*Article* **The Protein Composition Changed the Quality Characteristics of Plant-Based Meat Analogues Produced by a Single-Screw Extruder: Four Main Soybean Varieties in China as Representatives**

**Bo Lyu 1,2,3 , Jiaxin Li 1,2, Xiangze Meng 1,2, Hongling Fu 1,2, Wei Wang 2,4, Lei Ji 1,2, Yi Wang 1,2 , Zengwang Guo 2,3,\* and Hansong Yu 1,2,\***


**Abstract:** Plant-based meat analogues (PBMs) are increasingly interesting to customers because of their meat-like quality and contribution to a healthy diet. The single-screw extruder is an important method for processing PBMs, and the characteristics of the product are directly affected by the composition of the raw materials; however, little research focuses on this issue. To explore the effect of protein composition on the quality characteristics of PBMs produced by a single-screw extruder, four soybean varieties used in China (Heihe 43 (HH 43), Jiyu 86 (JY 86), Suinong 52 (SN 52), and Shengfeng 5 (SF 5)) were selected. The 11S/7S ratios for these varieties ranged from 1.0: 1 to 2.5: 1 in order to produce PBMs with different protein compositions. The structure, processing, nutrition, and flavor characteristics were explored to analyze their differences. The results showed that protein composition affected the structure of PBMs, but the correlation was not significant. Meanwhile, a lower 11S/7S ratio (HH 43) did not prove to be a favorable characteristic for the processing of PBMs. From the perspective of nutrition and flavor, it seems acceptable to use a moderate 11S/7S ratio (JY 86 and SN 43) to produce PBMs. This study proved that the protein composition of raw materials affects the characteristics of PBM products produced by a single-screw extruder. To produce PBMs of higher quality, soybeans with a markedly different 11S/7S ratio should not be selected.

**Keywords:** extrusion technology; textured soy protein; protein subunit composition; processing applicability; plant-based meat analogues

### **1. Introduction**

With improvements in living standards, great changes have taken place in people's dietary structures, including the increased intake of animal-derived foods [1]. However, the increase in the intake of animal-derived foods and the decrease in vegetarian intake is one of the causes of many diseases, such as intestinal and cardiovascular diseases [2,3]. This result may be caused by the excessive intake of fats, drug residues, or other factors [4,5]. To maintain the excellent taste and good processing characteristics of meat products while preventing the potential health risks caused by the excessive intake of animal-based food, plant-based meat analogues (PBMs) came into being. The intake of PBM products not only does not cause health risks but also can reduce land use and resource consumption [6,7]. PBMs can also meet the requirements of modern people for food diversity [8]; this has gradually increased the acceptability of PBM products, as well as the demand for them [9].

**Citation:** Lyu, B.; Li, J.; Meng, X.; Fu, H.; Wang, W.; Ji, L.; Wang, Y.; Guo, Z.; Yu, H. The Protein Composition Changed the Quality Characteristics of Plant-Based Meat Analogues Produced by a Single-Screw Extruder: Four Main Soybean Varieties in China as Representatives. *Foods* **2022**, *11*, 1112. https://doi.org/10.3390/ foods11081112

Academic Editor: Yonghui Li

Received: 23 March 2022 Accepted: 12 April 2022 Published: 13 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

An important aspect of PBM products is the fact that the process of manufacturing textured plant protein mainly depends on the changes in protein structure caused by the high temperature and pressure produced by screw extruders [10]. Mainstream textured plant protein production equipment includes the twin-screw extruder and the single-screw extruder [11]. The twin-screw extruder is widely used because of its excellent processing capacity; as a result, there are more theoretical studies based on it [10,12]. However, the single-screw extruder is also used on a large scale because of its lower cost and ability to process a wider variety of raw materials, such as insoluble dietary fiber, starch, etc. [13]. Therefore, consumers may encounter products manufactured using two different kinds of extrusion technologies in the market at the same time. Certainly, there are differences in the quality of the products because of the differences in chemical cross-linking and molecular aggregation [14].

Soybean protein is considered to be a good choice for producing PBMs because of its excellent gelation, superior nutritional value, low cost, and safety as a raw material [15]. The composition of soybean protein affects the processing and nutritional characteristics of soybean products directly [16]. In particular, the proportions of soy glycinin (11S) and soy β-conglycinin (7S) directly affect the key processing characteristics, such as gel and foaming properties [17,18]. It is generally believed that high 7S content is related to hydration characteristics, such as emulsification and foaming properties [17,19]. A high 11S content means a higher protein structural strength, as manifested in characteristics such as gelation [20]. However, the relationship between them is not strictly linear. There is reason to believe that the texture of PBMs is also related to the gel properties of soybean protein [21]. However, no study has compared the properties of PBM products produced using different varieties of soybean with different protein compositions, and no study has demonstrated what kind of protein composition is more suitable for PBM products produced by singlescrew extruders. Previous studies on PBMs tend to analyze the composition of soybean protein isolate (SPI). Currently, under the guidance of "Whole-Soybean Processing", a great deal of PBMs are processed directly from defatted soybean powder. As such, we should pay more attention to the protein composition of soybean. In addition, soybean protein should be perceived as a safe raw material for producing PBM products. Soybeans used in the production of protein products are required not to use transgenic soybeans in many countries [22–24]; similar regulations also avoid some of the risks of processing livestock products, such as hormones [25]. Although soybean protein has a certain potential to aggravate food sensitivities, high temperatures and pressures, such as those the singlescrew extruder provides, can reduce the allergen content [26], which can improve the acceptability of soybean products. Therefore, soybean protein can be regarded as the best raw material for the production of PBMs.

In this study, to explore the potential impact of the soybean protein composition of the raw materials on PBM characteristics, we selected four soybean varieties, representative of the main planting varieties in China, with different compositions and ratios of 11S and 7S (11S:7S = 1, 1.5, 2, 2.5), which were processed into defatted soybean flours (DSFs) with different protein compositions. A single-screw extruder was used to produce textured soybean protein as a representative of PBM products. The structure (basic composition, sulfhydryl content, secondary structure, functional group composition, and microstructure), processing characteristics (water-absorption capacity, water-holding capacity, water-swelling capacity, tensile strength, breaking elongation, and texture characteristics), nutritional characteristics (dietary fiber, reducing sugar, phytic acid, trypsin inhibitor, plant lectin, amino acid composition, and isoflavone content) and flavor characteristics were used to measure the quality of PBM products, to determine the impact of different protein compositions on their quality.

#### **2. Materials and Methods**

#### *2.1. Selection of Soybean Varieties and Preparation of Defatted Soybean Flour (DSF)*

By searching the database of the China Agriculture Research System (CARS): Soybean Processing Division, we selected four kinds of soybeans with different protein subunit compositions as the experiment raw materials: Heihe 43 (HH43), Jiyu 86 (JY 86), Suinong 52 (SN 52), and Shengfeng 5 (SF 5), for which the ratios of 11S/7S were 1.0, 1.5, 2.0, and 2.5, respectively. All four varieties are under large-scale cultivation in China. After soybean dehulling, the oil was removed by an oil press, and then defatted soybean flour (DSF) was obtained after crushing (oil content <7%). A SH-28 single-screw extruder (Shandong Yuya Soybean Machinery Manufacturing CO., Ltd., Zaozhuang, China) was used to produce PBMs under the following conditions: the ratio of DSF to water was 2:3; the temperature was 240 ◦C in the first zone, 220 ◦C in the second zone, 200 ◦C in the third zone, and 180 ◦C in the fourth zone; the screw rotated at 70 rpm.

#### *2.2. Analysis of Soybean Protein Composition by SDS-PAGE*

The analysis of the soybean protein composition was measured according to the method described by Song et al. [27]. The presence and absence of glycinin and β-conglycinin subunits in the soybean seeds were confirmed by SDS-PAGE. The total seed proteins were extracted from a small portion of cotyledon tissues with an SDS sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 5 M urea, and 62.5 mM Tris aminomethane) and then centrifuged at 15,000× *g*. Then, 10 µL of the supernatant was separated on 4.5% stacking and 12.5% separating polyacrylamide gels and stained with Coomassie Brilliant Blue R250. The gels were scanned by an Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the number of protein subunits was quantified according to the gray value.

#### *2.3. The Structure of PBMs*

#### 2.3.1. The Basic Composition of Four Kinds of PBMs

The moisture, protein, ash, and fat content of four kinds of PBMs were determined according to the AOAC Official Method (AOAC 2007.04).

#### 2.3.2. The Sulfhydryl Content of Four Kinds of PBMs

Four kinds of PBMs were crushed into 80 mesh after being fully dried, and a method employing Ellman's reagent (10 mm DTNB, 0.2 mm EDTA) was used to determine the content of SH in the samples [28]. The preparation of Tris-Glycine buffer was as follows: 0.086 M tris + 0.09 M Glycine + 4 mM EDTA, pH = 8.0. The samples were dispersed in the Tris-Glycine buffer to obtain 2 mg/mL solutions. Then, 0.03 mL Ellman's reagent was added to 3 mL solution, and the solution was mixed immediately and stored for 15 min at room temperature before measuring the absorbance at 412 nm. A buffer solution without a protein sample was used as a reagent blank.

#### 2.3.3. Circular Dichroism Spectrum (CD)

The crushed PBM samples (80 mesh) were prepared with 1 mM phosphate-buffered saline (PBS, pH = 7.0) into a 1 mg/mL solution, placed in a 1 mm optical path length quartz cell, and measured with a J-810 CD spectrometer (JASCO, Tokyo, Japan). The sensitivity was set to 2 mdeg/cm. The 185–260 nm CD spectrum was recorded at 20 ◦C.

#### 2.3.4. Fourier Transform Infrared Spectroscopy (FT-IR)

Fourier transform infrared spectroscopy (FT-IR) analyses of four kinds of PBMs were performed using a Nicolet iS5 spectrometer (Thermo Fisher, Waltham, MA, USA). The dried samples were mixed with KBr powder (1:100, *w*/*w*) after being crushed into 80 mesh, and the spectra were read over the range of 4000–400 cm−<sup>1</sup> with a resolution of 4 cm−<sup>1</sup> .

