Development and investigating parameters of fermented milk products based on various types of vegetable milk

. Plant-based milks and fermented plant-based milk products are gaining popularity but may not possess the same physicochemical characteristics as the traditional fermented dairy products. The different chemical composition of plant-based milk affects the physicochemical and consumer acceptability of fermented plant-based milk. To compare their characteristics to the traditional dairy yoghurt and consumer acceptability, different fermented plant-based milk products were prepared as well as their two-component mixtures and their physicochemical and consumer acceptability evaluated. The single component fermented plant-based milk sample (soy milk) did not vary in terms of physicochemical parameters with respect to the traditional fermented dairy product, yoghurt. The physicochemical parameters including pH, water holding capacity, syneresis improved when the milk samples were composited and are comparable to the traditional fermented dairy product (yogurt). The results indicated that the use of two-component plant-based milk for fermentation provides the best nutritional and energy values for consumers rather than using just one component. Also, the high consumer acceptability for fermented plant-based milk was demonstrated from the findings.


Introduction
Globally, the demand for an alternative food protein source that could potentially replace animal proteins has been on the rise in recent years.This dietary shift has been because of sustainability, health, and ethical considerations (vegetarians, cultural values).To ensure food security, the threat of climate change must not be overlooked and as a result the world's growing population moving towards plant-based diet is laudable and inevitable [1,2].Other factors that might influence this increment in the demand for plant-based milk might be the increase in lactose intolerance particularly in old age population, limited availability of milk and milk products, high price and presence of some pathogenic microorganisms (Salmonella spp.and Escherichia coli O157:H7) that may lead to disease outbreaks and affect human health [3].
Present consumers expect more healthy and palatable food products which has made the dairy industry switched their attention to the production of non-conventional milk with health benefits beyond the conventional milk products [4].Studies have revealed that plant-based milk improve the immune system, exhibit antimicrobial effects, reduce the risk of cardiovascular and gastro-intestinal diseases with improved physiological activities, reduced the risk of low bone mass and antioxidant activities.Plant milk can undergo fermentation either naturally or controlled, this improves the bioavailability of bioactive compounds they contain.Again, plant-based milk can be fortified to improve its nutritional bioavailability and components [5].Generally, legumes, cereals and oilseeds are the major targets for plantbased milk production due to their high nutritional profile.Usually, these seeds contain specific bioactive compounds that make them comparable to animal milk.Soybean, oat, coconut, rice, almond just to mention few, are used to produce milk and flavored with either vanilla or straw berry or chocolate and many others.Sweeteners are also added to improve their organoleptic properties.
The global market of the dairy industry, due to high demand for the dairy products, has always been in the top position.However, dairy products involve the use of lots of resources and present a lot of environmental hazards.This makes the process very stressful and timeconsuming.Most of the feed by the livestock is converted into other metabolic processes such as bones, muscles etc. formation, and only a few portions of the feed consumed are converted to milk.This leads to high input to produce less products as compared to plantbased milk.Also, the rearing of livestock such as cattle production has contributed to the release of methane gas to the environment leading to global warming.The production of plant-based milk in this case is more advantageous to the conventional animal milk when compared.It is making its way into the global market, capturing the attention of consumers.Soy milk is the leading produced plant-based milk, about 58.0% global production in 2017 and almond, rice, coconut and oat milk also contribute significantly [6].
Different food products including beverages are prepared and preserved by fermentation from age till date [7].During fermentation process, complex carbohydrates such as starch and sugars are converted into simple products like alcohols and acids with the production of carbon dioxide concurrently using microorganisms such as yeasts and or bacteria under anaerobic conditions.The process of fermentation and the product of the fermentation is determined by the microbial biomass that is used, ranging from small scale production thus experimental purposes to large scale production thus industrial production [8].It is known that in the fermentation process of raw materials, simpler, easily digestible molecules and biologically active compounds are formed.In this regard, the study of vegetable milk fermentation is relevant.Thus, research has shown that the extraction of biomolecules and improvement of phytochemical profile of plant-based milk products are made possible through the fermentation process using Lactobacillus acidophilus [9].
The objective of this study is to obtain fermented products using different plant milk types, their composites and characterize the physicochemical and consumer acceptability compared with the traditional dairy yoghurt.

