Preparation, characterization, identification, and antioxidant properties of fermented acaí (Euterpe oleracea)

Abstract Fermentation technology was used to prepare the acaí (Euterpe oleracea) fermentation liquid. The optimal fermentation parameters included a strain ratio of Lactobacillus paracasei: Leuconostoc mesenteroides: Lactobacillus plantarum = 0.5:1:1.5, a fermentation time of 6 days, and a nitrogen source supplemental level of 2.5%. In optimal conditions, the ORAC value of the fermentation liquid reached the highest value of 273.28 ± 6.55 μmol/L Trolox, which was 55.85% higher than the raw liquid. In addition, the FRAP value of the acaí, as well as its scavenging ability of DPPH, hydroxyl, and ABTS free radicals, increased after fermentation. Furthermore, after fermentation treatment, the microstructure, basic physicochemical composition, amino acid composition, γ‐aminobutyric acid, a variety of volatile components, and so on have changed. Therefore, fermentation treatment can significantly improve the nutritional value and flavor of the acaí. This provides a theoretical basis for the comprehensive utilization of acaí.


| INTRODUC TI ON
Oxidative stress is closely related to the human body. When the human body is stimulated by exogenous or endogenous factors, the imbalance in the antioxidant system leads to oxidative stress, which can cause direct or indirect oxidative damage to biological macromolecules, such as DNA, proteins, and lipids, leading to physiological and pathological reactions (Hussain et al., 2003). Because free radicals have a single electron, they quickly attach to DNA, causing DNA strand breaks, DNA and protein cross-links, and purine oxidation.
Furthermore, free radicals destroy the peptides in the protein chain, facilitate protein cross-linking, change spatial protein structures, and lead to protein inactivation. In addition, lipids containing a large amount of unsaturated fatty acids, such as phospholipids on cell membranes, are also vulnerable to free radicals, triggering a chain reaction of continuous free radical amplification. Malonaldehyde products can react to form lipofuscin, resulting in impaired biological macromolecule functionality (Fearon & Vogelstein, 1990).
Sustained oxidative stress leads to chronic inflammation, which in turn contributes to most chronic diseases such as cancer, diabetes, cardiovascular, neurological, and lung diseases. In addition, higher intracellular oxidative stress aggravates the aging of the body (Reuter et al., 2010). Therefore, oxidative stress prevention is essential since it can cause cell damage and pathological reactions.
Acaí (Euterpe oleracea), also known as Brazil berry, has attracted considerable attention due to its strong antioxidant capacity and can be used as a functional food. It is a grape-sized, purple-black, protein-rich berry that tastes like a combination of wild berries and chocolate (Lichtenthäler et al., 2005). Acaí is a major food source for local residents in Brazil, Peru, Colombia, and Suriname. Its juice is rich in bioflavonoids, anthocyanins, and other antioxidants and functional substances such as unsaturated fatty acids. These berries offer antioxidant (Alpert & Patricia, 2016) and anti-inflammatory properties (Kang et al., 2010), lower blood lipids and blood pressure (de Souza et al., 2012;Silva et al., 2018), protect the liver (Carvalho et al., 2019), promotes bone health, and enhances pain relief (Brito et al., 2016;Jensen et al., 2011).
Due to higher health awareness, the quality of fermented food has improved in recent years, increasing in popularity with consumers. Fermentation involves using microbial technology for food production, and it is also the oldest method of producing and preserving food products. Fermentation retains active components in raw food, such as polysaccharides, dietary fiber, bioflavonoids, and other substances beneficial to the body. It also changes various raw nutritional components in food products to produce a unique flavor while decomposing factors unfavorable to human health, such as oligosaccharides and flatulence elements in beans. Furthermore, many metabolites produced via microbial metabolism can regulate the physiological functions of the body and inhibit the production of harmful substances. This study used acaí fruit pulp as raw material to examine the changes in the organic acid content, phenols, and other bioactive substances, chemical compositions, and antioxidant activity before and after fermentation. The fermentation technology was optimized to provide technical support and theoretical guidance for the production and development of fermented acaí products.