#### 2.3.5. Scanning Electron Microscopy (SEM)

The PBMs were cut into strips of appropriate size for sample pre-treatment. The samples were immersed in a glutaraldehyde solution (2.5%, pH = 7.2–7.4) for 24 h. After they were fixed, the samples were washed with a phosphate buffer (pH = 7.2) 3 times before eluting with 30%, 50%, 70%, 85%, 95%, and 100% ethanol solutions. The samples were made into 2 mm × 2 mm flakes, as thin as possible, after freeze-drying in order to observe the microscopic appearance of four kinds of PBMs using a SU8020 scanning electron microscope (SEM; Hitachi, Tokyo, Japan) after spraying with a gold–palladium alloy. The scanning images were captured at accelerating voltages of 5 kV and photographed at magnifications of 5000X (scale bar 10 µm).

#### *2.4. The Processing Characteristics of PBMs*

2.4.1. Water-Absorption Capacity (WAC), Water-Holding Capacity (WHC), and Water-Swelling Capacity (WSC)

WAC: After recording the weight of the fully dried sample (M1), it was soaked in 60 ◦C water for 5 h, then drained for 6 min. The samples were weighed (M2).

$$\text{WAC} = (\text{M2} - \text{M1}) / \text{M1} \times 100\% \tag{1}$$

WHC: 1.00 g crushed sample (M1) and 20 mL of water were mixed in a dry centrifuge tube (M0). The sample was kept at RT for 24 h and centrifuged at 4000 rpm for 20 min. The supernatant was removed, and the weight (M2) was recorded.

$$\text{WHC} = (\text{M2} - \text{M0}) / \text{M1} \times 100\% \tag{2}$$

WSC: 1.000 g crushed, fully dried sample (M) and 10 mL of water were mixed in a dry centrifuge tube and kept at RT for 24 h. The volume of the sample was recorded (V).

$$\text{WSC} = \text{V/M} \times 100\% \tag{3}$$

2.4.2. The Tensile Strength (TS) and Breaking Elongation (BE) of Four Kinds of PBMs

The WDW-200H electronic tensile testing machine (Hongtuo, Dongguan, China) was used to analyze the tensile strength of four kinds of PBMs. The experiment conditions were as follows. The wet PBM was cut to 10 cm × 6 cm for testing; the initial clamping distance was 40 mm, and the tensile speed was 5 mm/s. The following values were recorded: the maximum tension at break (P), the cross-sectional area of samples (S), the elongation at break (δL), and the original length (L). Values were calculated for the tensile strength (TS) and breaking elongation (BE).

$$\text{TS (MPa)} = \text{P/S} \tag{4}$$

$$\text{BE (\%)}=\\$\text{L/L}\times\text{100\%}\tag{5}$$

#### 2.4.3. Texture Profile Analysis (TPA)

The texture characteristics of four kinds of PBMs were analyzed by a Texture Analyzer (TA.new plus, Isenso, Shanghai, China). The samples were cut into a square with a length of 10 mm, and the conditions were as follows. The detection mode was TPA mode with the P/36R detector; the rate before the test was 2 mm/s; the rate during the test was 1 mm/s; the rate after the test was 2 mm/s; the compression degree was 50%.

#### *2.5. The Nutritional Characteristics of PBMs*

2.5.1. The Dietary Fiber, Reducing Sugar, Phytic Acid, Trypsin Inhibitor, Plant Lectin, and Isoflavone Content of Four Kinds of PBMs

The dietary fiber, reducing sugar, phytic acid, and isoflavone content of four kinds of PBMs were determined according to the AOAC Official Methods (AOAC 2017.16, AOAC 945.66, and AOAC 986.11). The amounts of trypsin inhibitor and plant lectin (Soybean agglutinin, SBA) found in the four kinds of PBMs were determined using the

Trasylol Elisa kit (Ml064289, Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) and the SBA Elisa kit (Ml002453, Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).

#### 2.5.2. The Amino Acid Composition

The analysis of the amino acid composition of four kinds of PBMs was performed according to the method described by Song et al. [27]. After the PBMs were fully dried, a meal was prepared by mill grinding through a 0.25-mm sieve and thoroughly mixing. Total amino acids were obtained by the hydrolysis of seed meal with an excess of 6 M HCl for 22 h in sealed evacuated tubes at a constant boiling temperature (110 ◦C). An L-8800 amino acid analyzer (Hitachi, Tokyo, Japan) was used to determine the amino acid compositions of the hydrolysates. The amino acid composition was expressed as relative content (%) on a dry basis.

#### *2.6. The Flavor Characteristics of PBMs*

The volatile flavor compounds found in four kinds of PBMs were measured by a 6890N-5975B Gas Chromatography-Mass Spectrometry workstation (GC-MS; Agilent, Palo Alto, CA, USA).

Sample pre-treatment was as follows. The samples were sealed in head-space bottles and warmed at 80 ◦C in a water bath for 30 min. A solid-phase microextraction needle (SPMEN; 100 µL PDMS; SUPELCO, Bellefonte, PA, USA) was used for extraction for 30 min at 80 ◦C before desorption for 5 min.

The experiment conditions for GC-MS were as follows. Chromatographic column: HP-5MS (30 m × 0.25 mm × 0.25 µm); split ratio: no split; carrier gas flow rate: 1.2 mL/min; injection port temperature: 250 ◦C; scanning mode: full scan; ion source temperature: 230 ◦C; quadrupole temperature: 150 ◦C; temperature program: initial temperature of 50 ◦C for 2 min, raised to 180 ◦C at the rate of 5 ◦C/min for 5 min, then raised to 250 ◦C at the rate of 10 ◦C/min for 5 min.

The mass spectra were searched using the NIST database to identify the volatile components in the samples, and the relative content of each component was analyzed by the area normalization method.

#### *2.7. Statistical Analysis*

All determinations were conducted at least three times, and all results were expressed as mean ± standard deviation (*x* ± SD). One-way analysis of variance (ANOVA) and Duncan's test were used to analyze the differences in the properties of four kinds of PBMs using IBM SPSS 25.0 (SPSS Inc., Chicago, IL, USA); *p* < 0.05 was considered significant, and all results were expressed as mean ± standard deviation (*x* ± SD). All statistical graphs were produced with Origin Pro 2018 (GraphPad Software Inc., San Diego, CA, USA).

#### **3. Results and Discussion**

#### *3.1. Effect of Protein Composition on the Structural Characteristics of PBMs*

The basic compositions of the four kinds of PBMs are shown in Table 1. Among them, the protein (49.18%) and oil contents (4.17%) of SF 5 were slightly higher than those of the other varieties. The SDS-PAGE spectra of four kinds of PBMs are shown in Figure 1a, and the quantitative results of different protein subunits are shown in Table 2. The content and ratio of 11S/7S differed significantly among the four kinds of PBMs, of which SF 5 showed the highest ratio of 11S/7S (2.50), and HH 43 showed the lowest (1.05). This result is consistent with the protein composition of the corresponding soybeans in the CARS database, which met the requirements of the experiment.


**Table 1.** The basic components of four kinds of plant-based meat analogues (PBMs) (%).

Different lowercase letters indicate a significant difference (*p* < 0.05).

**Figure 1.** Structural characteristics of four kinds of plant-based meat analogues (PBMs): (**a**) SDS-PAGE of four kinds of PBMs; (**b**) FTIR spectra of four kinds of PBMs; (**c**) Free sulfhydryl content of four kinds of PBMs; (**d**) CD spectra of four kinds of PBMs; (**e**) The microstructure of four kinds of PBMs [I: HH 43; II: JY86; III: SN 52; IV: SF 5]; (**f**) The outward appearances of four kinds of PBMs. Different lowercase letters indicate a significant difference [*p* < 0.05]). HH 43: Heihe 43, JY 86: Jiyu 86, SN 52: Suinong 52, SF 5: Shengfeng 5. **Figure 1.** Structural characteristics of four kinds of plant-based meat analogues (PBMs): (**a**) SDS-PAGE of four kinds of PBMs; (**b**) FTIR spectra of four kinds of PBMs; (**c**) Free sulfhydryl content of four kinds of PBMs; (**d**) CD spectra of four kinds of PBMs; (**e**) The microstructure of four kinds of PBMs [I: HH 43; II: JY86; III: SN 52; IV: SF 5]; (**f**) The outward appearances of four kinds of PBMs. Different lowercase letters indicate a significant difference [*p* < 0.05]). HH 43: Heihe 43, JY 86: Jiyu 86, SN 52: Suinong 52, SF 5: Shengfeng 5.

*3.2. Effect of Protein Composition on the Processing Characteristics of PBMs*  The water absorption capacity (WAC, WHC, and WSC) of the four kinds of PBMs is shown in Figure 2a. As shown, JY 86 showed the highest WAC (275%); a lower or higher ratio of 11S/7S results in a lower WAC. Meanwhile, WHC and WSC had a positive correlation with the 11S/7S ratio, wherein a higher content of 11S led to a higher WHC and WSC. Excellent water adsorption capacity could make PBMs more like meat [40]. Therefore, the higher WHC (274%) and WSC (135%) might give SF 5 superior processing characteristics. Tensile strength (TS) refers to the maximum tensile capacity that food can bear and The FTIR spectra of four kinds of PBMs are shown in Figure 1b. As shown, the significant absorption peaks were located at 3281 cm−<sup>1</sup> , 2928 cm−<sup>1</sup> , 2850 cm−<sup>1</sup> , 2366 cm−<sup>1</sup> , 1740 cm−<sup>1</sup> , 1632 cm−<sup>1</sup> , 1527 cm−<sup>1</sup> , 1247 cm−1, and 1042 cm−<sup>1</sup> ; the spectra of the four kinds of PBMs showed minor differences. The spectra are determined by the combination of protein and dietary fiber in PBMs. The secondary structure of the protein was determined based on the amide I band analysis (1700–1600 cm−<sup>1</sup> ) [29], but there are also some functional groups similar to soybean dietary fiber in other components, such as some aldehyde and carboxyl groups [30]. Thus, the functional group composition of the four PBMs displayed little difference.

represents the toughness of food. Breaking elongation (BE) is the maximum length change of food before fracture and represents the elasticity. The results are shown in Figure 2b,c. The trends for TS and BE were similar: SN 52 (11S/7S = 2.0) showed the highest forward strength (TS = 0.013 MPa, BE = 23.38%) and JY 86 (11S/7S = 1.5) showed the highest reverse strength (TS = 0.030 MPa, BE = 32.41%). The forward strength represents the tightness of the textured structure of the PBM, while the reverse strength represents the strength of

addition, this result seems to be opposite to that of free SH content in Figure 1c, in that the PBM with the lowest SH content showed the best elasticity and toughness; this proves that a high SH content does not mean that the texture characteristics of a PBM are better.

