Litopenaeus vannamei fermentation using selected Lactobacillus spp. to reduce its allergenicity

ABSTRACT

Shrimp is a common cause of food allergies in allergic individuals. The management of patients suffering from shrimp allergy relies on avoidance of the specific food, which compromises nutritional requirements. In this study, fermentation was performed using Lactobacillus to reduce the allergenicity of Litopenaeus vannamei. The optimal fermentation conditions using Lactobacillus helveticus TS6024 were 1% glucose, 30% minced shrimp, 2.5% inoculum level, 3% NaCl, and 60 h of incubation at 37°C, whereas those using Lactobacillus acidophilus 6005 were 2% glucose, 20% minced shrimp, 15% inoculum level, 2% NaCl, and 48 h of incubation at 42°C. Under optimal conditions, Lactobacillus helveticus TS6024 and Lactobacillus acidophilus 6005 fermentation reduced the allergenicity of shrimp by 78.97% and 70.09%, respectively. SDS-PAGE analysis showed that the band intensity of tropomyosin (36KDa) reduced evidently after the selected Lactobacillus fermentation. The hypoallergenic shrimp/shrimp products will provide optimal nutrition with reduced risk of adverse reactions in hypersensitive individuals.

KEYWORDS:

• Litopenaeus vannamei
• minced shrimp
• Lactobacillus helveticus TS6024
• Lactobacillus acidophilus 6005
• allergenicity

Introduction

Shellfish is one of the leading causes of food allergy, with a prevalence of 2.8%–8% among all food allergies, and it is a common cause of food-induced anaphylaxis (El-Qutob, 2017). Shrimp, which is widely consumed worldwide for its delicate flavour and high nutritional value, is one of the shellfish with high allergenicity. Shrimp contains more than 10 allergenic components, such as tropomyosin (TM), arginine kinase, and myosin light chain (Pascal et al., 2015). Among these, TM, a myofibrillar protein, is the main allergen of shrimp (Xie et al., 2021). TM exhibits properties common in food allergens, including acidic isoelectric point, salt solubility, and small molecular weight of 34–38 kDa. Moreover, it can pass through the mucosal surface and is easily absorbed by the body (Leung et al., 2014), and has high thermal stability (Laly et al., 2019). As a result, heating and cooking do not eliminate the allergens.

At present, there are several ways to reduce the allergenicity of shrimp. Shimakura et al. (2005) found that the allergenicity of the extractives of shrimp was almost completely lost on digestion with proteases. Zhenxing et al. (2007) used γ-radiation to reduce the allergenicity of shrimp. The allergenicity of irradiated shrimp muscle decreased markedly when the dose exceeded 10 kGy. Dong et al. (2021) processed shrimp using microwaves. SDS-PAGE analysis showed that the band intensity of TM reduced with increase in processing temperature and duration. Treatment using microwaves at 125 °C for 15 min resulted in a significant reduction in the allergenicity of TM (approximately 75%). Moreover, Dong et al. (2020) investigated the impact of high-intensity ultrasonic waves on the physiochemical and allergenic properties of shrimp samples. The allergenicity decreased with increase in treatment time, and the best hypoallergenic effect was observed at 20 min, with a 76% reduction in TM. Xingxuan et al. (2018) investigated the effect of high hydrostatic pressure on the allergenicity of shrimp. Allergenicity was reduced by approximately 80% after sequential treatment for 30 min at 200.0 MPa, followed by additional treatment for 30 min at 600.0 MPa. Fu et al. (2019) used an approach to eliminate TM in Penaeus chinensis using Maillard reaction with reducing sugars, including ribose, galacto-oligosaccharide, and chitosan-oligosaccharide, which reduced the allergenicity by approximately 60%. Zhang et al. (2020) confirmed that the TM glycated by galacto-oligosaccharide, mannan-oligosaccharide, and maltopentaose had lower allergenicity and induced weaker mouse allergic responses. These methods inadequately reduce shrimp allergens to different degrees, with varying results. Moreover, bitter peptides are sometimes produced after protease treatment (Shimakura et al., 2005).

