Effects of Peanut Meal Enzymatic Optimization Process and its Products Replacing some Nitrogen Fertilizers on Potato Growth and Soil Environment

doi:10.21203/rs.3.rs-4827929/v1

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Research Article

Effects of Peanut Meal Enzymatic Optimization Process and its Products Replacing some Nitrogen Fertilizers on Potato Growth and Soil Environment

https://doi.org/10.21203/rs.3.rs-4827929/v1

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Background:

*Peanut meal is rich in proteins and minerals, and is commonly used in traditional agriculture for direct return to the field or compost fermentation and then applied. However, compost fermentation not only takes a long time and the products are unstable, but also tends to attract pathogens. Soil application of organic fertilizer is an effective method to maintain high crop yield and sustainable soil fertility. In this study, a 4-factor, 3-level Box-Behnken Design test was designed to optimize the process of alkaline protease hydrolysis of peanut meal, and used to prepare the enzymatic solution, and compared with the fermentation solution fermented by the traditional process, to evaluate the effects of both on potato growth and soil environment by replacing 5%, 10%, 15%, and 20% nitrogenous fertilizer under field conditions.

Results:

The Box-Behnken Design test showed that the optimum reaction conditions for alkaline protease hydrolysis of peanut meal were: reaction time of 4 h, initial reaction pH 9.0, reaction temperature of 55 ℃, and enzyme addition of 800 U·kg^− 1. The total nitrogen content of peanut meal after enzymatic hydrolysis was 5.10 g·kg^− 1, which was much higher than that of the peanut meal fermentation broth obtained from traditional fermentation (N 1.80 g·kg^− 1). The field experiment showed that the enzyme solution replacing 10% nitrogen fertilizer had the greatest increase in potato yield, which was 26.63% and 12.22% higher than that of the control group and the fermentation solution treatment group, respectively; in addition, the enzyme solution replacing nitrogen fertilizer could increase the nitrogen, phosphorus, potassium and dry matter content of potato tuber, and at the same time, it could effectively improve the physical and chemical properties of the soil, increase the diversity of the soil bacterial species in the rhizosphere, and change the structure of the bacterial community.

Conclusions:

The above results show that alkaline protease hydrolysis of peanut meal can greatly shorten the hydrolysis time of peanut meal and increase the nutrient content of the hydrolysate, which promotes the efficient use of peanut meal resources. The use of enzymatic hydrolysis solution to replace part of nitrogen fertilizer can not only reduce the use of chemical fertilizer, but also improve potato yield and quality, and improve the soil environment.

peanut meal

enzymatic digestion

nitrogen fertilizer replacement

potato

soil environment

Potato (Solanum tuberosum L.) is the fourth largest food crop in the world, and its tubers are rich in starch and are an important source of dietary fiber, vitamins, and minerals[1]. Numerous studies have shown that potato has the effect of lowering blood glucose and improving microflora in the intestinal tract[2]. The protease inhibitors extracted from potato can be used for the prevention and treatment of protease-induced perianal inflammation[3]. And the resistant starch contained in potato also has the effect of inhibiting the growth of certain tumor cells[4]. In addition, the phenolics substances in potato skin have inhibitory effects on Escherichia coli, Salmonella enteritidis and Staphylococcus aureus[5].

However, the utilization rate of chemical fertilizer in China is low, and the amount of fertilizer applied far exceeds the optimal application in the economic sense, which needs to suppress the amount of chemical fertilizer inputs[6]. Over-reliance on chemical fertilizers and unscientific application of fertilizers are very common in Chinese potato cultivation, resulting in serious nutrient loss, declining fertility of arable land and soil ecological degradation frequently. Organic fertilizer can provide a variety of nutrients needed for plant growth, improve crop yield and quality[7], and have a positive effect on regulating soil pH[8], enhancing soil biological activity[9, 10], and improving soil fertility [11, 12]. It was found that straw return to the field and application of organic fertilizers could significantly increase the activities of urease, acid phosphatase and nitrate reductase[13] and microbial biomass[14] in the soil, especially the greatest effect on the diversity of community Alpha[15]. However, in fact, Chinese farmers generally believe that organic fertilizers are low in nutrient content, large in input, slow in effect, and the application of solid organic fertilizers is cumbersome and labor cost is high, so farmers often ignore the application of organic fertilizers in the fertilization process.

China is a large producer of peanuts, with a steady production ranking first in the world[16], and a large volume of peanut meal, a byproduct of fat extraction from peanut kernels, amounting to 1.0 × 107 t per year[17]. Peanut meal is characterized by high protein content, complete range of minerals, low crude fiber and crude fat content [16]. The degradation methods of peanut meal can be categorized into four types: physical degradation, chemical degradation, biofermentation degradation[18] and bioenzymatic degradation[19]. Among them, the bioenzymatic degradation method has the advantages of high catalytic efficiency, strong specificity, mild reaction, safety and non-toxicity. At present, in the process of preparing organic liquid fertilizer by enzymatic degradation method, cellulase is usually used to hydrolyze polysaccharide macromolecules such as crude fiber in the material[20], which has a positive effect on the release of peanut meal protein. Researchers have found through soybean meal hydrolysis test that the addition of cellulase can significantly increase the yield of monosaccharides after pretreatment with citric acid (10 g·L^− 1) and heating at 90°C for 2 h[21], Peanut protein is rich in amino acids, vitamins, trace elements and minerals[22]. In order to release these effective substances from peanut protein, proteases are now commonly used for hydrolysis. Proteases can be categorized into alkaline proteases, neutral proteases and acidic proteases[23, 24]. Among them, alkaline protease is a class of enzymes with higher vitality and better stability in alkaline environment, which has the advantages of strong hydrolysis capacity and wide range of action compared with acidic and neutral proteases[25]. A test comparing the hydrolysis efficiency of defatted peanut meal and purified peanut proteins by different proteases found that alkaline proteases and flavor proteases were the most effective in hydrolyzing peanut meal and purified peanut proteins[26]。