*Foods* **2022**, *11*, 1112


**Table 2.** Subunit composition of the proteins in plant-based meat analogues (PBMs) (%).

The effect of the protein composition on free sulfhydryl (SH) content in PBMs is shown in Figure 1c. The SH content seems to have no obvious correlation with protein composition: HH 43 showed the highest, and JY 86 was the lowest. The SH content of 11S is higher than that of 7S [31]. However, in a mixed system, more 11S converts SH to disulfide bonds [32]. Therefore, this result might be caused by the mixed nature of the system and the protein denaturation process of PBMs. Studies have shown that the SH content in textured protein is related not only to raw materials but also to extrusion temperature, protein denaturation, and other factors [33,34]. Studies have shown that, with the extrusion process, the degree of protein cross-linking increases, the proportion of high-molecular-weight protein subunits increases, and the small-molecular-weight subunits decreases, resulting in a lower SH content [35]. Therefore, the high SH content does not mean that the texture characteristics of the PBMs were better; this needs to be discussed comprehensively in combination with future research.

The CD spectra of four kinds of PBMs are shown in Figure 1d. Combined with the analysis of the amide I band (1700–1600 cm−<sup>1</sup> ) in FTIR, the secondary structure compositions of the proteins in the four kinds of PBMs are shown in Table 3. There were significant differences in the protein secondary structures of the four kinds of PBMs. Compared to conventional soybeans or soy protein isolate (SPI), the ratios of the various secondary structures were also different [29,36]. The secondary structures of soybean proteins of different varieties should be very different, but the differences between them are significantly smaller after being processed into PBM. This shows that the screw extrusion process rearranges the secondary structures of the proteins, which should be regarded as the key factor for changing the secondary structures of proteins, rather than choosing different soybean varieties. In proteins, the existence of α-helix and β-sheet is mainly maintained by hydrogen bonds, while β-turn depends on the amino acid residues with charge [37]. This result shows that the extrusion process strengthens the rigidity of the protein structures, reduces the exposure to amino acid residues, and makes the protein structures more stable. A similar result has been found in other studies [38,39].


**Table 3.** Changes in the secondary structure of four kinds of plant-based meat analogues (PBMs) (%).

Different lowercase letters indicate a significant difference (*p* < 0.05).

The outward appearances of the four kinds of PBMs are shown in Figure 1f. As shown, the appearances of the four kinds of PBMs were not very different; among them, the surface of HH 43 was slightly dense. It should be noted that the wrinkles on the surface of PBMs result from the shear caused by the grinding head changing between different stages, rather than any difference in the apparent structure of the PBM itself. Figure 1e shows the differences in the microstructures of the four kinds of PBMs. JY 86 has an obvious lamellar structure and flat surface (II), the surface of HH 43 is uneven with torn lamellae (I), SN 52 has a smooth surface, less clearance, and an irregular shape (III), and SF 5 has the most obvious structure and is dense with an irregular shape (IV).

In summary, protein composition did affect the structural properties of the PBMs, but the correlation was not significant.

#### *3.2. Effect of Protein Composition on the Processing Characteristics of PBMs*

The water absorption capacity (WAC, WHC, and WSC) of the four kinds of PBMs is shown in Figure 2a. As shown, JY 86 showed the highest WAC (275%); a lower or higher ratio of 11S/7S results in a lower WAC. Meanwhile, WHC and WSC had a positive correlation with the 11S/7S ratio, wherein a higher content of 11S led to a higher WHC and WSC. Excellent

*Foods* **2022**, *11*, x FOR PEER REVIEW 9 of 16

water adsorption capacity could make PBMs more like meat [40]. Therefore, the higher WHC (274%) and WSC (135%) might give SF 5 superior processing characteristics. ture and high pressure provided by the extrusion process enhance the rearrangement of the protein subunits and the formation of the spatial structure.

The texture characteristics of four kinds of PBMs were determined by a texture analyzer, and the results are shown in Table 4. The results show that the five indexes were directly proportional to the 11S content; that is, SF 5 showed the best texture characteristics. Among them, the resilience and the springiness increased gradually as 11S content increased, but there was no significant difference (*p* > 0.05), whereas the hardness, adhesiveness, and chewiness were significantly different among different varieties (*p* < 0.05).

The 11S content is closely related to the textural properties of soy products. For protein gel especially, there is a correlation between 11S and textural properties [41]. Increasing the 11S content could improve the texture quality of protein products to a certain extent, which is attributable to the formation of disulfide bonds [42]. A study by Zheng et al. showed that a higher β sheet content and a high ratio of 11S/7S increases the quality of soy protein gel, and disulfide bonds might be one of the reasons [43]. In addition, in the process of soy texturization, the processing conditions also strengthen the texture characteristics of the product, such as temperature and pressure, among others. Research has shown that, in conventional soy product processing, adjusting the pressure and temperature of raw material processing directly affects the processing characteristics of the products [44]; this is caused by changes in the solubility, conformation, and protein aggregation of 11S. in the same is true of the extrusion process. During heating, individual subunits within globulins undergo dissociation, unfolding, and reaggregation to render them more functional by virtue of qualities such as higher gelation [45]. In essence, textured protein is another form of gelation, which also requires the rearrangement of different protein subunits. The higher 11S content also leads to the higher strength of the protein aggregates [46], which better withstand high pressure [47]. Therefore, the excellent texture characteristics of SF 5 may be due to the high 11S content; meanwhile, the high tempera-

This showed that 11S globulin plays an important role in the extrusion process.

**Figure 2.** Processing characteristics of four kinds of plant-based meat analogues (PBMs): (**a**) WAC, WHC, and WSC of four kinds of PBMs; (**b**) TS of four kinds of PBMs; (**c**) BE of four kinds of PBMs. Different lowercase letters indicate a significant difference [*p* < 0.05]). HH 43: Heihe 43, JY 86: Jiyu 86, SN 52: Suinong 52, SF 5: Shengfeng 5, WAC: Water-Absorption Capacity, WHC: Water-Holding Capacity, WSC: Water-Swelling Capacity,TS: Tensile Strength, BE: Breaking Elongation. **Figure 2.** Processing characteristics of four kinds of plant-based meat analogues (PBMs): (**a**) WAC, WHC, and WSC of four kinds of PBMs; (**b**) TS of four kinds of PBMs; (**c**) BE of four kinds of PBMs. Different lowercase letters indicate a significant difference [*p* < 0.05]). HH 43: Heihe 43, JY 86: Jiyu 86, SN 52: Suinong 52, SF 5: Shengfeng 5, WAC: Water-Absorption Capacity, WHC: Water-Holding Capacity, WSC: Water-Swelling Capacity, TS: Tensile Strength, BE: Breaking Elongation.

**Table 4.** Texture characteristics of four kinds of plant-based meat analogues (PBMs).  **Heihe 43 Jiyu 86 Suinong 52 Shengfeng 5**  Resilience 0.32 ± 0.03 a 0.35 ± 0.02 a 0.35 ± 0.03 a 0.39 ± 0.04 a Springiness 0.80 ± 0.02 a 0.83 ± 0.03 a 0.87 ± 0.04 a 0.92 ± 0.09 a Hardness (g) 606.29 ± 21.74 b 658.03 ± 19.11 a 693.82 ± 18.88 c 748.10 ± 20.38 d Adhesiveness (g·sec) 0.15 ± 0.02 ab 0.22 ± 0.06 a 0.25 ± 0.01 b 0.29 ± 0.01c Chewiness 510.34 ± 12.66 b 554.46 ± 12.11 a 578.33 ± 13.53 c 634.01 ± 10.91 d Different lowercase letters indicate a significant difference (*p* < 0.05). Tensile strength (TS) refers to the maximum tensile capacity that food can bear and represents the toughness of food. Breaking elongation (BE) is the maximum length change of food before fracture and represents the elasticity. The results are shown in Figure 2b,c. The trends for TS and BE were similar: SN 52 (11S/7S = 2.0) showed the highest forward strength (TS = 0.013 MPa, BE = 23.38%) and JY 86 (11S/7S = 1.5) showed the highest reverse strength (TS = 0.030 MPa, BE = 32.41%). The forward strength represents the tightness of the textured structure of the PBM, while the reverse strength represents the strength of the force between proteins. Therefore, JY 86 might have better toughness and elasticity. In addition, this result seems to be opposite to that of free SH content in Figure 1c, in that the PBM with the lowest SH content showed the best elasticity and toughness; this proves that a high SH content does not mean that the texture characteristics of a PBM are better.

The texture characteristics of four kinds of PBMs were determined by a texture analyzer, and the results are shown in Table 4. The results show that the five indexes were directly proportional to the 11S content; that is, SF 5 showed the best texture characteristics. Among them, the resilience and the springiness increased gradually as 11S content increased, but there was no significant difference (*p* > 0.05), whereas the hardness, adhesiveness, and chewiness were significantly different among different varieties (*p* < 0.05). This showed that 11S globulin plays an important role in the extrusion process.



Different lowercase letters indicate a significant difference (*p* < 0.05).