Preparation of fermented products
About 120 ml of milk was poured into a glass, placed in a water bath, heated to 80 ᴼC, and kept for 10 minutes to pasteurize the milk.The milk was then cooled to 40 ᴼC and in a sterile conditions of a laminar flow cabinet (BAVp-01-"Laminar-S"-1.2) 100 ml of milk was poured into a jar, 0.1 g of starter culture and 1 g of sugar were added, the contents of all jars were mixed with a sterile wooden stirrer.The milk mixture was fermented in a Memmert UN55 thermostat at 38 ᴼC for 6 hours.The samples were cooled in a refrigerator at 4 ᴼC for further experiments.

Water Holding Capacity, WHC
WHC of the samples were determine by centrifugation of about 5 g of the samples at 4500 rpm at 4 ᴼC for 30 minutes.The water suspended was removed and weighed.The WHC was calculated as a percentage using the equation:

Measurement of Syneresis
The syneresis was determined using the method described in the work of Raikos et al. [10].About 5 g of the samples were weighed onto a 2-V folded filter paper (Whatman) and placed on the top of a funnel.Syneresis was determined by gravity by measuring the weight (g) of liquid collected in a measuring cylinder of known weight.The drainage time was about 120 minutes and was done at room temperature.The percentage of syneresis was calculated according to the equation:

Measurement of pH and Titratable Acidity
The pH of the samples was measured at room temperature using FiveEasy pH meter (Mettler Toledo Ltd., Victoria, Australia).The titratable acidity (TA) was obtained through colorimetric titration method 942.15 (AOAC, 2016) [11] using a 2% (w/v) phenolphthalein solution in ethanol as an indicator.A 1 mL sample was mixed with 9 mL of purified water followed by addition of a few drops of indicator and titrated using 0.5 N NaOH solution.The TA, expressed as percentage lactic acid, was calculated as follows: Where 10 is the dilution factor, VNaOH is the volume of NaOH used to neutralize the lactic acid (ml), 0.009 is the conversion factor, 0.5 is the normality of NaOH, and W is the weight of sample.

Measurement of Dry Matter Content
The dry matter (DM) contents of the samples were determined by drying about 3 g of samples at 105 ˚C for 60 minutes in hot air oven.The final weight of the dried samples was measure and the DM content determined as a percentage using the equation:

Sensory evaluation
Sensory analysis was performed within 24 hours after production at a temperature of 4 ˚C by untrained panels (staff and graduate students at the Institute of Chemical Technology, Ural federal University, Yekaterinburg, Russia).Evaluating flavor, appearance, taste, and overall acceptability on a 5-point hedonic scale (1-dislike extremely, 2-dislike, 3-neither like nor dislike, 4-like, 5-like extremely) for each sample.Water was used as a palate cleanser in between samples by the panelists.

Calculation of nutritional value
The calculation of the nutritional value of samples of fermented milk products was carried out on the basis of the nutritional value of the components used in their preparation according to the formula: where  is the content of the i-th nutrient in 100 g of fermented milk product, g or mg; is the content of the i-th nutrient in 100 g of component j, g or mg;  is the mass of component j, g.
The energy value of samples of fermented milk products, kcal, was calculated by the formula: Where  is the energy value of samples of fermented milk products, kcal; 1−3 is the caloric coefficients of proteins, fats and carbohydrates, b is the mass of protein in 100 g of fermented milk product, g; g is the mass of fats in 100 g of fermented milk product, g; у is the mass of carbohydrates in 100 g of fermented milk product, g.

Statistical analysis
All statistical analyses were executed using GraphPad Prism 8 statistical software package.Two trials were made in parallel, and the outcomes are expressed as means ±standard deviation.The water holding capacity, syneresis, pH, titratable acidity, and dry matter were analyzed using One-way Analysis of Variance (ANOVA) and compared Dunnett's multiple comparisons.The sensory data were analyzed using Two-way ANOVA with Dunnett's multiple comparisons.

Results and discussions
The first part of the work is devoted to obtaining fermented milk products on the based whole plant-based milk, such as soy milk (Alpro), almond milk (Alpro), coconut milk (Alpro), and coconut milk (Aroy-D) and compare them with cow milk yoghurt.The second part includes the use of mixtures of different plant-based milk for fermentation.