Lactic acid bacteria fermentation is one of the oldest economical, natural, and safe methods of food processing. It has a variety of probiotic effects, such as bacteriostasis of pathogenic strains (Alvarez-Sieiro et al., 2016), prevention of cancer (Yu & Li, 2016), and improving the body's immunity (Rask et al., 2013). A previous study by Fu et al. (2020) suggested that oral administration of L. casei Zhang alleviated TM-induced shrimp allergy in mice, making it a potential immunomodulator and immunotherapy assistor. Recent studies show that lactic acid bacteria can degrade proteins to produce polypeptides using cell- envelope proteinases (CEPs) (Skrzypczak et al., 2018), with Lactobacillus showing a broad range of peptidase activity (Raveschot et al., 2020; Zhong et al., 2021). In recent years, the use of lactic acid bacteria to reduce food allergenicity has gained much attention. Zhao et al. (2021) found that L. helveticus and L. plantarum could reduce cow milk protein allergenicity through the combined action of CEP and peptidase on α-casein. The potential allergenicity of soybean meal was reduced via solid-state fermentation induced by a mixture of L. casei, yeast, and Bacillus subtilis (Yang et al., 2018). Rizzello et al. (2006) confirmed that the IgE bound low molecular weight protein of Lactobacillus-fermented bread was significantly lower than that of yeast-fermented bread. Furthermore, a notable decrease in the antigenicity and allergenicity of gliadins, glutenins, and α-amylase inhibitors were observed after fermentation of gluten by Lactococcus lactis LLGKC18 (El Mecherfi et al., 2022).

Litopenaeus vannamei is the most widely cultured shrimp species and extensively sold seafood product globally. However, to the best of our knowledge, the role of Lactobacillus fermentation in reducing the allergenicity of sea shrimp has not been evaluated. Therefore, the present study aimed to investigate the effects of Lactobacillus fermentation on the allergenicity of Litopenaeus vannamei, which will be useful for improving the safety of shrimp/shrimp products for human consumption.

Materials and methods

Bacterial strains

Lactobacillus helveticus TS6024 and Lactobacillus acidophilus 6005 were obtained from the Laboratory of Functional Ingredients and Human Health, Tianjin University of Commerce, Tianjin, China.

Shrimp and human serum

Shrimp (Litopenaeus vannamei) were obtained from Hanjiashu aquatic product market (Beichen District, Tianjin, China).

Twelve patients (seven male and five female patients aged 20–52) with clinically diagnosed shrimp allergy visiting Shidao People's Hospital of Rongcheng City (Shandong Province), Kailuan Hospital of Tangshan City (Hebei Province), and Tianshui First People's Hospital (Gansu Province) were recruited in this study. Control subjects (ten male and ten female aged 20–45 years) who showed no adverse reaction to any food allergen were from Tianjin University of Commerce School Hospital (Tianjin City). Skin prick test result that is greater than 1/2 of the positive control diameter range was graded as “++” (Frati et al., 2018). Blood sample was collected from each patient. Subsequently, the serum was separated, and all serum samples from patients were pooled in equal amounts. Similarly, serum samples from 20 control subjects were collected and pooled to serve as the negative control. All serum samples were frozen at −80°C until use. Informed consent was obtained from all participants. The study protocol was approved by the Ethics Committee of Tianjin University of Commerce, Tianjin, China.

Reagents

Horseradish Peroxidase-labelled goat anti-human IgE antibody (A9667) was purchased from Sigma Corporation, USA. All the other chemical reagents were analytically pure. Buffers and reagents used for competitive ELISA were as follows: PBS: 10 mM phosphate, pH 7.0, 0.15 M NaCl; coating buffer: 0.1 M bicarbonate buffer, pH 9.6; blocking buffer: 10 mM phosphate, pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.15 M NaCl; washing buffer (PBST): 10 mM phosphate, pH 7.4, containing 0.05% Tween 20; substrate solution: 0.1 M citric acid (4.86 mL), 0.2 M Na₂HPO₄ (5.14 mL), 4 mg phthalediamine, and 3% H₂O₂ (50 μL).