At present, peanut meal in agricultural production is mainly direct return to the field, compost decomposition or liquid fermentation with microbial agents. However, the crude fiber in peanut meal has high toughness and poor solubility[27], which is difficult to be degraded in the preparation of organic liquid fertilizer using the fermentation process. Meanwhile, during the fermentation process, the fermentation liquid exists for a long time, with a high content of pathogenic bacteria, and a low utilization rate of peanut meal proteins. But the use of bio-enzymes for hydrolysis of peanut meal to prepare organic liquid fertilizer has rarely been reported. The objectives of this study are as follows: (1) to optimize the enzymatic hydrolysis process of peanut protein through Response Surface Experiment, and prepare peanut meal enzymatic hydrolysis solution with the best reaction conditions, (2) to use the enzymatic hydrolysis solution to replace part of the chemical fertilizer, compared with the fermentation solution prepared by the traditional process to replace part of the chemical fertilizer in order to study the impact of both on potato growth and soil environment. Ultimately to provide a basis for the sustainable development of the actual production of potato planting and the efficient resource utilization of peanut meal.

Experiment 1: Process optimization for the preparation of peanut meal enzyme digest by enzymatic digestion

Test sites and materials

The experiment was conducted from March 2022 to October 2022 in the Crop Nutrition and Fertilization Research Laboratory, College of Resources and Environment, South China Agricultural University (113°21'33.12''E, 23°9'42.21 '').

Peanut meal was purchased from farmers' markets and the main substance contents are shown in Table 1; Bio-fermenter is Yuanqi No. 1 Fermenter (purchased from Guangzhou Yuanqi Farmland Biotechnology Co., Ltd.); Cellulase and complex alkaline protease were purchased from Dongheng Huadao Biotechnology Co.

Table 1 Main components of peanut meal (g·kg^-1)

Nitrogen

(chemistry)

Phosphorus

(chemistry)

Potassium

(chemistry)

Calcium

(chemistry)

Magnesium

(chemistry)

Crude Protein

Crude Fiber

Crude Fat

Crude Ash

67.18

10.19

14.39

1.50

1.90

482.20

45.22

11.30

57.12

Experimental design and implementation

Single-factor experimental design: Based on the sample solution obtained from the alkaline pretreatment and cellulase treatment of peanut meal, a single influence factor change was designed to test the effect of the composite alkaline protease on the hydrolysis of the protein contained in the peanut meal, and the influencing factors included: different reaction times (1 h, 2 h, 3 h, 4 h, 5 h, 6 h), different reaction pH (6, 7, 8, 9, 10, 11), different reaction temperatures (40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C), and different enzyme dosages (200, 400, 600, 800, 1000, 1200 U·g^-1). The degree of hydrolysis of peanut protein was calculated by determining the amino acid nitrogen content of the enzymatic products.

Box-Behnken Design (BBD) Experimental design: Based on the results of the single-factor test, a 4 factors and 3 levels BBD test was designed using Design Expert 2013 software (as shown in Table 2), with a total of 29 trials (as shown in Table 6, in which A, B, C and D represent the reaction time, the initial reaction pH, the reaction temperature, and the amount of enzyme added, respectively). The regression model was constructed by combining the experimental results, and the data were analyzed by ANOVA and Wangda analysis to screen out the optimal reaction conditions to prepare the peanut meal enzymatic solution and determine its nutrient content.

Table 2 Different factorial and horizontal designs in BBD trials

(math) factor		level (of achievement etc.)
(math) factor	-1	0	1
Reaction time	3 h	4 h	5 h
Initial reaction pH	8	9	10
Reaction temperature	50℃	55℃	60℃
Amount of enzyme added	600 U·g^-1	800 U·g^-1	1000 U·g^-1

Measurement items and methods

(1) Determination of peanut meal nutrients and nutrient content of enzyme solution: Kjeldahl method determination of total nitrogen content, molybdenum-antimony antioxidant colorimetric method for the determination of total phosphorus content, atomic absorption spectrophotometry for the determination of total potassium content, dry ashing-atomic absorption spectrophotometry for the determination of calcium and magnesium content, crude protein content determined with reference to GB/T5009.2003, crude fiber content determined with reference to GB/T5009.10-2003, crude fat content determined with reference to GB/T5009.4-2016, and crude ash content determined with reference to GB/T5009.4-2016.

(2) Degree of protein hydrolysis of peanut meal: Determination of amino acid nitrogen content by formaldehyde titration, calculated by the formula. Degree of protein hydrolysis (%) = (amino acid nitrogen of sample liquid after reaction - amino acid nitrogen before reaction) / total nitrogen content × 100%.

Experiment 2: Effects of replacing part of nitrogen fertilizer with peanut meal enzymatic solution and fermentation solution on potato growth and soil environment

Test site and materials

The experiment was conducted from December 2022 to March 2023 at En Town, Jiangmen City, Guangdong Province, China (112°21'4.22''E, 22°12'56.73' 'W) was conducted. The area has a subtropical oceanic climate, with an average annual rainfall of 1900 ~ 2000 mm. the experimental field has been under potato and rice crop rotation for a long time, and the basic physicochemical properties of the soil are shown in Table 3.

Table 3 Basic physical and chemical properties of soil

soil texture

value

conductivity (uS·cm^-1)

organic matter

(g·kg^-1)

alkaline nitrogen decomposition

(mg·kg^-1)

effective phosphorus (mg·kg^-1)

quick-acting potassium (mg·kg^-1)

loam soil

4.68

183.00

24.00

100.55

54.54

112.14

Test crop: Potato Joshiba No.1.