| Raw acaí liquid and reagents
The raw acaí liquid was provided by Heilongjiang Feihe Dairy Co., Ltd.

| Pretreatment of raw acaí liquid
Here, 1.5% wheat oligopeptide and 0.5% glucose were added to the raw acaí liquid and mixed evenly, after which the pH was adjusted to 6.4-6.8 by addition of 1 mg/ml citric acid and calcium carbonate, and the mixture was sterilized at 115°C for 15 min.

| Expanded strain culture
Larger scale single-and mixed-culture fermentations were performed to better evaluate the effects of selected Lactobacillus strains on the physical, chemical, and volatile aroma components and aroma characteristics of Acaí fermentation liquid. The growth cycle, growth characteristics, and stability of the fermented strains under temperature and pH were screened to screen out the optimal strains and optimal process conditions for the fermentation of Acaí raw materials.

| Orthogonal test
The inoculation amount, strain proportion, fermentation time, and nitrogen source addition quantity were selected according to previous studies. Orthogonal experiments with four factors and three levels were performed (Table 1). The oxygen radical absorbance capacity (ORAC) value of the acaí fermentation liquid was considered the evaluation index to determine the influence of various factors and their interaction with the ORAC value of acaí fermentation liquid. The optimal fermentation process conditions were also established. The experimental design and analysis were realized by using SPSS software.

| Electronic tongue detection
A 30 ml liquid sample was measured, after which distilled water was added until reaching a constant volume of 60 ml. After stirring for 3 min, 50 g reference liquid was added and centrifuged for 3000 g for 10 min. The clear liquid was removed and detected using a TS-5000Z taste analysis system (INSENT Company) .

| Scanning electron microscopy (SEM)
The freeze-dried powder samples were smeared on the doublesided adhesive of the sample tray, followed by nitrogen-blowing treatment. After the processed samples were vacuumed via SEM, a certain voltage was applied, and the beam spot size was adjusted until the focus was clear. The images were obtained at 500×, and 1000× magnification, respectively, and the differences were observed (Müller et al., 2011). 2.5 | Determination of the physical and chemical composition 2.5.1 | Determination of the basic physical and chemical components The protein, ash, fat, and moisture content of the COPs were measured using the official AOAC method (AOAC, 2002). The total polyphenol content was determined using an Evolution-201 Ultraviolet spectrophotometer (Thermo Fisher Technology Co., Ltd.) according to a method described by Abeysinghe et al. (2021).

| Determination of the amino acid composition
The amino acid composition was analyzed using an A300 automatic amino acid analyzer (Membrapure, Germany) according to Yang et al. (2007).

| Determination of the procyanidin content
The procyanidin content was determined using an LC-20A HPLC (Shimadzu Company) according to a method described in a previous study Yang et al. (2007).

| Determination of the flavonoid content
Here, 50 mg (accurate to 0.001 g) samples were weighed into a centrifuge tube, after which 5 ml methanol was added, shocked for 30 min, ultrasonicated for 30 min, and shocked again for

| Determination of the volatile aroma components
The volatile aroma components were determined via Clarus SQ8 gas chromatography-mass spectrometry (GC-MS) with an EI ion

| Chemical composition analysis via liquid chromatography-mass spectrometry (LC-MS)
The freeze-dried powder sample was dissolved in ultrapure water, Mass spectrometry analysis was operated in positive (spray voltage of 4.0 kV) and negative ion (spray voltage of 3.2 kV) modes, respectively. The air warping rate and auxiliary gas rate were set to 40 and 10 ml/min, respectively. The capillary temperature was set to 300°C, and S-lens was 50% . After reacting for 1 h, 200 μl reaction liquid was placed in a 96well plate, and the absorbance value was measured at 510 nm (Liu et al., 2015).