Nutritional composition of milk types
Table 1 shows the nutritional composition of the various milk types used in the experiment according to the manufacturer.It can be observed that the protein content in soy milk (Alpro) is similar to that of cow milk, 3.0 g.This provides the probability of replacement in the production of fermented products.However, other milk samples, almond milk (Alpro), coconut milk (Alpro), and coconut milk (Aroy-D) have lesser protein content, and this may have effect on the fermented product formation.Other compounds such fat and carbohydrates are necessary for a better curd formation and stability of the fermented products.
Table 1.Nutritional composition of milk types (100 g of raw material).

Carbohydrates, g
Fats, g

Energy, kcal
Organic acid, g

Dry matter
The dry matter content of the fermented soy (Alpro) and coconut milk (Alpro) did not differ significantly (p > 0.05) from the control sample as shown in fig. 3.However, coconut milk (Aroy-D) and almond milk (Alpro) differs significantly from the control sample with the coconut milk (Aroy-D) having the highest dry matter content (25.68 ±0.96) whiles coconut milk (Alpro) has the least dry matter (3.95 ±0.40).

Consumer acceptability
The results of the consumer acceptability as shown in table 3, indicates that, considering all the characteristics tested by the panelists (flavor, appearance, taste, consistency, and overall acceptability), soy milk (Alpro) and coconut milk (Aroy-D) did not differ significantly, (p > 0.05) from the control sample.These make them a perfect replacement for the traditional fermented dairy product (yoghurt).The low scores observed in the coconut milk (Alpro) and almond milk (Alpro) may be due to the inability of the product to conform physicochemically with the control sample as shown in the earlier results.where ns = no significant difference (p > 0.05) and * = significant difference (p < 0.05).1-dislike extremely, 2-dislike, 3-neither like nor dislike, 4-like, 5-like extremely.

Fermented products based on mixtures of different plant milk types
It was shown that soy and coconut fermented products have the best organoleptic and physicochemical parameters.For this reason, we used these milk types as two composite fermented products.This was done in the ratios (soy milk (Alpro): coconut (Aroy-D)) of 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0.Thus, the best result was found in fermented soy (Alpro) and coconut (Aroy-D) milk in the proportion 80:20 and 90;10 respectively.This was selected based on the consumer preferences and the similarity of the physicochemical properties with the traditional fermented cow milk (yoghurt) as shown in table 4 as well as consumer preferences.To establish the degree of satisfaction of the daily human need for macro-and micronutrients, the nutritional value of the resulting product was calculated.Table 5 represents the results of calculating the energy and nutritional value of a fermented milk product from a composite of soy (Alpro) and coconut (Aroy-D) milk (80:20) and (90:10) respectively.The results in table 5 shows that the energy value (59.41 kcal / 100 g) of a two-component product from a mixture of soy (Alpro) and coconut (Aroy-D) milk is higher than that of a one-component product from soy milk (39.00 kcal / 100 g) due to the higher content of fats and carbohydrates in milk mixture.According to the results of the energy and nutritional value of the resulting product, it can be concluded that the fermented milk product from a mixture of soy and coconut milk (80: 20) is the best option considering the percentage covering daily requirement.In addition, the degree of satisfaction of the daily requirement of potassium and phosphorus has increased.

Conclusions
In this study, the use of composited plant-based milk products for fermented product formation is most preferred on nutritional and energy requirement bases.Physicochemical properties of fermented plant-based milk affect its consumer acceptability.Soy milk (Alpro) and coconut milk (Aroy-D) did not differ in many cases from the control sample and hence their high acceptability by consumers.However, their individual energy values are much lesser compared to cow milk and hence the need to composite them.The different plant-based fermented products have provided information about how to compose these products for fermentation based on their physicochemical characteristics.The results show that the change in the nutritional content of the samples due to fermentation and composition needs to be assessed in further studies and the optimal and best type of milk composition.

Fig. 3 .
Fig. 3. Bars represent the mean values (n = 2) of the dry matter of the various milk samples.Significant difference ** p < 0.01, * is p < 0.05 and where ns means no significant difference.

Table 5 .
Energy and nutritional value of fermented products from different plant milk types.
The outcome of the work is beneficial to the plant-based milk industry for the production of composite plant-based fermented milk product for consumers.Overall, this work produced valuable insights into the potential benefits and drawbacks of fermenting different plant-based milks and could help inform future research in this area.The research funding from the Ministry of Science and Higher Education of the Russian Federation (Ural Federal University Program of Development within the Priority-2030 Program) is gratefully acknowledged.