The 11S content is closely related to the textural properties of soy products. For protein gel especially, there is a correlation between 11S and textural properties [41]. Increasing the 11S content could improve the texture quality of protein products to a certain extent, which is attributable to the formation of disulfide bonds [42]. A study by Zheng et al. showed that a higher β sheet content and a high ratio of 11S/7S increases the quality of soy protein gel, and disulfide bonds might be one of the reasons [43]. In addition, in the process of soy texturization, the processing conditions also strengthen the texture characteristics of

the product, such as temperature and pressure, among others. Research has shown that, in conventional soy product processing, adjusting the pressure and temperature of raw material processing directly affects the processing characteristics of the products [44]; this is caused by changes in the solubility, conformation, and protein aggregation of 11S. in the same is true of the extrusion process. During heating, individual subunits within globulins undergo dissociation, unfolding, and reaggregation to render them more functional by virtue of qualities such as higher gelation [45]. In essence, textured protein is another form of gelation, which also requires the rearrangement of different protein subunits. The higher 11S content also leads to the higher strength of the protein aggregates [46], which better withstand high pressure [47]. Therefore, the excellent texture characteristics of SF 5 may be due to the high 11S content; meanwhile, the high temperature and high pressure provided by the extrusion process enhance the rearrangement of the protein subunits and the formation of the spatial structure. *Foods* **2022**, *11*, x FOR PEER REVIEW 11 of 16 **Table 5.** Composition and content of soybean isoflavones (ng/g).  **Heihe 43 Jiyu 86 Suinong 52 Shengfeng 5**  Daidzin 0.413 ± 0.03 b 0.456 ± 0.02 a 0.418 ± 0.02 b 0.389 ± 0.03 c Glycitin 0.113 ± 0.03 b 0.106 ± 0.02 b 0.118 ± 0.03 b 0.236 ± 0.05 a Genistin 0.911 ± 0.07 a 0.821 ± 0.08 a 0.877 ± 0.08 a 0.668 ± 0.05 b

#### *3.3. Effect of Protein Composition on the Nutritional Properties of PBMs* Glycitein 0.432 ± 0.06 a 0.302 ± 0.04 b 0.254 ± 0.05 c 0.312 ± 0.03 b

We chose to use the content of dietary fiber, reducing sugar, phytic acid, phytohemagglutinin, trypsin inhibitor, isoflavone, and amino acid composition to analyze the nutritional characteristics of PBMs comprehensively. The results are shown in Figures 3 and 4 and Table 5. Genistein 0.117 ± 0.01 a 0.095 ± 0.01 b 0.136 ± 0.01 a 0.098 ± 0.00 b Total 2.005 ± 0.13 a 1.797 ± 0.16 b 1.827 ± 0.15 c 1.721 ± 0.10 d Different lowercase letters indicate a significant difference (*p* < 0.05).

Daidzein 0.019 ± 0.01 a 0.017 ± 0.01 a 0.024 ± 0.01 a 0.018 ± 0.01 a

**Figure 3.** Nutritional properties of four kinds of plant-based meat analogues (PBMs): (**a**) The dietary fiber content; (**b**) The reducing sugar content; (**c**) The phytic acid content; (**d**) The phytohemagglutinin content; (**e**) The trypsin inhibitor content. Different lowercase letters indicate a significant difference [*p* < 0.05]). **Figure 3.** Nutritional properties of four kinds of plant-based meat analogues (PBMs): (**a**) The dietary fiber content; (**b**) The reducing sugar content; (**c**) The phytic acid content; (**d**) The phytohemagglutinin content; (**e**) The trypsin inhibitor content. Different lowercase letters indicate a significant difference [*p* < 0.05].

**Figure 4.** Composition of amino acids (%). Different lowercase letters indicate a significant difference (*p* < 0.05). **Figure 4.** Composition of amino acids (%). Different lowercase letters indicate a significant difference (*p* < 0.05).


*3.4. Effect of Protein Composition on the Flavor of PBMs*  **Table 5.** Composition and content of soybean isoflavones (ng/g).

components of soybean products [62], and acetic acid and hexanal are considered to be Different lowercase letters indicate a significant difference (*p* < 0.05).

beany flavor (10.52%), which means that it should be more accepted by consumers. There is a relationship between flavor and the protein composition of soybean products, but it is very complex, and even the preparation process is closely related to the flavor [64]. Similarly, the flavor characteristics of PBMs are closely related not only to the composition and structure of the proteins but also to the processing and the characteristics The dietary fiber and reducing sugar content of four kinds of PBMs are shown in Figure 3a,b. In PBMs, neither have a strong relationship with protein composition, in theory. Dietary fiber in soybean products is derived from cellulose, hemicellulose, and lignin in soybean [30], while reducing sugar is derived from the destruction of polysaccharides, including dietary fiber, during extrusion [48].

the main contributors to the beany flavor [63]. In this study, SN 52 showed the lowest

of the products [39]. Compared to protein composition, we believe that the structure of PBMs has a greater impact on flavor. In this study, the structural characteristics of SN 52 demonstrated average features (all indexes were in the middle reaches), which may mean that the soybean varieties suitable for preparing PBMs are not those varieties with a notable difference in their 11S/7S ratios (too high or low). In addition, whether the bad flavor of PBMs can be reduced by adjusting the processing parameters is an important research direction. The findings for three antinutritional factors in PBMs are shown in Figure 3c–e. As these are substances that have negative effects on processing and nutritional characteristics, a lower content of these antinutritional factors in PBMs may improve the quality. The results show that there was no significant relationship between the antinutritional factor levels and the protein composition, with the exception of trypsin inhibitors. HH 43 showed the highest levels of phytic acid (135.62 mg/g) and trypsin inhibitors (16.09 µg/mg), and SF 5 showed the highest phytohemagglutinin content (822.80 pg/mg). Overall, the levels of antinutritional factors for JY 86 and SN 52 were slightly lower.

Trypsin inhibitors mainly exist in 2S globulins [49]. However, excessive intake leads to the decline of protein digestion, absorption, and utilization in the intestine [50]. Phytohemagglutinin mainly exists in 7S [51] and may cause a decrease in digestive ability [52]. Compared with the two other antinutritional factors, the content of phytic acid depends more on the existence of enzymes necessary for its biological process. The key enzymes in phytic acid biosynthesis, myo inositol-3-phosphate synthase (MIPS) and phosphatidylinositol kinase (IPK), are mainly located in 7S [53]. However, the molecular weight of many enzymes is still uncertain and complex [54], and the effect of the 11S/7S ratio on phytic acid metabolism cannot currently be determined. However, the content of antinutritional factors

in soybean products changes significantly with the processing process [55]. Therefore, the extrusion process may greatly change the content of antinutritional factors in PBMs.

The composition of isoflavones and the levels found in the four kinds of PBMs are shown in Table 5. Similar to other nutrients, there was no significant correlation between isoflavone content and protein composition. HH 43 showed the highest total isoflavone content (2.005 ng/g), and SF 5 showed the lowest (1.721 ng/g). During extrusion, the bioactivity and stability of isoflavones in PBMs are affected by the processing conditions [56,57]. Isoflavones may degrade, especially at high temperatures or high pressure [58]. One study showed that isoflavones transform into daidzein groups under high temperatures [59]. However, in this study, the daidzein, glycitein, and genistein levels were significantly lower than that of daidzin, glycitin, and genistin, which means that the isoflavones were significantly transformed during extrusion.

The amino acid compositions and contents for four kinds of PBMs are shown in the Supplementary Materials (Table S1) and in Figure 4. Generally, the amino acid compositions of the four kinds of PBMs displayed little difference, but SF 5 was dominant with a higher content of essential amino acids (39.66%). Among all amino acids, the glutamate content was the highest, and it was higher in SN 52 than in the other three (20.08%). As an amino acid that can enhance flavor [60], the high glutamate content may change the flavor of PBMs; analysis of flavor characteristics is required. In addition, we found that the levels of many amino acids increased with a higher amount of 11S, such as threonine, alanine, and arginine; however, some decreased, such as aspartate. This may be due to the differences in amino acid composition between 11S and 7S [61].

Briefly, soybean varieties with a low 11S/7S ratio should not be selected if nutritional characteristics are used as the standard to judge the quality of PBMs.

#### *3.4. Effect of Protein Composition on the Flavor of PBMs*

The results pertaining to the flavor compounds for the four kinds of PBMs are shown in the Supplementary Materials (Tables S2–S5). Additionally, we plotted the results in Figure 5 according to the classification of substances. As shown, the relative amounts of flavor components in different kinds of PBMs were different, but the main components in all cases were aldehydes, alcohols, alkanes, etc. SN 52 had the highest aldehyde content, and HH 43 had the lowest; the highest alcohol compound content was found in SF 5, and the lowest was SN 52; SN 52 had the highest alkane content, and HH 43 had the lowest. Some of the flavor substances, including acetic acid, hexanal, benzaldehyde, 1-octene-3 alcohol, 2,4-decadienal, trans-2-heptenenal, etc. are defined as typically negative flavor components of soybean products [62], and acetic acid and hexanal are considered to be the main contributors to the beany flavor [63]. In this study, SN 52 showed the lowest beany flavor (10.52%), which means that it should be more accepted by consumers.

There is a relationship between flavor and the protein composition of soybean products, but it is very complex, and even the preparation process is closely related to the flavor [64]. Similarly, the flavor characteristics of PBMs are closely related not only to the composition and structure of the proteins but also to the processing and the characteristics of the products [39]. Compared to protein composition, we believe that the structure of PBMs has a greater impact on flavor. In this study, the structural characteristics of SN 52 demonstrated average features (all indexes were in the middle reaches), which may mean that the soybean varieties suitable for preparing PBMs are not those varieties with a notable difference in their 11S/7S ratios (too high or low). In addition, whether the bad flavor of PBMs can be reduced by adjusting the processing parameters is an important research direction.

**Figure 5.** Flavor components of four kinds of plant-based meat analogues (PBMs). **Figure 5.** Flavor components of four kinds of plant-based meat analogues (PBMs).