| Determination of the ABTS free radical scavenging ability
Here, 100 μl ABTS liquid and 100 μl oxidant liquid were mixed to obtain the ABTS working mother liquid, which was left to stand overnight at room temperature in the dark to yield the ABTS free radical reserve liquid. The ABTS working liquid was diluted 35 times with 0.1 mol/L phosphate buffer (pH 7.4) before use, while the absorbance value at 734 nm was 0.70 ± 0.02, after which 200 μl ABTS working liquid was added to each well. Next, 10 μl of Trolox standard liquid at different concentrations was added to the standard curve detection well, and 10 μl of the sample liquid was added to the sample detection well, mixed gently, and incubated for 6 min at room temperature, after which the absorbance value was determined at 734 nm. The standard curve was drawn using the Trolox liquid concentration as the abscissa and the absorbance as the ordinate. The antioxidant capacity of the final sample was expressed as mmol/g Trolox (Corsetto et al., 2020

| Statistical analysis
All the tests were done in triplicate (n = 3), and statistical analysis was performed by one-way analysis of variance (anova) using Originpro 8.0 (OriginLab Corp.). Data were presented as means with standard deviation (SD). Statistical significance was set at p < .05.

| Electronic tongue analysis
The sensors used in the electronic tongue include six taste attributes of bitter, sweet, salty, sour, fresh, and astringent, and two comprehensive attributes of aftertaste-A and aftertaste-B.
In addition, the difference in taste indicators between each sam-

| SEM analysis
SEM was used to examine the micromorphology of the samples before and after acaí fermentation in terms of overall morphology and particle size and observe the freeze-dried powder of acaí liquid and fermentation liquid at 500× and 1000× magnification, respectively, as shown in Hydroxyl radical scavenging rate be due to the homogenization, enzymatic hydrolysis, and fermentation of the compound lactic acid bacteria during the fermentation process. Studies have shown that the structures and states of acaí treated with different drying methods vary. IRFD-dried samples were slightly darker than FD-dried samples, while those exposed to far-radiation heating were more compact and harder (Oliveira et al., 2021).
The protein content in the acaí liquid increased significantly to 2.77 ± 0.06 (g/100 g) after fermentation. This may be due to the enzymes produced by microorganisms during the fermentation process to promote the hydrolysis of proteins with larger molecular weights (Yang et al., 2020). This was consistent with the results of Frias et al. (2008), who used Lactobacillus plantarum to ferment soybeans. The small molecular weight proteins also increased, promoting the formation of various flavor substances. Fermentation significantly reduced the total fat, total polyphenol, and anthocyanin content in the acaí. The reduction in the fat content may be due to the fat metabolism by microbial activity. The loss of anthocyanins during fermentation is affected by a variety of factors, for example, during fermentation, anthocyanins may interact with other flavonoids to form more stable anthocyanidins, reducing the polarity and solubility of these compounds (Benito et al., 2011). In addition, anthocyanins and phenolic compounds can react to form complexes, which may also be the reason for the decrease in total polyphenols and anthocyanins (Klopotek et al., 2005).

| Amino acid composition
Amino acids can provide a sour, sweet, bitter, or fresh taste and play a role in the flavor of food. Table 2 shows the hydrolyzed amino acid and free amino acid content in the raw and fermented acaí liquid.
After fermentation, the acaí amino acid content increased, which was consistent with the protein determination results. In the case of hydrolyzed amino acids, the contents of glutamic acid, aspartic acid, leucine, lysine, and proline were higher in the raw acaí liquid, while the fermentation liquid displayed higher glutamic acid, proline, leucine, valine, and phenylalanine levels. In terms of free amino acids, the contents of alanine, glutamic acid, serine, phenylalanine, and leucine were higher in the raw acaí liquid, while the fermentation liquid exhibited higher leucine, glutamic acid, alanine, valine, and phenylalanine levels. These amino acids provided the acaí products with a sweet and sour taste.
After fermentation, the total amino acids in the acaí products increased from 1.012 to 1.727 (g/100 g). The highest nonessential amino acid content in the acaí stock solution was glutamic acid, followed by aspartic acid, lysine, proline, serine, alanine, and arginine. The highest content of essential amino acids is leucine, followed by lysine, phenylalanine, valine, valine, threonine, and the lowest is methionine. Yang Research has shown that the amino acid composition of seaweed can be changed by microbial fermentation treatment (Norakma et al., 2022). Therefore, fermentation can increase the amino acid concentration in acaí products. Studies have shown that amino acids such as tyrosine, histidine, cysteine, and methionine display antioxidant activity. Histidine exhibits strong free radical scavenging activity due to the decomposition of its imidazole ring, while cysteine acids display strong reducibility due to the presence of sulfhydryl groups (Virtanen et al., 2007). The antioxidant activity of fermented acaí in this study may be due to the presence of these amino acids.