Peanut meal fermentation liquid: according to the traditional process in the fermentation tank, add brown sugar powder, peanut meal, Yuanqi one fermenter and water, mix well and then aerobically fermented for 40 d, filtered liquid part, and then take the supernatant as the peanut meal fermentation liquid after overnight resting. The basic physical and chemical properties of the fermentation broth are shown in Table 4.

Table 4 Nutrient profile of peanut meal fermentation broth (g·kg^-1)

N	P₂O₅	K₂O	Ca	Mg	Organic matter
1.80	0.40	1.40	0.88	0.55	23.20

Peanut meal enzyme solution: prepared by 2.1.3 enzyme solution process.

Inorganic fertilizer: Auter compound fertilizer (16-6-24, produced in Hubei Auter Chemical Co., Ltd.), Jia Shang urea-formaldehyde slow-release compound fertilizer (16-8-22, produced in Foshan City Jia Shang Fertilizer Co., Ltd.), urea, ammonium dihydrogen phosphate, potassium sulphate.

Experimental design and implementation

The experiment was set up with control MCK treatment (pure application of chemical fertilizer), group M treatments including four treatments of M5, M10, M15 and M20 (respectively, peanut meal enzyme solution replacing 5%, 10%, 15% and 20% inorganic nitrogen) and group F treatments including four treatments of F5, F10, F15 and F20 (respectively, fermentation solution replacing 5%, 10%, 15% and 20% inorganic nitrogen), with nine treatments in total, each with three replications, randomly distributed in 27 blocks, planted in monoculture with two rows, theoretical planting density of 46,500 plants·hm^-2, and irrigated with shallow buried drip irrigation on the monoculture. All treatments were applied with equal amount of bottom fertilizer before sowing, and the follow-up fertilizer was as shown in Table 5, which was carried out in three times, starting from the expansion period of potato stolons. Other agronomic measures were implemented and managed uniformly according to local farmers' habits. Samples were collected on March 31st.

Table 5 Fertilizer regimen of different treatments (kg·hm^-2)

treatment	base Fertilizer		additional fertilizer
treatment	compound fertilizer (16-6-24)	compound fertilizer (16-8-22)	digestate	fermentation broth	urea	potassium dihydrogen phosphate	potassium sulfate
CK	262.5	525.0	—	—	135.0	71.6	134.8
M5	262.5	525.0	1764.7	—	112.5	53.7	108.1
M10	262.5	525.0	3529.4	—	90.0	35.8	81.4
M15	262.5	525.0	5294.1	—	67.5	17.9	54.7
M20	262.5	525.0	7058.8	—	45.0	—	28.0
F5	262.5	525.0	—	5000.0	112.5	67.8	93.6
F10	262.5	525.0	—	10000.0	90.0	64.0	83.1
F15	262.5	525.0	—	15000.0	67.5	60.2	72.6
F20	262.5	525.0	—	20000.0	45.0	56.3	62.1

Note: digestate nutrients are (0.51-0.53-0.97), fermentation broth nutrients are (0.18-0.04-0.14).

Measurement items and methods

(1) The fresh weight of each graded potato tuber, the total output and the dry weight of representative tubers were calculated by the formula: fresh weight of each graded tuber/total output×100%, and the percentage of different graded tubers was calculated; the nutrient contents of the tubers such as total nitrogen, total phosphorus and total potassium were determined by Kjeldahl method, molybdenum antimony antimonial colorimetry and atomic absorption spectrophotometry respectively; the protein content, soluble sugar content and vitamin C content of the tubers were determined by bis-acetonitrile method, anthracene-ketone colorimetry and solid-blue-salt colorimetry respectively.

(2) Soil physicochemical properties: pH (electrode potential method), conductivity, organic matter (potassium dichromate volumetric method-external heating method), alkaline dissolved nitrogen (alkaline dissolved diffusion method), effective phosphorus (molybdenum-antimony antimony colorimetric method), and fast-acting potassium content (ammonium acetate leaching-atomic absorption spectrophotometry).

(3) Soil microorganisms were sent for 16SrRNA amplification and sequencing by Shanghai Meiji Biomedical Technology Co.

Statistics and analysis of data

The BBD experiment was designed with Design Expert 2013 software, and data analysis and graphing were performed; WPS Office 2019 software was used to organize and calculate the data; then SPSS 25.0 software was used to analyze the data for differences, and Origin 2019 software was used to make graphs. Data organization and graphing of soil bacterial indicators were completed in Meiji Science and Service Network (provided by Shanghai Meiji Biomedical Technology Co., Ltd.).

Optimization of the process for the preparation of peanut meal enzyme digest by enzymatic hydrolysis method

One-way test of hydrolysis conditions by composite alkaline protease

As shown in Figure 1, the most suitable one-factor reaction conditions of peanut protein hydrolysis in the one-factor test were screened to be: reaction time 4 h, initial reaction pH 9.0, reaction temperature 55 ℃ and enzyme addition of 800 U·g^-1, considering the time cost and economic cost, respectively.

Response surface results and analysis of variance (ANOVA)

The corresponding test results under different conditions are shown in Table 6.