#### **4. Conclusions**

**4. Conclusions**  In this study, defatted soybean powder was prepared from four of the main soybean varieties in China, and four kinds of PBMs with different protein compositions were prepared. According to our analysis of the structure, processing characteristics, nutritional characteristics, and flavor characteristics, it may be advisable to prepare PBMs using a soybean with a moderate 11S/7S ratio (1.5:1 to 2.0:1) in order to achieve better quality characteristics. In addition, although the protein composition affects the quality characteristics of PBMs significantly, alterations in the processing process may cause even greater changes, which may play a key role in actual production. This study provides a basic theory for selecting the raw materials for PBMs, and in-depth research would be conducive to further development, including the manufacturing of special equipment, special-use soybean varieties for PBMs, bioactive substances—PBM complex reconstruction, and products with different processing applications. However, these potential applications require a more in-depth study of the production mechanisms of PBMs, including not only the selection of raw materials but also the transformation of bioactive substances, the conformational transition of proteins at different stages, the effects of different additives on the characteristics of PBMs, and even methods of sensory gaining. The above In this study, defatted soybean powder was prepared from four of the main soybean varieties in China, and four kinds of PBMs with different protein compositions were prepared. According to our analysis of the structure, processing characteristics, nutritional characteristics, and flavor characteristics, it may be advisable to prepare PBMs using a soybean with a moderate 11S/7S ratio (1.5:1 to 2.0:1) in order to achieve better quality characteristics. In addition, although the protein composition affects the quality characteristics of PBMs significantly, alterations in the processing process may cause even greater changes, which may play a key role in actual production. This study provides a basic theory for selecting the raw materials for PBMs, and in-depth research would be conducive to further development, including the manufacturing of special equipment, special-use soybean varieties for PBMs, bioactive substances—PBM complex reconstruction, and products with different processing applications. However, these potential applications require a more in-depth study of the production mechanisms of PBMs, including not only the selection of raw materials but also the transformation of bioactive substances, the conformational transition of proteins at different stages, the effects of different additives on the characteristics of PBMs, and even methods of sensory gaining. The above problems need further research to promote the improved development of PBM products.

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: The amino acid composition of four kinds of plant-based meat analogues (PBMs) (%); Table S2: Volatile flavor compounds of the PBM produced by Heihe 43; Table S3: Volatile flavor compounds of the PBM produced by Jiyu 86; Table S4: Volatile flavor compounds of the PBM produced by Suinong 52; Table S5: Volatile flavor compounds of the PBM produced by **Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11081112/s1, Table S1: The amino acid composition of four kinds of plant-based meat analogues (PBMs) (%); Table S2: Volatile flavor compounds of the PBM produced by Heihe 43; Table S3: Volatile flavor compounds of the PBM produced by Jiyu 86; Table S4: Volatile flavor compounds of the PBM produced by Suinong 52; Table S5: Volatile flavor compounds of the PBM produced by Shengfeng 5.

problems need further research to promote the improved development of PBM products.

Shengfeng 5. **Author Contributions:** B.L.: conceptualization, software, writing—original draft; J.L.: visualization, software, writing—original draft; X.M.: methodology; H.F.: investigation; W.W.: methodology; L.J.: **Author Contributions:** B.L.: conceptualization, software, writing—original draft; J.L.: visualization, software, writing—original draft; X.M.: methodology; H.F.: investigation; W.W.: methodology; L.J.: visualization; Y.W.: investigation; Z.G.: funding acquisition, project administration; H.Y.: funding

visualization; Y.W.: investigation; Z.G.: funding acquisition, project administration; H.Y.: funding

acquisition, project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by: China Agriculture Research System of MOF and MARA, grant number CARS-04; Young & Middle-Aged Technological Innovation Outstanding Talent (Team) Project (Innovation), grant number 20210509015RQ; Major Science and Technology Innovation Projects in Shandong (Major Scientific & Technological Innovation), grant number 2022CXGC010603.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Raw data can be provided by the corresponding author on request.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


## *Review* **Applications of Plant Protein in the Dairy Industry**

**Ge-Ge Hu <sup>1</sup> , Jing Liu <sup>1</sup> , Yi-Hui Wang <sup>1</sup> , Zhen-Nai Yang 1,\* and Hong-Bo Shao 2,3,\***


**Abstract:** In recent years, a variety of double protein dairy products have appeared on the market. It is a dairy product made by replacing parts of animal protein with plant protein and then using certain production methods. For some countries with limited milk resources, insufficient protein intake and low income, double protein dairy products have a bright future. More and more studies have found that double protein dairy products have combined effects which can alleviate the relatively poor functional properties of plant protein, including solubility, foaming, emulsifying and gelling. In addition, the taste of plant protein has been improved. This review focuses on the current state of research on double protein dairy products. It covers some salient features in the science and technology of plant proteins and suggests strategies for improving their use in various food applications. At the same time, it is expected that the fermentation methods used for those traditional dairy products as well as other processing technologies could be applied to produce novelty foods based on plant proteins.

**Keywords:** plant protein; double protein dairy; process flow; production; health effects; taste; flavor

#### **1. Introduction**

Protein is the material basis of all life and plays a very important role in maintaining the normal growth, metabolism and immune regulation of the body. According to the source of intake, protein can be divided into animal protein and plant protein. Animal protein is rich in a lot of essential amino acids, but excessive intake of animal protein will greatly increase fat intake. Saturated fatty acids as the main component will lead to increased plasma cholesterol levels, which is not conducive to human health [1]. Due to the improvement of people's living standards and the gradual increase in protein intake, the supply of animal protein is not enough to meet people's growing demand [2]. Plant protein has the advantages of abundant resources, being cheap and easy to obtain, having no cholesterol, and it can prevent diseases [3]. It is a green and safe food raw material, which can make up for the insufficient supply of animal protein.

In 2006, the "Shanghai Declaration" was released at the "Second China Soybean Food Industry Roundtable Summit" held in Shanghai, which was the first time that China proposed the concept of "double protein". The concept of "double protein" and the "double protein" strategy were put forward for the first time, emphasizing the combination of soy protein and milk protein to meet the health needs of comprehensive protein supplementation. It is pointed out that efforts should be made to develop new nutritional and healthy foods such as nutritionally fortified foods and double protein foods. With people's further

**Citation:** Hu, G.-G.; Liu, J.; Wang, Y.-H.; Yang, Z.-N.; Shao, H.-B. Applications of Plant Protein in the Dairy Industry. *Foods* **2022**, *11*, 1067. https://doi.org/10.3390/ foods11081067

Academic Editor: Yonghui Li

Received: 20 March 2022 Accepted: 5 April 2022 Published: 7 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

awareness of nutrition and health, high-nutrition and high-protein products are more and more attractive to consumers [4].

Dairy products are liquid or solid products made from milk as the main raw material through heating, drying and fermentation processes. Dairy products are rich in protein, fat and carbohydrates, which are easy to digest and absorb. In particular, it contains vitamins and calcium, which are an excellent source of nutrients needed by the human body. As a nutritious and comprehensive ideal food, dairy products occupy a very important position in the dietary structure [5]. Due to the existence of healthy long-chain unsaturated fatty acids, the development of dairy products based on plant protein endows dairy products with new nutrition and health care connotations. It not only realizes the functionalization and popularization of dairy products, but also promotes the diversified development of the dairy processing industry.

#### **2. Double Protein Dairy**

### *2.1. Double Protein Yogurt*

Yogurt is a fast acid-producing product, which is made from raw cow (goat) milk or milk powder after high-speed homogenization, sterilization and fermentation. Due to its unique flavor and texture, it is currently the most widely distributed and consumer favorite fermented dairy product in the world. Double protein yogurt uses plant protein and animal protein as the main nutritional bases. Through probiotic fermentation, it has a unique flavor and high nutritional value, which helps to improve nutrition and improve human health.

During the fermentation process of yogurt, the performance of the starter greatly affects the quality of yogurt. Therefore, the development of probiotic strains with excellent fermentation performance is the key to the preparation of a highly active starter [6]. At present, dairy production enterprises mainly use *Streptococcus thermophilus* and *Lactobacillus bulgaricus* as starters for producing yogurt. In recent years, research on plant protein yogurt starter has been increasing. In the fermentation of suitable strains of plant-based yogurt, one or more functional strain combinations can be included.

Sertovic et al. [7] mixed *Streptococcus thermophilus*, *Lactobacillus bulgaricus* and *Lactobacillus acidophilus* to ferment milk and soymilk, and they found that the acidity of fermented milk was higher than that of fermented soymilk. This showed that the optimal starter for soy protein yogurt was slightly different from that of ordinary milk yogurt. Havas et al. [8] fermented pure soymilk using human-derived bacteria *Bifidobacterium* B3.2 and *Bifidobacterium* B7.1. The results showed that the acid-producing ability of the two kinds of bifidobacteria in soymilk was similar to that of cow's milk, and there was no unpleasant odor. Li et al. [9] found that both *Lactobacillus plantarum* fs-4 and *Lactobacillus casei* 05-20 had protease activity. They could utilize nutrients such as sugar and protein in peanut milk and were suitable for fermenting peanut milk. The obtained peanut yogurt was white in color and had a peanut and milk flavor. Wan et al. [10] used rice-milk double-protein yogurt fermented by *Lactobacillus furfur* or *Lactococcus lactis* subsp. *lactis*. When the degree of hydrolysis was 7.5%, the sensory score was the highest, which showed that the color was bright, the curd state was stable, the taste was fine and there was no bad flavor. When the degree of hydrolysis exceeded 7.5%, the bitterness of the product became heavier. The main reason was that the rice protein was hydrolyzed to a certain extent and produces bitter peptides. Wang [11] found that mixed strain fermentation had a better effect on the overall quality of coconut yogurt than single strain fermentation. When the addition amount of coconut milk was 30% and the addition amount of whey protein and sodium caseinate was 2%, the starter was mixed and fermented with *Streptococcus thermophilus*, *Lactobacillus bulgaricus*, *Lactobacillus casei* and *Lactobacillus plantarum* in the same proportion. Coconut yogurt was the best quality.