| Flavonoid content
The liquid chromatographic diagram of the flavonoid standard is shown in Figure 3a. The liquid chromatographic diagrams of the nine flavonoids in the raw and fermented acaí liquid are shown in Figure 3b,c, while the flavonoid content results are shown in Table 3.
The total content of the nine flavonoids in the acaí decreased after fermentation. The 4′OH-nobiletin, 5′OH-tan levels, 4′OH-tan, and nobiletin in the raw acaí liquid were higher, while that of sinensetin, tangeretin, 3′,4′OH-nobiletin, and 5′OH-nobiletin increased after fermentation. The reason for the decrease in the total content of flavonoids may be as mentioned above. During fermentation, TA B L E 2 Physicochemical and amino acid composition of the raw and fermented acaí liquid.

| Analysis of the volatile aroma components
The chromatograms of the raw and fermented acaí liquid are shown in Figure 3d,e. The results of the volatile aroma components in the two acaí products are shown in Table 3. Four volatile aroma components were identified in the raw liquid, which was represented from high to low by acetaldehyde, n-propanol, isoamyl alcohol, and ethyl acetate. Seven volatile aroma components were produced in fermentation liquid, which was represented from high to low by  Yan et al. (2018) found that the volatile components in strawberry-flavored products mainly comprised esters and alcohols, among which methyl butyrate and ethyl butyrate were the key flavor compounds.

| Chemical composition identification via LC-MS
The chemical composition of acaí before and after fermentation was examined via LC-MS. The results are shown in Figure 4a-d and

H-ORAC value
Oxygen radical absorbance capacity (ORAC) refers to an antioxidant capacity index, which is also known as antioxidant radical scavenging capacity or antioxidant capacity index measurement.
ORAC is an evaluation method used in the field of antioxidant research. The H-ORAC value was used to evaluate the ORAC value of the water-soluble antioxidants. The dynamic fluorescence attenuation and standard curves of different Trolox concentrations are shown in Figure 5a,b, y = 0.08737x−0.07947, R 2 = .99983.
According to the standard curve, the H-ORAC values of the freeze-dried powder derived from the raw and fermented acaí liquid were 453.64 ± 24.28 μmol/g Trolox and 708.77 ± 12.46 μmol/g Trolox, respectively. After fermentation, the H-ORAC value of the acaí increased by 56.24%. The water-soluble antioxidant capacity of the fermented acaí product was similar to that of sage spice (987.14 μmol/g Trolox), 30 times that of wolfberry (31.70 μmol/g Trolox), and 60 times higher than that of red grapes (16.40 μmol/g Trolox) (Buratto et al., 2021).