Table 6 Analysis of Box-Behnken response surface test results

serial number	A	B	C	D	DH (%)
1	-1	-1	0	0	18.17
2	1	-1	0	0	23.25
3	-1	1	0	0	13.34
4	1	1	0	0	14.86
5	0	0	-1	-1	17.83
6	0	0	1	-1	17.22
7	0	0	-1	1	22.41
8	0	0	1	1	22.31

continuation sheet 6

serial number		A	B	C	D	DH (%)
	9	-1	0	0	-1	14.73
	10	1	0	0	-1	18.24
	11	-1	0	0	1	22.90
	14	0	1	-1	0	13.41
	15	0	-1	1	0	20.62
	16	0	1	1	0	12.88
	17	-1	0	-1	0	18.46
	18	1	0	-1	0	22.68
	19	-1	0	1	0	17.95
	20	-1	0	1	0	22.58
	21	0	-1	0	-1	16.95
	22	0	1	0	-1	11.46
	23	0	-1	0	1	21.20
	24	0	1	0	1	16.28
	25	0	0	0	0	23.12
	26	0	0	0	0	24.95
	27	0	0	0	0	24.01
	28	0	0	0	0	23.54
	29	0	0	0	0	23.80

A response regression model was established based on the experimental results to obtain the quadratic multiple regression model equation for the hydrolysis degree of peanut protein:

DH = - 669.0911 + 23.9143A + 89.6385B + 7.5216C + 0.1051D - 0.8900AB + 0.0205AC - 3.2000AD + 6.5000BC + 7.1250BD + 1.2750CD - 1.6016A² - 5.0153B² - 0.0710C² - 5.7633D²

The analysis of variance (ANOVA) was performed on the quadratic multiple regression equation and the results are shown in Table 7, the P-value of the total model < 0.0001 indicates that the quadratic multiple regression model reached significance and the P-value of the dislocation term = 0.2611 > 0.05 indicates that at the level of dislocation term α = 0.05 the effect of non-testing factors on the results of the test is not significant. The coefficient of determination of the model, R² = 0.9748, and the modification coefficient, R_ajd² = 0.9496, indicate that the variations in the experimental data can be explained by the quadratic multiple regression model and that the significance of the model is good. The above results indicate that the regression model established in this experiment fits well, the regression is significant and reliable, the model is valid, and the model can be used to predict the experimental results of the peanut protein hydrolysis process by alkaline protease.

The F-value in the table is proportional to the effect of response value, the larger the F-value, the greater the effect on the response value, the F-value of A is 41.19, the F-value of B is 159.99, the F-value of C is 0.65, the F-value of D is 109.18, i.e., B > D > A > C, which indicates that the strength of the effect of each factor on the degree of hydrolysis of peanut proteins is as follow: the initial reaction pH > amount of enzyme addition > reaction time > reaction temperature.

Table 7 Box-Behnken test regression model ANOVA

source of variance	squares sum	degrees of freedom (physics)	mean square	F-value	P-value	significance
general model	433.98	14	31	38.65	< 0.0001	**
A-reaction time	33.03	1	33.03	41.19	< 0.0001	**

continuation sheet 7

source of variance		squares sum	degrees of freedom (physics)	mean square	F-value	P-value	significance
	B-initial pH	128.31	1	128.31	159.99	< 0.0001	**
	C-reaction temperature	0.53	1	0.53	0.65	0.432	—
	D-enzyme addition	88.13	1	88.13	109.88	< 0.0001	**
	residual	11.23	14	0.8
	lost proposal	9.37	10	0.94	2.01	0.2611	—
	pure error	1.86	4	0.47
	aggregate	445.21	28

Note：* denotes significant difference（P < 0.05）; ** denotes highly significant difference（P < 0.01）; —denotes insignificant difference（P > 0.05）.

Response surface factor interactions

Response surface Design Expert 2013 software was used to fit the results of 29 groups of experiments to obtain the response surface three-dimensional plot of the two factors affecting the degree of hydrolysis of peanut protein, in order to explore the effect of the interaction between the reaction time, the initial reaction pH, the reaction temperature and the enzyme addition amount, the results are shown in Figure 2. Combined with the comparison of the significance analysis in Table 7, it shows that the initial reaction pH has the greatest effect on the degree of hydrolysis of peanut protein, while the reaction temperature has the least effect.

Validation of the regression model

The resulting regression equation model was subjected to DH Wangda analysis, and the enzymatic hydrolysis process was optimized by response surface analysis, which showed that the theoretical hydrolysis degree of peanut protein was 25.596% under the conditions of reaction time of 4.519 h, initial reaction pH of 8.635, reaction temperature of 54.855 °C, and enzyme addition of 900.773 U·g^-1. The reaction conditions were adjusted according to the actual situation, and the modified optimal enzymatic hydrolysis process conditions were as follows: reaction time of 4.5 h, initial reaction pH of 8.6, reaction temperature of 55.0°C, and enzyme addition of 900.0 U·g^-1. Three replicated validation tests were carried out with the optimized process, and the hydrolysis degrees were obtained as 24.96%, 25.13%, and 24.97%, respectively. The average hydrolysis degree was 25.02%, and the standard deviation (SD) was calculated to be 0.00095, indicating that the regression model can better predict the actual hydrolysis situation and has better practical value.

Determination of Enzymatic Solution Product Composition

The peanut meal enzyme digest was prepared using a complex alkaline protease under the optimal reaction conditions obtained by optimizing the hydrolysis process in a regression model, and the resulting product was subjected to compositional determination, which was converted to the results shown in Table 8.

Table 8 Composition of Enzymatic Solution (g·kg^-1)

N	P₂O₅	K₂O	Ca	Mg	Organic matter
5.10	5.30	9.70	0.92	0.67	50.40

Effects of peanut meal enzymatic solution and fermentation solution replacing part of nitrogen fertilizer with on potato growth and soil environment

Potato yield and yield components

As shown in Figure 3, compared with MCK treatment, the total potato yield of group M treatments were significantly increased, of which M10 treatment had the best yield increase effect, which was significantly increased by 26.63% compared with MCK treatment; the treatments that played a role in yield increase effect in group F treatments were F5 and F10 treatments, which had a significant increase in yield by 11.38% and 4.58%, respectively. However, the total potato yield of M10 treatment was significantly higher than that of F5 treatment.

As shown in Figure 4 (a) and (b), under the test conditions, with the increase of the proportion of peanut meal enzyme solution replacing inorganic nitrogen fertilizer, the effect on the increase of big potato yield showed a trend of increasing and then decreasing, of which the M10 treatment had the best effect on the increase of big potato yield, which was significantly increased by 30.90% compared with the MCK treatment; the M20 treatment had the highest rate of big potato, which was significantly increased by 5.97% compared with the control.