Seed and fermentation media

The seeding medium consisted of MRS medium components (5 g yeast extract, 10 g peptone, 10 g beef extract, 20 g glucose, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 0.58 g MgSO₄.7H₂O, 0.25 g MnSO₄.4H₂O, 2 g K₂HPO₄, and 1 mL Tween 80) (Hu et al., 2007), whey liquid (100 mL), and distilled water (900 mL) with pH 6.8. Whey liquid was prepared by adding 1L distilled water to 120 g whey powder and sterilised at 112°C for 30 min. Subsequently, the solution was filtered and the filtrate was preserved.

Following the orthogonal experimental design, nine types of fermentation media with different concentrations of minced shrimp, glucose, and NaCl were prepared. The natural pH was approximately 6.0, which is close to the optimal growth pH for the selected Lactobacillus. To maintain the natural flavour of shrimp, natural pH was used in this study. Except for shrimp, the ingredients were sterilised at 115°C for 30 min using an autoclave (Yamato, Chongqing, China).

Minced shrimp preparation

Briefly after washing and removing the shrimp shell, head, tail and gut, each gram of shrimp tissue was homogenised with 1 mL sterile deionised water. The whole process was performed under hygienic environmental conditions required for food processing.

Seed culture preparation

L. helveticus TS6024 and L. acidophilus 6005 were inoculated in seed medium at an initial concentration of approximately 6.90 log CFU/ mL and 6.74 log CFU/ mL, respectively, and incubated at 37°C for 24 and 17 h, respectively. After incubation, the viable bacterial counts were 8.20 log CFU/mL and 8.04 log CFU/mL, respectively. Subsequently, the cultures were centrifuged, and the precipitate was suspended in sterile normal saline to obtain a 10-fold concentrated seed culture.

Determining lactic acid bacteria concentration

The concentration of lactic acid bacteria was determined during minced shrimp fermentation. At different stages of fermentation, the minced shrimp was stirred well, and 5 g of the sample was collected. After grinding, the sample was put into 95 mL sterile normal saline and shaken for 30 min. Subsequently, 1 mL of the supernatant was diluted 10-fold, and three dilutions were selected. Solidified seed medium was poured into Petri plates, each dilution (10⁻⁴–10⁻² for control, 10⁻⁷–10⁻⁵ for incubated with Lactobacillus) was plated (1 mL), and bacterial colonies were counted after 2 d of incubation at 37°C.

Orthogonal test to optimise fermentation conditions

To investigate the optimal fermentation conditions using L. helveticus TS6024 and L. acidophilus 6005, orthogonal array was employed. L₉ (3⁴) orthogonal test was performed using four factors: shrimp concentration, glucose concentration, inoculum level, and NaCl. In the previous preparation experiments, we found that the growth of L. helveticus TS6024 and L. acidophilus 6005 would be notably inhibited when the concentration of NaCl exceeded 4% (w/v). In the study, sterilisation was not performed before fermentation to maintain the natural texture of shrimp. Considering that a certain concentration of NaCl inhibits the growth of putrid bacteria (Hu et al., 2007) and that the main allergen in shrimp (TM) is salt-soluble, the concentrations of NaCl were set as 1%, 2%, and 3% respectively. The design is shown in Table 1.

Table 1. Orthogonal test to optimise fermentation conditions L₉ (3⁴).