Taking peanut yogurt as an example, peanuts are rich in protein and essential amino acids, and the nutritional composition of peanut protein and animal protein is similar. If the peanut milk is simply fermented with lactic acid bacteria, the fat content in the peanut kernel is too high, which will lead to oil circles. The high fiber content in peanuts will also affect the formation of peanut yogurt gel. Therefore, milk powder can be added to the peanut yoghurt, so that the peanut yoghurt gel can be formed stably. Peanut yogurt is made of peanuts and milk or milk powder as the main raw materials. After lactic acid bacteria fermentation, it has the characteristics of complementary animal and plant protein and reasonable nutritional structure [12]. The process flow of peanut milk and peanut yogurt is shown in (Figure 1). The researchers studied the ratio of peanut protein and milk or milk powder and obtained peanut yogurt with unique flavor and good taste. the peanut milk is simply fermented with lactic acid bacteria, the fat content in the peanut kernel is too high, which will lead to oil circles. The high fiber content in peanuts will also affect the formation of peanut yogurt gel. Therefore, milk powder can be added to the peanut yoghurt, so that the peanut yoghurt gel can be formed stably. Peanut yogurt is made of peanuts and milk or milk powder as the main raw materials. After lactic acid bacteria fermentation, it has the characteristics of complementary animal and plant pro‐ tein and reasonable nutritional structure [12]. The process flow of peanut milk and peanut yogurt is shown in (Figure 1). The researchers studied the ratio of peanut protein and milk or milk powder and obtained peanut yogurt with unique flavor and good taste.

dium caseinate was 2%, the starter was mixed and fermented with *Streptococcus thermoph‐ ilus*, *Lactobacillus bulgaricus*, *Lactobacillus casei* and *Lactobacillus plantarum* in the same pro‐

Taking peanut yogurt as an example, peanuts are rich in protein and essential amino acids, and the nutritional composition of peanut protein and animal protein is similar. If

*Foods* **2022**, *11*, x FOR PEER REVIEW 3 of 14

portion. Coconut yogurt was the best quality.

**Figure 1.** Process flow chart showing some common methods and steps used for production of pea‐ nut protein and peanut yogurt. **Figure 1.** Process flow chart showing some common methods and steps used for production of peanut protein and peanut yogurt.

Qin et al. [13] reported the best production process parameters for peanut protein yogurt: the ratio of peanut protein powder to water was 1:9.4, pure milk 15%, whey pro‐ tein powder 2%, white sugar 10%, starter 3% and fermentation time 7.4 h. The obtained sensory score was 38.02% higher than that before the previous optimization. Compared with the conventional nutritional content of commercially available yogurt, peanut pro‐ tein yogurt was found to have at least 81.07% higher protein content, at least 25.08% lower fat content, and no significant difference in acidity. According to the acidity, taste and product status, Yang et al. [14] found that the optimal inoculum of lactic acid bacteria‐ fermented peanuts was about 3%, and the mass ratio of peanut milk, white granulated sugar and milk powder was 90:5:3.5. The obtained peanut yogurt had both the rich aroma of peanut and the flavor of yogurt, and the curd was dense and shiny. Tong [15] found that the ratio of peanut protein to milk protein was 1:2, and then added 0.02% konjac flour and 0.1% soybean polysaccharide for compounding. The peanut yogurt was glossy, evenly curd, moderately sweet and sour, and had a peanut flavor. Ma et al. [16] found that the optimal formula for sprouted peanut yogurt was 8% sugar and 2% sprouted pea‐ nut freeze‐dried powder. Sprouted peanut yogurt had higher protein content than regular yogurt and slightly lower moisture content. Fang et al. [17] selected *Lactobacillus Delbruckii* as the strain for fermentation, and used purple potato, peanut milk and milk powder as the main raw materials. The peanut and purple potato were organically fermented, and the nutrients of the two were retained. The prepared purple potato peanut yogurt had a mellow taste, full color and greatly improved antioxidant activity. Cao et al. [18] used shelled ginkgo nuts and peanuts as the main raw materials to determine the optimal for‐ mula of ginkgo peanut yogurt. The mass ratio of ginkgo to peanut was 1:6, the volume ratio of peanut milk to milk was 6:4, the added sugar was 7% and the inoculum of *Bacillus* Qin et al. [13] reported the best production process parameters for peanut protein yogurt: the ratio of peanut protein powder to water was 1:9.4, pure milk 15%, whey protein powder 2%, white sugar 10%, starter 3% and fermentation time 7.4 h. The obtained sensory score was 38.02% higher than that before the previous optimization. Compared with the conventional nutritional content of commercially available yogurt, peanut protein yogurt was found to have at least 81.07% higher protein content, at least 25.08% lower fat content, and no significant difference in acidity. According to the acidity, taste and product status, Yang et al. [14] found that the optimal inoculum of lactic acid bacteria-fermented peanuts was about 3%, and the mass ratio of peanut milk, white granulated sugar and milk powder was 90:5:3.5. The obtained peanut yogurt had both the rich aroma of peanut and the flavor of yogurt, and the curd was dense and shiny. Tong [15] found that the ratio of peanut protein to milk protein was 1:2, and then added 0.02% konjac flour and 0.1% soybean polysaccharide for compounding. The peanut yogurt was glossy, evenly curd, moderately sweet and sour, and had a peanut flavor. Ma et al. [16] found that the optimal formula for sprouted peanut yogurt was 8% sugar and 2% sprouted peanut freeze-dried powder. Sprouted peanut yogurt had higher protein content than regular yogurt and slightly lower moisture content. Fang et al. [17] selected *Lactobacillus Delbruckii* as the strain for fermentation, and used purple potato, peanut milk and milk powder as the main raw materials. The peanut and purple potato were organically fermented, and the nutrients of the two were retained. The prepared purple potato peanut yogurt had a mellow taste, full color and greatly improved antioxidant activity. Cao et al. [18] used shelled ginkgo nuts and peanuts as the main raw materials to determine the optimal formula of ginkgo peanut yogurt. The mass ratio of ginkgo to peanut was 1:6, the volume ratio of peanut milk to milk was 6:4, the added sugar was 7% and the inoculum of *Bacillus bulgaricus* and *Streptococcus thermophilus* was 3%. It was possible to obtain nutritious yogurt with a unique taste and delicate organization.

At present, in addition to the scientific research and manufacturing of double-protein yogurt with peanuts as raw materials, double-protein yogurt with beans, nuts, cereals and other plants as raw materials are also emerging one after another. Bruckner-Guhmann et al. [19] reported that the gel strength and elasticity of fermented milk added with oat protein were lower than those of pure fermented milk. However, the sensory evaluation results showed that the fermented milk containing oat protein had better taste, more

delicate texture, and had a significant oat flavor. Su et al. [20] mixed pecan milk and milk to develop pecan milk yogurt. Pecan milk yogurt had higher antioxidant properties than regular yogurt. Total solids, fat, crude protein, and amino acids were also higher. Appearance and flavor scores were not significantly different from regular yogurt, but texture scores were higher. Li et al. [21] developed coagulated pea yogurt with pea protein powder and milk as the main raw materials. When the added amount of pea protein powder was 6%, the obtained yogurt had moderate acidity and good quality characteristics. The sensory evaluation was the highest, with a mixed aroma of pea protein powder and yogurt. Chang et al. [22] fermented quinoa milk with 5% puffed quinoa flour and milk as raw materials. At this time, the consistency and acidity of quinoa yogurt had reached the optimal level. Compared with ordinary yogurt, 30 new flavor substances were detected in quinoa yogurt, including six plant-derived terpene compounds and alpha-terpineol. Therefore, the addition of puffed quinoa powder made yogurt have certain advantages in terms of nutrition and flavor. Yang et al. [23] found that the fat content of quinoa yogurt was much lower than that of ordinary yogurt, but the protein content was higher than that of ordinary yogurt. At the same time, it was rich in 8 essential amino acids, among which the content of essential amino acids such as isoleucine and leucine were significantly higher than other yogurt. Gao et al. [24] found that adding about 0.2% soybean and corn combination peptide and 87.5% trehalose to milk powder, the obtained soybean and corn combination peptide fermented milk had the best quality. The smell was refreshing, the taste was delicate, the ingredients were uniform and it had the nutritional and health care functions of soybean polypeptide and rice polypeptide.

Therefore, combining the advantages of plant protein and yogurt can not only enrich the product variety of yogurt products, but also more in line with contemporary people's pursuit of health and nutrition.

#### *2.2. Double Protein Beverages*

With the development of science and technology, people's pursuit of health is getting higher and higher. The development of beverages has entered a new stage, from the original scale growth to the quality upgrade. As a result, the market share of carbonated beverages has continued to decline, and the consumption trend of healthy and natural beverages such as plant protein beverages, fruit and vegetable juices and tea beverages has risen.

Today, plant-based cereal and nut beverages are the newcomers to the dairy industry, but there are some technical challenges in making new dairy products from cereals and nuts. Compared with natural milk, some grains and nuts are rich in starch and fiber. The suspension stability in milk beverages is poor, so the phenomenon of particle suspension and stratification precipitation is easy to occur. In addition, the taste of beverage products is light and bitter. In order to solve these problems, researchers have conducted related research in recent years, including adding stabilizers and thickeners to stabilize product quality; adding flavor substances to improve taste; enriching and strengthening nutrients to improve nutritional value, etc.

Yang et al. [25] studied the compounding scheme of emulsion stabilizer in oat milk beverage. Orthogonal test results showed that the compound stabilizer ingredients included 0.3% microcrystalline cellulose, 0.012% carrageenan, 0.10% mono- and diglyceride fatty acid esters and 0.06% sodium stearoyl lactylate. The stabilizer could effectively suspend product particles and had a good effect of controlling product fat floating. Li et al. [26] determined that the optimal ratio of peanut pulp and milk was 1:2, the stabilizers were sucrose fatty acid ester (SE) 0.05%, glycerol monostearate (GMS) 0.1%, carboxymethyl cellulose sodium (CMC-Na) 0.025%. The produced beverage had good stability. Han et al. [27] found that the addition of walnut juice was 15%, the addition of peanut juice was 25%, the addition of milk was 30%, and the addition of sucrose was 6%. The developed compound milk beverage had the best taste flavor. Under the optimal process conditions, when sodium alginate, gum arabic and CMC were selected as stabilizers, the precipitation

rate of the composite beverage decreased and its stability was the best. Huang et al. [28] added 0.04% pectin, 0.02% gellan gum and 0.6% CMC in the production of fermented walnut milk beverages, which not only solved the problem of layered precipitation, but also gave the beverage rich taste.

Enzymatic hydrolysis is the use of amylase or protease to enzymatically hydrolyze macromolecular substances such as starch and protein in grains or nuts under certain conditions. This will refine the granules in the drink, decompose some insoluble starch and protein into soluble sugar, dextrin, polypeptide and amino acid, thereby improving the stability of the drink [29]. Hou et al. [30] used oat as the main raw material and added 0.15% α-amylase for enzymatic hydrolysis. Then, added 1.50% whole milk powder, 3.0% white sugar, 0.15% citric acid, 0.10% pectin and 0.05% xanthan gum to develop the best production process of a new type of cereal beverage. Li et al. [31] used walnut pulp and pea milk as the main raw materials, the addition of milk was 10%, and the addition of white sugar was 3%. The amount of α-amylase added was 0.4%, and the enzymatic hydrolysis was carried out at 70 ◦C for 3 h. The walnut and pea milk produced under this condition was stable and had the aroma of walnut kernels and peas.