The accumulation of macro-elements and dry matter in potato tubers

The results are shown in Figure 5 (a), (b), (c) and (d). The effects of the four treatments in group M on the accumulation of nitrogen, phosphorus, potassium, and dry matter in potato tubers showed a trend of increasing and then decreasing, which were better than the MCK treatments as a whole, with the best effect of the M10 treatment, which was significantly more than the MCK treatments by 19.04%, 22.47%, 29.32%, and 31.86%, respectively. Compared with MCK treatment, F10 treatment had the best effect on nitrogen and phosphorus, which increased by 9.8% and 17.51%, respectively, and F5 treatment had the best effect on dry matter accumulation, which effectively increased by 15.62%. Both of them significantly increased the accumulation of potassium with small differences.

Potato quality

As shown in Figure 6, compared with MCK treatment, the protein and vitamin C contents of potato tubers from M10, M15 and M20 treatments in group M treatments were significantly increased, and the vitamin C content of M20 treatment was 253.95 mg·kg^-1, which was significantly increased by 20.54%; the soluble sugar content of tubers was higher than that of MCK treatment, and reached the highest in M15 treatment, which was significantly higher than MCK treatment by 24.10%. Among the treatments in group F, the protein content of F15 treatment was significantly increased by 26.40% compared with that of MCK treatment, while in terms of vitamin C content of potato, only F15 treatment was significantly higher than that of MCK treatment, and there was no significant difference among the other treatments.

Soil physical and chemical properties

As shown in Figure 7(a), (b), (c), (d) and (e), soil pH, EC, effective phosphorus, quick-acting potassium and organic matter content reached their highest values at M20 and F20 treatments, which were significantly higher than MCK treatments by 5.70% and 4.43%, 40.06% and 23.22%, 31.65% and 22.70%, 14.28% and 10.53%, 13.48% and 17.83%, respectively; as shown in Figure 7(f), soil alkaline dissolved nitrogen content reached its highest value at M15 and F20 treatments, which were 106.26 mg·kg^-1 and 97.23 mg·kg^-1, respectively.

Sequencing information of soil bacteria from different treatments

The microbial sequencing information of soil samples under different treatments is shown in Table 9, the sequence number (Seq_num) of each treatment was in the range of 62601 ~ 84437, the number of sequence bases (Base_num) was in the range of 25823612 ~ 34861824, and the average optimized sequence length (Mean_length) was in the range of 412.113672 bp ~ 413.942411 bp, the shortest sequence length (Min_length) is in the range of 200 bp ~ 201 bp, and the longest average sequence (Max_length) is in the range of 474 bp ~ 515 bp.

Table 9 Sequencing information of soil microorganisms in different treatments

treatment	Seq_ num		Base_ num	Mean_ length	Min_ length	Max_ length
MCK_1	62601	25823612		412.511174	200	475

continuation sheet 9

treatment		Seq_ num		Base_ num	Mean_ length	Min_ length	Max_ length
	MCK_2	66732	27533267		412.594662	201	475
	MCK_3	73979	30513706		412.464429	201	486
	M5_1	68257	28166848		412.658746	201	486
	M5_2	63703	26319866		413.165251	201	475
	M5_3	66614	27492707		412.716651	201	475
	M10_1	68667	28397532		413.554284	201	475
	M10_2	68520	28302419		413.053400	201	474
	M10_3	73183	30222052		412.965470	201	475
	M15_1	63710	26292872		412.696154	201	475
	M15_2	64670	26693092		412.758497	201	487
	M15_3	67496	27939457		413.942411	201	481
	M20_1	67487	27906646		413.511432	201	486
	M20_2	71325	29489591		413.453782	201	475
	M20_3	68223	28125699		412.261246	201	491
	F5_1	77530	31951173		412.113672	200	475
	F5_2	78863	32560251		412.871068	201	475
	F5_3	71458	29480399		412.555613	201	482
	F10_1	82144	33938081		413.153499	201	475
	F10_2	81387	33562282		412.378906	200	502
	F10_3	83191	34286274		412.139222	201	477
	F15_1	79725	32927029		413.007576	201	515
	F15_2	78632	32418199		412.277432	201	492
	F15_3	73793	30487328		413.146613	201	475
	F20_1	78788	32517041		412.715655	201	475
	F20_2	81659	33682505		412.477559	201	475
	F20_3	84437	34861824		412.873788	201	475

Venn diagrams of soil bacterial communities at the OTU level for different treatments

As shown in Figure 8, the nine treatments had a total of 2,272 bacterial OTUs, or 15.32% of the total, with the F10 treatment having the largest number of unique bacterial OTUs at 910, or 6.13% of the total, and the M10 treatment having 855 unique bacterial OTUs, or 5.76% of the total.

Alpha diversity index of soil bacterial communities in different treatments

As shown in Table 10, the Coverage index of all treatments exceeded 96%, indicating that the sequencing results of soil samples from all treatments were reliable and could truly reflect the characteristics of soil bacterial communities. In group M and F treatments, the Ace index, Chao index and Sobs index of soil bacterial community of M10 and F10 treatments were significantly higher than the remaining three treatments in the group, indicating that the species of soil bacteria were more abundant and the status of dominant species was more prominent under M10 and F10 treatments, which were not significantly different.

