bonds between proteins and calcium solubility during the draining period.According to [9], soymilk protein coagulation occurs due to a decrease in pH caused using rennet, GDL, and LAB enzymes with a low draining pH (4.5) result in considerable calcium loss in the whey fraction.Instead, increasing pH during ripening may reduce the calcium bridges, which results in an improvement in protein solubility and decreasing viscosity during the ripening phase [11].
The hedonic scoring for spread-ability acceptance of control made from commercial dairy milk had a score 7.82 (like moderately); PBCA 1 made from soymilk, Gc, and bromelain had a score 7.48 (like moderately); PBCA 2 made from soymilk, GDL, and bromelain had a score 7.40 (like moderately), PBCA 3 made from soymilk, Gc, and GL had a score 7.78 (like moderately) and PBCA 4 made from soymilk, Gc, GDL, and bromelain had a score 7.25 (like moderately).The results showed that PBCA has lower spread-ability than control.Even so, PBCA's spread-ability acceptance gained moderate likes.This is because dairy cheese has more saturated fat than PBCA.According to [9], the most common fatty acids in dairy milk are saturated (70%), mono-unsaturated (25%), and polyunsaturated (2.3%), while in soymilk contain substantial amounts of unsaturated fatty acids so they tend to be liquid at ambient temperature and cannot form fat crystal networks.The addition of Gc and GDL to the formulation has an impact on reducing spread-ability due to coagulation that occurs in soymilk protein (p<0.05).The decrease in spread-ability is strengthened by the addition of the enzyme bromelain to the formula (PBCA 4).This is due to the ability of proteolityc possessed by the enzyme bromelain.
The hedonic scoring for overall acceptance of control made from commercial dairy milk had a score 8.05 (like very much); PBCA 1 made from soymilk, Gc, and bromelain had a score 7.68 (like moderately); PBCA 2 made from soymilk, GDL, and bromelain had a score 7.33 (like moderately), PBCA 3 made from soymilk, Gc, and GL had a score 7.67 (like moderately) and PBCA 4 made from soymilk, Gc, GDL, and bromelain had a score 7.71 (like moderately).

Best treatment of PBCA
In this study, the best treatment of PBCA was made from dairy cheese commercial and soymilk determined by de Garmo method (Table 3).The best treatment determination in making PBCA was carried out by using the effectiveness index test based on sensory acceptance, fat, salt, and protein content.The score of the PBCA0, PBCA1, PBCA2, PBCA3, and PBCA4 product value was 1.00; 0.46; 0.53; 0.00; and 1.00 respectively.Sample with Gc 3%, GDL 0.25%, and bromelain 8x10 5 U/g has the highest effectiveness index value so that PBCA4 was chosen as the best formulation in this study.Although PBCA 4 had the quite-low protein content (17.0%) of all treatments, its overall sensory value was similar to control (8.03:8.44)and had the lowest fat content (24.4%) and salt content (0.28%).According to [13], the sensory of a food affects consumer acceptance significantly.In addition, low levels of fat and salt content in a food is a separate desire from consumers as an effort to minimize the risk of non-communicable diseases [29].

Figure 1 .
Figure 1.Flowchart how to prepare Geotrichum candidum 2.2.Preparation of soymilk Raw soymilk cooked under 85 o C in 10 minutes.The cooked soymilk was added with 0.1% (w/v) carrageenan (Indo Food Chem, Indonesia) and 2.5% (w/v) olive oil with a melting point at 23 o C, and then homogenized at 25 MPa.Soymilk was prepared according to Figure 2.

( 9 =
Like extremely; 8 = Like very much; 7 = Like moderately; 6 = Like slightly; 5 = Either like or dislike; 4 = Dislike slightly; 3 = Dislike moderately; 2 = Dislike very much; 1 = Dislike extremely).Ethical clearance regarding the use of panelists based on Guidelines for the Code of Ethics for Research and Community Service Universitas Brawijaya.

Table 1 .
The result of chamical assay from PBCA

Table 2 .
The result of sensory analyses from PBCA