In addition, homogenization can make fat globules smaller. The miniaturization and homogenization of suspended particles can prevent the separation of finished fat and the precipitation of protein particles, thereby improving the emulsification and stability of liquid grain dairy products. Two important parameters of homogenization are homogenization pressure and homogenization temperature. Ma et al. [32] determined the optimal homogenization conditions in the stability study of black glutinous rice milk beverage, that is, homogenized twice under the conditions of 60 ◦C and 20–30 MPa. When the homogenization temperature was too high, the protein in the system might denature and cause flocculation. When the homogenization pressure exceeded 40 MPa, the number of collisions of suspended particles in the system increased, resulting in polymerization, which eventually led to an increase in the precipitation rate of the system.

From the perspective of raw materials, the taste of beverages can be improved by adding natural raw materials. Zheng et al. [33] used barley and buckwheat as raw materials. Passion fruit juice (10%), xylitol (10%) and citric acid (0.05%) were added to enhance the taste, and the resulting compound grain beverage was rich in aroma and sweet in taste. Wang et al. [34] used walnut juice and milk as the main raw materials. With the addition of 10% macadamia juice, the drink tasted best and had a special aroma of walnuts and macadamia nuts. Zhang et al. [35] used peanut, wolfberry and milk as raw materials, white granulated sugar and xanthan gum as ingredients to develop peanut and wolfberry milk. The study found that the best roasting temperature for peanuts was 120 ◦C, and the best roasting time was 20 min, the peanuts had the strongest aroma.

From the perspective of preparation, suitable flavor substances can be derived by means of fermentation. Tavares et al. [36] pointed out that organic acids such as lactic acid and acetic acid are released during fermentation and refrigeration of corn beverages fermented with probiotics and yeast. Maintaining the pH of beverages at around 4.0 had an important impact on food safety, taste and aroma. Tue et al. [37] dried and ground germinated brown rice into powder, and fermented after adding honey, corn germ oil and yeast. After adding milk, white sugar and citric acid, it was homogenized to make a brown rice enzyme milk drink with unique flavor.

The double protein beverage has rich raw material resources, meets the individual needs of consumers, conforms to the development trend of market consumption and has a certain health care value, so it has broad development prospects.

#### *2.3. Double Protein Cheese*

Cheese is made from cow or goat milk. Adding an appropriate amount of starter and rennet can make the protein coagulate, discharge part of the whey, and finally ferment and mature after a certain period of time. During the stage of cheese fermentation, proteins and fats are enzymatically decomposed into tiny substances that are easily absorbed in the human digestive system, which improves the absorption and utilization rate of cheese. Therefore, it has the reputation of milk gold in the industry.

In recent years, a mixed cheese has appeared on the market, which is a cheese made by replacing part of the protein in animal milk with protein extracted from plants. Using plant protein to replace part of animal protein can not only reduce the cost of cheese production, but also improve the nutritional value of cheese. In the cheese research and development field, the experiment of replacing part of animal protein with plant protein has become a new research and development direction [38].

Among them, mixed soybean cheese is the most studied double protein cheese. Soybean contains 35~40% protein, which is a high-quality source of plant protein. It has high nutritional value, contains various amino acids and unsaturated fatty acids necessary for the human body, and is also rich in minerals and vitamins. Soy protein plays an important role in the diet structure of many countries. The development of mixed soybean protein cheese can not only reduce the production cost of cheese, alleviate the shortage of milk source, but also promote the deep processing of soybean and increase the added value of soybean products.

Under the same ripening conditions, compared with ricotta cheese, mixed soybean cheese has larger pores and looser texture. Large particles of soy protein can reduce curd stability and affect the compactness of the casein structure [39]. Therefore, the current research on the quality of mixed cheese mainly focuses on the addition amount of soymilk, the processing method and the improvement of production technology.

Yang [40] reported that when the content of soybean protein isolate was controlled at 4%, the muted taste of mixed cheese was greatly reduced, and the milky aroma was stronger. When the content of soy protein was more than 4%, the taste of milk cheese became rougher, the aroma of milk decreased, and the aroma of soy increased (Figure 2). Zhao et al. [41] found that the addition of soymilk resulted in a higher yield of Mozzarella mixed cheese and significantly reduced the fat content and firmness of the cheese samples. However, the addition of soymilk also made the cheese waterier, especially when the addition exceeded 10%. Its stretchability was significantly reduced, which was detrimental to its application on pizza. Bai et al. [42] found that with the increase in black soybean milk addition in the range of 2–6%, the water activity of cheese increased, the pH decreased, the hardness, elasticity, adhesiveness and chewiness increased. Based on the analysis of each index, the cheese made with black soybean milk 4% had a special flavor and proper indexes including color, texture, protein degradation and so on. *Foods* **2022**, *11*, x FOR PEER REVIEW 7 of 14

**Figure 2.** Process flow chart showing the production of mixed soybean cheese using soybean protein isolate and milk. **Figure 2.** Process flow chart showing the production of mixed soybean cheese using soybean protein isolate and milk.

On the premise of not reducing the cheese yield, the addition of enzymatically hy‐ drolyzed soymilk has a better effect on improving the texture of the mixed cheese. Li et

Some researchers had pointed out that an important reason why consumers do not accept blended cheeses containing soymilk was its soy flavor [45]. At present, there are three main methods to remove the beany smell. The first is to discover and cultivate new soybean varieties through the improvement of raw materials; the second is to reduce the beany smell during processing by inactivating or inactivating the activity of lipoxygenase in soybeans; the third is to improve storage conditions [46]. Ali et al. [47] used protease and peptidase to produce flavored enzyme‐modified cheese. The results showed that after enzymatic hydrolysis, the contents of amino acids, free fatty acids and volatiles in cheese were significantly increased, and the sensory properties were significantly improved. Han et al. [44] found that compared with ordinary refining, the soymilk obtained by anaerobic refining had lower overall volatile flavor substances, especially beany flavor substances. The types and contents of beany flavor components in the spread‐type soybean cheese prepared by anaerobic refining were significantly reduced, and the sensory evaluation

At present, in addition to soy cheese, other plant‐based mixed cheeses are also emerg‐ ing. Shi et al. [48] developed a hazelnut processed cheese with an optimal dosage of 30% hazelnut, and the emulsifier included 1.2% sodium citrate and 1.2% compound phos‐ phate. The prepared processed cheese had a sweet taste, fine texture and mellow hazelnut aroma. Wu [49] reported that almond pulp and milk were mixed at 45:55%, 5% starter was

cheese matures, hardness and cohesion increased, while elasticity decreased. However, the cheese made by adding soymilk had poor shape and brittleness. Adding enzymolyzed soymilk could improve this phenomenon, and the protein structure formed by adding enzymolyzed soymilk was more compact. Han et al. [44] used 0.3% papain to hydrolyze soybean milk for 15 min before processing, which could significantly reduce the particle size of soybeans, reduce product hardness and smear work. Adding complex emulsified salt (sodium citrate: sodium tripolyphosphate: sodium hexametaphosphate) to the spread‐type mixed soybean cheese could significantly improve the fineness and stability

of the product.

was higher.

On the premise of not reducing the cheese yield, the addition of enzymatically hydrolyzed soymilk has a better effect on improving the texture of the mixed cheese. Li et al. [43] found that after adding soymilk and enzymatically hydrolyzed soymilk to cheddar cheese, the moisture content increased, and the fat content decreased significantly. As cheese matures, hardness and cohesion increased, while elasticity decreased. However, the cheese made by adding soymilk had poor shape and brittleness. Adding enzymolyzed soymilk could improve this phenomenon, and the protein structure formed by adding enzymolyzed soymilk was more compact. Han et al. [44] used 0.3% papain to hydrolyze soybean milk for 15 min before processing, which could significantly reduce the particle size of soybeans, reduce product hardness and smear work. Adding complex emulsified salt (sodium citrate: sodium tripolyphosphate: sodium hexametaphosphate) to the spread-type mixed soybean cheese could significantly improve the fineness and stability of the product.

Some researchers had pointed out that an important reason why consumers do not accept blended cheeses containing soymilk was its soy flavor [45]. At present, there are three main methods to remove the beany smell. The first is to discover and cultivate new soybean varieties through the improvement of raw materials; the second is to reduce the beany smell during processing by inactivating or inactivating the activity of lipoxygenase in soybeans; the third is to improve storage conditions [46]. Ali et al. [47] used protease and peptidase to produce flavored enzyme-modified cheese. The results showed that after enzymatic hydrolysis, the contents of amino acids, free fatty acids and volatiles in cheese were significantly increased, and the sensory properties were significantly improved. Han et al. [44] found that compared with ordinary refining, the soymilk obtained by anaerobic refining had lower overall volatile flavor substances, especially beany flavor substances. The types and contents of beany flavor components in the spread-type soybean cheese prepared by anaerobic refining were significantly reduced, and the sensory evaluation was higher.

At present, in addition to soy cheese, other plant-based mixed cheeses are also emerging. Shi et al. [48] developed a hazelnut processed cheese with an optimal dosage of 30% hazelnut, and the emulsifier included 1.2% sodium citrate and 1.2% compound phosphate. The prepared processed cheese had a sweet taste, fine texture and mellow hazelnut aroma. Wu [49] reported that almond pulp and milk were mixed at 45:55%, 5% starter was added for fermentation, 0.8% rennet and 0.06% CaCl2 were added for processing. The almond cheese with milky white color, smooth and uniform, moderate sour and sweet, and rich flavor could be obtained. Tian et al. [50] developed a fermented spread walnut cheese with walnut kernels as the main raw material. The additions of lipase and flavor protease were 0.2%, and the additions of whey protein, cream, and sucrose were 1.56%, 0.81%, and 6.37%, respectively. Walnut cheese was full of flavor, high nutritional value, and had better sensory qualities. Zhang [51] used red dates and skim milk as raw materials, added starter and rennet, and made red date cheese through curdling. The experiment found that adding 4% of jujube puree, the curd time was relatively short and the curd strength was the greatest. At this time, the cheese was rich in flavor, pure in frankincense, and of the best quality. Chen [52] invented a preparation method of whole grain cheese with mild flavor and tender taste. Purple potato, sweet potato, wheat, red bean and oat were milled to make multigrain juice, mixed with skim milk, maltose, fructo oligosaccharide and honey, and then inoculated with lactic acid bacteria to ferment the curd.