Table 10 Alpha diversity indices of soil bacterial communities

treatment	Coverage Index	Ace Index	Chao Index	Sobs Index
MCK	0.969±0.0 abc	4709.5±78.5 cd	4529.9±47.5 cde	3562.3±71.0 bc
M5	0.970±0.0 ab	4651.7±88.0 d	4491.7±71.0 de	3527.3±68.8 bc
M10	0.967±0.0 bcd	5083.9±120.8 ab	4872.4±126.1 ab	3898.0±56.6 a
M15	0.969± 0.0 abc	4737.7±126.9 cd	4578.1±144.2 bcde	3540.3±76.1bc
M20	0.971±0.0 a	4442.3±10.4 d	4290.5±30.3 e	3370.0±16.1 c
F5	0.969±0.0 abc	4770.1±92.3 bcd	4614.2±76.1 bcd	3570.0±83.2bc
F10	0.966±0.0 d	5175.1±83.2 a	4962.3±63.5 a	3813.0±138.6 ab
F15	0.967±0.0 cd	5002.0±160.6 abc	4833.9±154.2 abc	3708.7±125.7 ab
F20	0.969±0.0abc	4751.2±98.6 cd	4554.6±62.5 cde	3545.3±72.2 bc

Note: The data in the table are the mean ± standard error of 3 repetitions, and those with the same letter at the end of the data in the same column indicate that the difference is not significant (P > 0.05).

Structural composition of soil bacterial communities at different treatment genus levels and analysis of significance tests for differences between groups

As shown in Figure 9, there were 10 genera that ranked in the top 20 in terms of abundance and could be accurately categorized, and the abundance of these 10 genera varied to different degrees under the treatments of Groups M and F. Among them, four genera with abundance greater than 1% and could be accurately categorized with highly significant or significant differences among the treatments of MCK, Groups M and F were Sinomonas (P ≤ 0.01), Terrabacter (P ≤ 0.05), Sphingomonas (P ≤ 0.05) and Arthrobacter (P ≤ 0.05), respectively, Terrabacter (P ≤ 0.05), Sphingomonas (P ≤ 0.05), and Arthrobacter (P ≤ 0.05). As shown in Figure 10, the abundance of Sinomonas increased by 149.8% in the F20 treatment compared to the M20 treatment, and the abundance of Terrabacter, Sphingomonas, and Arthrobacter increased by 36.1%, 91.8%, and 173.9%, respectively, in the M5 treatment compared to that at F5.

As shown in Figure 11, based on the results of the sample clustering tree analysis, the MCK treatment was more similar to the M10 treatment, the M20 treatment to the M15 treatment, and the M20 treatment to the F20 treatment in terms of soil dominant genus composition, respectively.

Heat map analysis of correlation between soil bacterial communities and environmental factors at different treatment genus levels

Five genera could be accurately categorized in the figure as shown in Figure 12. Arthrobacter and Terrabacter showed significant or highly significant negative correlation (P<0.05 and P<0.01) with soil effective phosphorus; fast potassium was significantly negatively correlated with Arthrobacter (P<0.05) and significantly positively correlated with Bacillus; Arthrobacter, Terrabacter and Sphingomonas were significantly negatively correlated with organic matter, pH and EC, while organic matter was significantly positively correlated with Sinomonas.

Study on the process of preparing peanut meal enzyme solution by enzymatic hydrolysis method

The protein content of peanut meal is over 45%[28] , which is the highest content of peanut meal. How to release the effective nutrients from peanut meal proteins has become the key to research. Peanut meal contains about 38% total carbohydrates, including 12.5% starch, 4.0% hemicellulose, 4.5% cellulose, and about 13.90% sucrose[29] , and the presence of cellulose increases the difficulty of hydrolysis of peanut meal protein. In this experiment, before the peanut meal protein was hydrolyzed, peanut meal was hydrolyzed with cellulase enzyme, aiming at destroying the structure of sugar macromolecules, such as crude fiber, so that the peanut meal protein was better exposed, which was conducive to the next step of hydrolysis by alkaline protease. Alkaline protease is derived from microbial extracts, which can hydrolyze the peptide chains of protein molecules to produce peptides or amino acids[30]. Alkaline protease is the most effective protease in the hydrolysis of peanut meal proteins compared to acidic and neutral proteases[31], as it is more active and stable under relatively harsh conditions and can be used for specific applications depending on its properties[32] . Researchers have found that Alcalase protease showed higher activity in alkaline environments and produced shorter peptides with higher yields compared to peptides released by trypsin[33]. The solubility of peanut meal proteins was significantly increased by 273% at pH 8.0, hydrolysis temperature of 40°C, protease to peanut meal ratio of 3.5% (v/w), and hydrolysis time of 1 h[34]. Shu et al. in exploring the effect of flavored enzyme and alkaline protease treatments of peanut proteins in mice, found that under specific conditions, compared to single enzyme treatments, the complex protease treatment ( 2% flavored enzyme + 5% alkaline protease) significantly reduced the sensitization of peanut proteins as well as increased the hydrolysis degree of peanut proteins, which was attributed to the fact that the composite protease possessed both endonuclease activity and exonuclease activity[35]. In this experiment, the hydrolysis process of peanut meal protein was optimized by Response Surface optimization method, and it was found that the hydrolysis degree of peanut meal protein was the highest under the conditions of enzyme addition of 900.0 U·g^-1, reaction time of 4.5 h, reaction pH of 8.6, reaction temperature of 55.0°C, and solid-liquid ratio of 1 : 12, and the average degree of hydrolysis was 25.02, which was lower than that of the previous study[36], and it may be One of the reasons was that the compound alkaline protease for trial contains only endonuclease without exonuclease, and the second was that peanut meal was not purified of peanut proteins, therefore the hydrolysis reaction environment had more debris, which affected the effective combination of alkaline protease and peanut protein and resulted in a low degree of hydrolysis. Nevertheless, the total nitrogen content of the peanut meal digest prepared by enzymatic hydrolysis (N 5.10 g·kg^-1) was much higher than that of the peanut meal fermented by microbial agent (N 1.80 g·kg^-1). At the same time, the fermentation broth prepared by bio-fermentation takes about 30 days under suitable conditions, while the enzymatic broth prepared by enzymatic hydrolysis in this experiment can be prepared in less than two days, which greatly improves the decomposition efficiency of peanut meal.