L-ORAC value
RMCD was used to measure the L-ORAC value and evaluate the ORAC of fat-soluble substances by incorporating them into the cavity to increase water solubility. The dynamic fluorescence attenuation curves and standard curves of the different Trolox concentrations are shown in Figure 5c, (Kang et al., 2011). Some studies have found that heat treatment can reduce the free radical scavenging ability of acaí juice (da Silveira et al., 2019), while Tadapaneni et al. (2014) found that the free radical scavenging ability of heat-treated strawberry juice and strawberry milk-based beverages lost up to 40%. Therefore, the fermentation process of acaí products can be used as a reliable method for further processing, while it is suggested that consuming fermented acaí products may provide antioxidative protection to promote human health.
3.6.2 | DPPH radical scavenging ability DPPH free radical scavenging ability is an important index to evaluate the antioxidant capacity of substances based on electron transfer and charge neutralization principles. A DPPH free radical is a stable N ion radical in an organic environment with a maximum absorption peak at 517 nm (Ganguly et al., 2020). When a radical scavenger is present in the system, the single electron of the DPPH radical is neutralized by the scavenger, and the color of the liquid changes from purple to light yellow or colorless, directly indicating a decrease in the absorbance value. As shown in Figure 5e, the two acaí products displayed significant DPPH radical scavenging ability in a range of 0.0625-2.00 mg/ml, while the scavenging rate was dose dependent with the mass concentration. The DPPH free radical scavenging ability of the acaí fermentation liquid was stronger than that of the raw acaí liquid. The DPPH radical scavenging rate of the acaí samples before and after fermentation was compared, yielding IC 50 values of 0.58 and 0.16 mg/ml, respectively. The acaí scavenging ability improved after fermentation.
Previous research has shown that acaí displays strong antioxidant activity, and compared with the results of this study, the hydroalcoholic extract from acaí seeds exhibits higher antioxidant activity, corresponding to the EC50 of water extract at 8.8 μg/ml (DPPH assay) (Barros et al., 2015). Martins et al. (2020)

| Hydroxyl radical scavenging ability
A hydroxyl radical is an extremely active free radical formed in a biological system that can cause lipid peroxidation of cell membranes and is extremely harmful to the human body. The hydroxyl radical scavenging capacity of the two acaí samples is shown in Figure 5f. The hydroxyl radical scavenging ability of the acaí samples showed an obvious dose-dependent relationship and was positively correlated with the experimental concentration range.
The acaí samples displayed distinct hydroxyl radical scavenging ability, while the scavenging rate increased at a higher mass concentration, showing a significant dose-dependent relationship.
The hydroxyl radical scavenging ability of acaí fermentation liquid was higher than that of raw acaí liquid. After fermentation, the IC 50 value of the acaí hydroxyl radical scavenging rate was 0.97 and 0.49 mg/ml, respectively, indicating an improved scavenging ability. The hydroxyl radical scavenging ability is related to its capacity as a hydrogen donor to reduce free radicals and stop the free radicals chain reaction.
Studies have shown that the phenolic compound content is proportional to increased hydroxyl radical levels. Márquez et al. (2019) studied the generation of hydroxyl radicals in white wine and their relationship with phenolic compounds and concluded that the changes in the hydroxyl radicals were related to the phenolic constituents in the wine. Therefore, the reason for the improvement in the hydroxyl radical scavenging ability of the acaí products after fermentation may be due to a reduction in the total phenolic content, decreasing the hydroxyl radicals, and increasing the reducing amino acid content.
3.6.4 | ABTS free radical scavenging ability ABTS was used as a color initiator in this method. The principle was that after adding an oxidant, the ABTS reagent could generate stable blue-green radical ABTS + in the liquid, yielding maximum absorption at 734 nm. The addition of antioxidant substances neutralized the ABTS + charge, causing the color to become lighter and decreasing the absorbance value. The decreasing trend reflected the strength of the antioxidant of the detected substance. Figure 5g shows the ABTS curve drawn based on the Trolox standards. Calculations showed that the ABTS radical scavenging ability of the freezedried powder derived from the raw and fermented acaí liquid was 0.44 ± 0.03 mmol/g Trolox and 0.45 ± 0.08 mmol/g Trolox, respectively. The acaí fermentation liquid displayed a better scavenging ability than the raw acaí liquid. After fermentation, the ABTS radical scavenging rate of the acaí was slightly higher.
The ABTS method determines the ability of antioxidants to quench ABTS + free radicals via electron transfer reactions. Studies have shown that the phenolic compounds in the acaí are excellent electron donors and can quench ABTS + free radicals (Garzón et al., 2017). Therefore, the fermented acaí product displayed a satisfactory ABTS + free radical scavenging rate.