At present, the research on double protein cheese is very extensive and in-depth, and the scientific research results have been applied to actual production. The development of various forms of cheese is of great benefit to the cultivation of the cheese market. The growing double protein cheese has great development potential and wide application prospects in the dairy industry.

#### *2.4. Calf Double-Protein Milk Replacer*

In order to wean the calves early, the calves should be fed with milk replacer (also known as artificial milk) instead of regular milk about 10 days after birth. At present, the use of milk replacer to cultivate and implement the early weaning technology of sucking calves has become a common technical means in the world dairy farming industry. Milk replacer raw materials are mainly composed of dairy by-products such as skim milk, whey protein concentrate, dry whey, etc. [53]. With the deepening of research and the development of milk replacer processing technology, low-cost and high-quality plant protein has become the main research direction for the development of milk replacer protein sources. High-quality plant protein and high proportion of milk protein have obvious effects on preventing and reducing calf diarrhea, and also on increasing daily weight gain of calves. Good economic benefits have been achieved by saving feeding costs [54].

The most widely used plant protein in calf milk replacer is soybean protein, wheat protein, rice protein, etc. Plant protein sources are rich and high in crude protein. The crude protein content of feed-grade soybean protein isolate, wheat hydrolyzed protein and rice protein provided in China can reach about 90%, 85% and 65%, respectively. Different sources of plant protein have different nutritional characteristics because of their different protein components and amino acid compositions and have different effects on the growth function of sucking calves [55]. Although plant protein has a slightly poorer amino acid balance, certain functional amino acids are abundant in plant protein. For example, wheat protein is rich in glutelin, accounting for 30% of the total amino acid [56]. In addition to synthesizing proteins to meet the needs of animal growth and maintenance, these functional amino acids are also necessary for the synthesis of various biologically active substances.

In the past studies, plant protein mainly had adverse factors such as poor solubility, low digestibility, poor amino acid balance and containing anti-nutritional factors [57]. However, with the development of science and technology, it is now possible to remove anti-nutritional factors through modification and processing, add different plant proteins to achieve amino acid balance, and add some enzymes or probiotics to improve the digestibility of plant proteins in animals (Table 1). Therefore, after using soybean protein isolate, wheat hydrolyzed protein and rice protein isolate as the protein source of milk replacer to partially replace milk-derived protein to feed calves, it can achieve a feeding effect similar to that of milk-derived protein [58].


**Table 1.** Main functional properties of plant protein components.

The broad concept of "double protein" does not mean that there can only be one type of plant-derived protein added. In terms of human nutrition, the purpose of proposing the "double protein" project is to balance the diet, optimize the dietary structure, and improve the nutritional status. Therefore, adding two or more plant-derived proteins into milk replacer is particularly important for balancing amino acids and optimizing the dietary structure of livestock [71]. More and more studies have found that milk replacer composed of a variety of plant proteins have a combined effect, and the feeding effect is better than the combination of a single plant-derived protein and milk-derived protein.

Huang [72] reported that the feeding combination of 30% milk protein + 23.4% soybean protein concentrate + 23.3% rice protein isolate + 23.3% peanut protein concentrate was more in line with the nutritional needs of calves. For the same 30% milk protein retention, Liu et al. [73] found that the combined milk replacer with 40% soybean protein isolate, 10% wheat hydrolyzed protein and 20% rice protein isolate could give calves better growth performance. Raeth et al. [74] found that when 50% of milk-derived protein was replaced by soy protein isolate and wheat hydrolyzed protein in the same proportion, it would cause a decrease in daily weight gain and feed efficiency of calves. Studies had shown that high proportions of gliadin and glutelin in calf milk replacer can lead to reduced growth performance. However, as long as no less than 40% of retained colostrum was added or no less than 60% of whey protein was added, the normal growth and development of calves could be ensured.

Sucking calves are in the stage of rapid growth and development, and the level of energy intake directly affects the growth rate and nutrient metabolism of the body. It is very important to ensure an appropriate energy supply for sucking calves. Different protein sources of milk replacer have different energy utilization rates in animals. Compared with milk-derived protein, plant protein can reduce the metabolic rate of energy, nitrogen, calcium and phosphorus in calves. However, the effect of plant protein on the metabolic rate of energy, nitrogen, calcium and phosphorus in calves has a downward trend with the increase in age, and the adaptability of calves to plant protein is also continuously improved [72,75]. This is because plant protein contains a certain amount of fiber and rich nutrition, which has a significant promoting effect on the development of digestive organs such as the rumen and intestinal tract of the calf, and it lays a good foundation for the high production performance in the later period [76].

There were also differences in the effects of protein sources in milk replacers on the immune function of calves. Huang et al. [72] found that from the serum IgG, IgA, IgM and 1L-2 levels of calves, the stress caused by milk-derived protein, soybean protein and rice protein to calves was significantly lower than that of wheat protein and peanut protein. At the same time, compound plant protein could also increase the body's deposition of nitrogen by increasing the levels of GH and IGF-1 in serum and improve the ability of tissue growth and development [73].

With the in-depth research on the development of plant protein, the development of milk replacer with plant protein as the protein source has a very good prospect. However, research on double-protein milk replacer is not sufficient, and other nutrients other than protein and amino acids have not been systematically studied. There is also a lack of relevant reports on the specific effects of feeding double-protein milk replacer on the microecology of the calf's digestive tract and digestion and absorption function, as well as on the subsequent production performance. Therefore, more research is needed to reveal the nutritional potential of double-protein milk replacers, so as to provide more of a theoretical basis for precision feeding in the breeding stage of calves.

#### *2.5. Other Double Protein Dairy Products*

Cereal milk powder is more nutritious and functional than milk powder, and supplements the dietary fiber needed by the human body without changing the taste of milk powder. Cereal milk powder can be consumed in breakfast or other meals as a staple functional beverage. When drinking milk on an empty stomach for breakfast, the protein in

the milk will be converted into sugar to release energy, resulting in a waste of protein. The grain-added milk can be eaten directly as a staple food, so that the protein can be absorbed well, and the convenience and staple food of dairy products can be realized. Wang [77] invented a method for preparing oat milk powder. After the oat flakes were extruded by a twin-screw extruder, milk powder, xylitol and fructo oligosaccharide were added in a certain proportion to make oat milk powder. Zhang [78] invented a preparation method of corn milk powder. The corn flour was puffed and then pulverized, and 30% of the puffed corn flour, 68% of the milk powder, 1.8% of the sugar and 0.2% of the edible essence were mixed in a mixer to make the corn milk powder.

According to the growth and development characteristics of infants and young children, timely and reasonable addition of complementary foods plays an important role in promoting the healthy development of infants and young children. Rice protein is a recognized hypoallergenic protein, and various clinical studies have also shown that rice protein can be used as a hypoallergenic protein resource, especially suitable for infant food ingredients. Liu et al. [79] obtained a series of formulas through orthogonal experiments based on the nutritional characteristics of infants and young children in different periods. Taking 100 g as the standard, the nutritional rice flour for infants and young children in 0–6 months contained 46 g of rice flour, 41 g of first-stage milk powder, 7.2 g of vegetable and fruit powder, 3.6 g of FOS, and 2 g of various trace elements. Nutritional rice flour for infants aged 6–12 months contained 46 g of rice flour, 32 g of second-stage milk powder, 12.6 g of multigrain flour, 6.3 g of FOS, and 3 g of various trace elements.

Ice cream is a popular dairy product for summer, and the production of new, safe and healthy ice cream has become an industry trend. Wu et al. [80] used flaxseed meal as raw material, extracted flax protein with enzyme preparation, added flax protein content of 3%, skim milk powder content of 13%, cream content of 15% and sucrose content of 16%. The finished product was light brown, with pure fragrance and fine texture. It could be seen that adding flax protein can improve the quality of ice cream. Zheng et al. [81] used USPI-PLW to replace part of the milk powder. Compared with ordinary low-fat ice cream, the expansion rate of ice cream was increased by 94.84%, the melting rate of ice cream was reduced by 26.86%. The product was in a stable condition, with a good appearance and smooth taste, which made its flavor more popular.

#### **3. Perspectives for the Future**

Based on the current research and development status of double-protein dairy products, several suggestions are put forward on the research and development ideas of this category of products in China in the future, which should be followed as below:


can be purposefully developed for different consumer groups. For infants and young children, fruit and vegetable raw materials rich in vitamins and minerals can be developed and strengthened; for middle-aged and elderly people, some medicinal and food homologous ingredients rich in antioxidants and anti-aging substances can be added.

#### **4. Conclusions**

Concerns about environmental impact and sustainability, animal welfare and personal health issues have fueled consumer demand for plant protein. However, the transition towards greener diets is being hampered by the poor acceptance of vegan foodstuffs among consumers. Mixed animal/plant products to familiar dairy products offer a new field of innovation. Therefore, plant-based proteins were used in a variety of dairy products. This comprehensive review presents the research and application of plant protein in the dairy industry, a distinctive and interesting topic for researchers in food technology, nutrition and dietetics. The continuous development of new blended products and the expansion of the application of plant protein in dairy products can promote a greater role of plant protein in human society. The current focus is on possible ways to improve nutritional properties through processing, such as the use of enzymes, the selection of raw materials based on their protein quality, advanced processing and technological interventions. There is also a need to ensure the palatability and acceptability of double-protein dairy for the population.

**Author Contributions:** G.-G.H. contributed to the study design and drafted the article. J.L. collected information. Y.-H.W. contributed to the final version edited. H.-B.S. contributed to the conception of the article design. Z.-N.Y. revised the article. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by National Key Research and Development Program (No. 2017YFE0131800): National Natural Science Foundation of China (Project No. 31871823).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