Effects of replacing part of nitrogen fertilizer with peanut meal enzymatic solution and fermentation solution on potato growth and soil environment

Effect of replacing part of nitrogen fertilizer by peanut meal enzymatic solution and fermentation solution on the growth of potato

Studies have shown that replacing part of inorganic nitrogen fertilizer with organic nitrogen can promote the growth of crops and have the effect of increasing yields and improving quality, and that different substitution percentages different trends in crops varies[37]. After replacing 50% of nitrogen fertilizer with organic fertilizer, wheat and rice yields increased by 23.27% and 13.20%, respectively[38]; replacing 30% of nitrogen fertilizer resulted in an increase in the yield and quality of maize, as well as an increase in maize kernels yield and the content of nitrogen[39]; when replacing 20% of chemical fertilizer, the yields, vitamin C, and soluble protein of cabbage increased by 30.11%, 5.66%, and 16.02%, respectively[40]. The present experimental study comparing the effects of peanut meal enzymatic solution and fermentation solution replacing some inorganic nitrogen fertilizers in potato production showed that compared with peanut meal fermentation solution, the two treatments of peanut meal enzymatic solution replacing 10% and 15% inorganic nitrogen fertilizers significantly increased potato yield, rate of large potatoes, nitrogen, phosphorus and potassium content of tubers, and dry matter accumulation, and at the same time, improved the quality of the potato. Surprisingly, when the percentage of peanut meal fermentation solution replacing inorganic nitrogen fertilizer reached 15% and above, potato tuber's content of large elements and dry matter accumulation were significantly lower than those of the control group. It is speculated that peanut meal may be susceptible to microbial contamination during fermentation process, which is likely to increase the number of soil pathogenic bacteria after excessive application to the soil, while incomplete fermentation may also increase the probability of aflatoxin infection in peanut meal[41] , which may indirectly or directly inhibit the activity of soil beneficial microorganisms, thus hindering the accumulation of macro-elements and dry matter in potato tubers.

Amino acids are the main form of soluble organic nitrogen that can be directly absorbed and utilized by plants[42] , and there are high affinity absorption system (HATS) and low affinity absorption system (LATS) for amino acids in plants[43]. Hu et al.[44] found that the free amino acid content of tubers was significantly increased by spraying amino acid fertilizers on the leaves of potato, and the highest total potato tuber yield, tuber number could be obtained when treated with compound amino acid fertilizers[45]. The four treatments of peanut meal enzymatic solution replacing nitrogen fertilizer in this experiment significantly improved potato yield and quality, with the M10 treatment having the best effect of increasing yield and quality among all treatments. This may be due to the fact that the peanut meal enzymatic solution is rich in amino acids and polypeptides, and the amino acid nitrogen contained therein can be directly used as a source of nitrogen for potatoes to promote the synthesis and accumulation of various organic substances in potatoes, thus improving potato yield and quality. Among the four treatments of replacing nitrogen fertilizer with peanut meal fermentation solution, only F5 and F10 treatments had better yield increase effect on potato, and F15 and F20 treatments even reduced potato yield and tuber quality, the specific reasons for which have to be explored subsequently.

Effects of replacing part of nitrogen fertilizer by peanut meal enzymatic solution and fermentation solution on soil environment

Long-term application of large quantities of chemical fertilizers while neglecting the application of organic fertilizers will result in lower soil organic matter content, soil acidification, lower nutrient effectiveness, lower soil fertility[46] , exacerbate the spread of soil diseases[47] impede plant growth, and even become a cause of greenhouse gas emissions[48]. Researchers monitoring nutrient inputs to the Anthrosols area in Zhangjiagang County, China, for 20 years found that soil pH in the Anthrosols area decreased by 1-2 pH units compared to the previous period, and the study found that long-term increases in fertilizer inputs in the area, especially ammonium bicarbonate and urea, were the main cause of severe acidification and accelerated rates of nitrogen mineralization and nitrogen leaching from the soil[49]. In contrast, partial replacement of N fertilizer treatment by organic fertilizer significantly improved the physicochemical properties of the soil. After 20 years of application of organic fertilizer replacing 30% of nitrogen fertilizer, soil pH increased by 1.0 unit from the initial value[50]; replacing chemical fertilizers in red soil could both reduce soil exchangeable acidity and exchangeable aluminum content and alleviate soil acidification[51]. The results of this experiment showed that replacing part of the nitrogen fertilizer with peanut meal enzymatic solution and fermentation solution had different degrees of positive effects on improving soil physicochemical properties, with the former having a more pronounced effect. This may be because the peanut meal enzyme solution is rich in organic matter, applied to the soil can make the soil organic matter content rise. Organic matter can provide soil organisms with a large amount of available energy, which can increase the activity of soil enzymes, thereby improving the effectiveness of soil nutrients[52].

It was found that replacing some chemical fertilizers with organic fertilizer could increase the inter-root soil bacterial diversity of rice, change the structure of soil microbial community, and improve the soil bacterial ACE index, Chao index and Shannon index, which significantly increased the bacterial species richness by 6.53% (P<0.05), and the Shannon index significantly increased by 5.64% (P< 0.05) compared with the treatment of organic fertilizers and chemical fertilizers alone[53], which was similar to the results of this experimental study. The composition and abundance of soil bacterial community at the genus level were changed under peanut meal fermentation solution and enzyme solution substitution treatments, indicating that the treatments of peanut meal enzyme solution and peanut meal fermentation solution substituting part of nitrogen fertilizer could change the compositional structure of soil bacteria at the genus level, which made the bacterial community in the soil more species-rich, among which, the treatment of peanut meal enzyme solution and fermentation solution substituting 10% nitrogen fertilizer was more species enriched and the status of dominant species was more prominent.