| FRAP value
FRAP is a rapid, simple indicator of antioxidant capacity and can be used to determine the reducing capacity of substances. The standard curve of FeSO 4 is shown in Figure 5h, which was used to calculate the FRAP values of the two acaí samples. The FRAP values of freeze-dried powder derived from the raw and fermented acaí liquid were 0.43 ± 0.01 and 0.55 ± 0.02 mmol/g FeSO 4 , respectively.
A higher FRAP value typically indicates a stronger antioxidant capacity. The FRAP value of the acaí fermentation liquid was higher than the raw liquid.
Although studies have indicated that phenolic compounds can reduce Fe(III) to Fe(II), this has nothing to do with the generation of hydroxyl radicals, which is a relatively complicated process (Márquez et al., 2019). Therefore, the increased FRAP value of the acaí products after fermentation may be due to a higher total phenolic content.
Phenolic compounds interact with Fe(III) in different ways, depending on their chemical structures (Perron & Brumaghim, 2009 and flavonoids) and volatile aroma components were analyzed and identified.
The fermentation process showed that the order of the influencing factors on the ORAC value of acaí after fermentation via compound lactic acid bacteria was as follows: inoculation amount > nitrogen source addition amount > fermentation time > strain proportion.
The optimal fermentation condition combination was A1B1C1D3, that is, the inoculation amount was 1%. The parameters included a strain ratio of Lactobacillus paracasei: Leuconostoc mesenteroides: Lactobacillus plantarum = 0.5:1:1.5, a fermentation time of 6 days, and a nitrogen source supplemental level of 2.5%. The results verified that in optimal conditions, the ORAC value of the fermentation liquid reached the highest value of 273.28 ± 6.55 μmol/L Trolox, which was 55.85% higher than the raw liquid. Although there was no peculiar smell, a specific acaí flavor was present.
The antioxidant activity evaluation results showed that the ORAC value (H-ORAC value+L-ORAC value) of the lyophilized powder derived from the raw and fermented acaí liquid were 595.45 ± 30.71 μmol/g Trolox and 877.05 ± 20.58 μmol/g Trolox, respectively, and increased by 47.29% after fermentation. In addition, the FRAP value of the acaí, as well as its scavenging ability of DPPH, hydroxyl, and ABTS free radicals, increased after fermentation.
By comparing the changes in microstructure and physicochemical properties of acaí before and after fermentation treatment, it can be concluded that after fermentation treatment, the microstructure, basic physicochemical composition, amino acid composition, γ-aminobutyric acid, a variety of volatile components, and so on have changed; furthermore, it is worth noting that the contents of γ-aminobutyric acid, lactic acid, and a variety of volatile components have significantly increased. Therefore, fermentation treatment can significantly improve the nutritional value and flavor of the acaí. The content of the volatile and various chemical components increased, while fermentation significantly improved the nutritional value and flavor of the acaí.
In summary, the DPPH, ABTS, hydroxyl radical scavenging ability, ORAC, and FRAP data results provide evidence that fermented acaí products display strong antioxidant capacity and can interact with different free radicals. These products may present food health and biomedical applications for reducing oxidative stress in the body. In addition, the antioxidant capacity of any food sample results from the synergistic effect of a mixture of compounds, including phenols, carotenoids, and vitamins C and E. Although the vitamin content of acaí is relatively low, polyphenols play a substantial role in its antioxidant properties (Maria do Socorro et al., 2011).

| CON CLUS ION
In this study, acaí fermentation liquid was prepared by fermentation technology. The antioxidant activities of acaí stock solution and fermentation liquid were evaluated from eight aspects. At the same time, the samples before and after fermentation were analyzed by scanning electron microscopy, electronic tongue detection, physical and chemical components, volatile aroma components, and so on. The results showed that acaí fermentation liquid had a strong antioxidant capacity after fermentation treatment, and the fermentation treatment changed the microstructure and flavor of acaí and increased the content of volatile components and various chemical components. In conclusion, fermentation treatment of acaí can improve its quality and nutritional value, which provides a theoretical basis for the comprehensive utilization of acaí.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author upon reasonable request.