Numerous studies have shown that the composition of microbial communities is closely related to the environment[54, 55, 56]. The application of organic matter has an important effect on soil microbial composition, abundance and activity, which can promote the physiological and biochemical activities of soil microorganisms[51], significantly increase the number of various types of microorganisms, especially those related to soil nutrient transformation[57]^. In this study, among the four bacterial genera with bacterial abundance greater than 1%, which could be accurately categorized with significant differences, Sinomonas had the highest relative abundance in the F20 treatment, with an increase of 149.8% compared to the M20 treatment, while Terrabacter, Sphingomonas and Arthrobacter had the highest relative abundance in the MCK treatment, followed by the M5 treatment, and compared with the F5 treatment M5 treatment increased by 36.1%, 91.8% and 173.9%, respectively. The above indicates that the presence of peanut meal enzyme digest and fermentation solution decreased the abundance of Terrabacter, Sphingomonas and Arthrobacter, while increased the abundance of Sinomonas, and this effect was more pronounced in the F20 treatment. This is similar to the results of correlation between soil bacterial communities and environmental factors at the genus level. The activities of Terrabacter, Arthrobacter, and Sphingomonas were inhibited by environmental factors such as soil effective phosphorus, where soil pH highly significantly inhibited the activity of Arthrobacter. One of the reasons may be that the peanut meal enzyme digest and fermentation solution are rich in amino acids and polypeptides, which can be directly absorbed and utilized by potatoes, reducing nutrient conversion and loss, and therefore reducing the activity of functional bacteria, such as Arthrobacter which is a functional bacterium related to nitrification and denitrification of soil nitrogen[58]. when the content of ammonium nitrogen and nitrate nitrogen in the soil was reduced, the activity of Genus Arthrobacter was also reduced accordingly, thus decreasing the Genus Arthrobacter relative abundance of Arthrobacter Increase in soil organic matter increased the activity of Sinomonas. The organic matter content reached its highest value at M20 and F20 treatments, which were significantly higher than MCK treatment by 13.48% and 17.83%, respectively. This may be due to the fact that Sinomonas belongs to chemoenergetic heterotrophic microorganisms[59], which require organic compounds as a carbon source and obtain energy from the process of oxidizing organic matter, and thus the higher the organic matter content the higher its activity.

Compared with the peanut meal fermentation solution prepared by fermentation of microbial agents, alkaline protease enzyme digestion of peanut meal to prepare enzyme solution is shorter, more stable, and higher in organic nutrients, and the replacement of nitrogen fertilizers into the soil, the soil physicochemical properties, bacterial community composition and abundance of the soil changed significantly. Potato yield was significantly affected by total soil nutrients, fast-acting nutrients and bacteria, and potato yield and quality were significantly better under the 10% and 15% substitution treatments of peanut meal enzymatic solution than the other treatments. Soil physicochemical properties are closely related to bacterial community composition, and the application of peanut meal enzyme digest and fermentation solution can reduce soil acidification, replenish soil nutrients, and increase soil organic matter content and beneficial microbial abundance. Therefore, we suggest using alkaline protease to hydrolyze peanut meal and replace 10% or 15% of nitrogen fertilizer, which is conducive to both the improvement of potato yield and quality, as well as the high-value resource utilization of peanut meal.

Availability of data and materials

The data presented in this study are available in the article.

Acknowledgements

Not applicable.

Funding

This study was supported by the National Natural Science Foundation of China (No. 42377211) and the National Key Research and Development Program of China (2023YFD2300805). The funders were not involved in study design, data collection and analysis, publication decision, or manuscript writing. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author cotributions

QZ and ZX designed the experiment and wrote the manuscript, and determined the experimental indicators and analyzed the data; QZ, ZX and WB revised the manuscript; QZ, ZX, WB, MY, FZ, HD, and HK were involved in the enzymolysis and field trials; and DL supervised and provided funding.

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Competing interests

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You are reading this latest preprint version

Effects of Peanut Meal Enzymatic Optimization Process and its Products Replacing some Nitrogen Fertilizers on Potato Growth and Soil Environment

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Experiment 1: Process optimization for the preparation of peanut meal enzyme digest by enzymatic digestion

Test sites and materials

Experimental design and implementation

Measurement items and methods

Experiment 2: Effects of replacing part of nitrogen fertilizer with peanut meal enzymatic solution and fermentation solution on potato growth and soil environment

Test site and materials

Experimental design and implementation

Measurement items and methods

Statistics and analysis of data

Results

Optimization of the process for the preparation of peanut meal enzyme digest by enzymatic hydrolysis method

One-way test of hydrolysis conditions by composite alkaline protease

Response surface results and analysis of variance (ANOVA)

Response surface factor interactions

Validation of the regression model

Determination of Enzymatic Solution Product Composition

Effects of peanut meal enzymatic solution and fermentation solution replacing part of nitrogen fertilizer with on potato growth and soil environment

Potato yield and yield components

The accumulation of macro-elements and dry matter in potato tubers

Potato quality

Soil physical and chemical properties

Sequencing information of soil bacteria from different treatments

Venn diagrams of soil bacterial communities at the OTU level for different treatments

Alpha diversity index of soil bacterial communities in different treatments

Structural composition of soil bacterial communities at different treatment genus levels and analysis of significance tests for differences between groups

Heat map analysis of correlation between soil bacterial communities and environmental factors at different treatment genus levels

Discussion

Study on the process of preparing peanut meal enzyme solution by enzymatic hydrolysis method

Effects of replacing part of nitrogen fertilizer with peanut meal enzymatic solution and fermentation solution on potato growth and soil environment

Effect of replacing part of nitrogen fertilizer by peanut meal enzymatic solution and fermentation solution on the growth of potato

Effects of replacing part of nitrogen fertilizer by peanut meal enzymatic solution and fermentation solution on soil environment

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1