Level	A-glucose (g/100 g)	B-shrimp concentration (g/100 g)	C-inoculum level (mL/100 g)	D-NaCl (g/100 g)
1	1%	20%	2.5%	1%
2	2%	30%	15%	2%
3	3%	40%	25%	3%

Fermentation was conducted at 37°C and 70 r/min for 48 h, and the OD_492nm recorded in ELISA was obtained from each experiment. The significance of each factor was analysed to determine the optimal medium composition and inoculum level that reduce allergenicity of minced shrimp.

Comparative assessment of supernatant and precipitated allergens of fermented shrimp

Comparative assessment of the supernatant and precipitated allergens was performed after fermentation of the minced shrimp. The concentrations of minced shrimp, glucose, and NaCl were 30%, 2%, and 2% (w/w), respectively. L. helveticus TS6024 and L. acidophilus 6005 were inoculated into the minced shrimp at 15% inoculum level (mL/100 g) for fermentation at 37°C and 70 r/min for 24 and 48 h. Then, the fermented minced shrimp was centrifuged at 12,000 × g at 4°C for 15 min to collect the supernatant and precipitate.

Extraction of shrimp protein containing antigen

The supernatant of the fermented ground shrimp was filtered using a filter paper (Whatman Grade 4, Particle retention: 25 μm), and the filtrate was dialysed (3.5 kD) overnight at 4°C against 10 mM PBS (pH 7.0). The dialysed solution was lyophilised and used as the antigen obtained from the supernatant of fermented shrimp.

Antigen extraction from the precipitate of fermented and unfermented shrimp followed a similar method described previously (Zhenxing et al., 2007), with minor modification. Cold acetone (pre-cooled overnight at −20°C) was mixed in the minced shrimp for precipitation of antigenic proteins. After centrifugation at 10,000 × g at 4°C for 15 min, the precipitates were collected. After air drying at 20–28°C, acetone powder was prepared and were extracted using an extraction buffer (1 M KCl and 0.5 mM 1, 4-dithiothreitol, pH 7.0) at a ratio of 1:10 (m/V) overnight with constant stirring. After centrifugation at 12,000 × g for 15 min at 4°C, the supernatant was dialysed (3.5 kD) overnight at 4°C against 10 mM PBS (pH 7.0), lyophilised, and considered as allergen obtained from the precipitate.

Allergen activity assay

Allergen activity was quantified using indirect ELISA described previously (Zhenxing et al., 2007), with minor modification. Microtiter plates were coated with 100 μL/well of antigen extract solution (stock concentration: 10 mg/mL) for 26 h at 4°C. The plates were washed with PBST, and free binding sites were blocked using a blocking buffer for 2 h at 37°C. The blocking buffer was removed, and the plates were rinsed thrice with PBST. Pooled serum (100 μL) from shrimp allergic patients (diluted 1:5) was subsequently added in each well, and the plates were incubated for 2 h at 37°C and then washed thrice with PBST. Then, 100 μL of goat anti-human IgE peroxidase conjugate (1:2000 in blocking buffer) was added into the wells of the microtiter plate, with subsequent incubation of 2 h at 37°C. The plates were washed thrice with PBST. Finally, 100 μL substrate solution was added into each well and placed at 37°C in dark for 15 min, followed by the addition of termination solution (2 M H₂SO₄; 50 μL). Absorbance was measured at 492 nm using ELISA reader (ThermoFisher, Walsham, MA, USA). Unfermented shrimp and normal human serum were used as positive and negative controls, respectively. The reduction rate of sensitisation to minced shrimp was determined using the following equation: $\begin{array}{rl}& \mathrm{Reduction}\mathrm{rate}(\text{\%})\\ & =[(\mathrm{positive}\mathrm{control}\mathrm{OD}-\mathrm{fermentation}\mathrm{sample}\mathrm{OD})/\mathrm{positive}\mathrm{control}\mathrm{OD}]\times 100\end{array}$

Sodium-Dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

Sample solutions (5 mg/mL) were diluted 1:1 in loading buffer (10% 1M Tris-HCl (pH6.8) (v/v), 20% glycerol (v/v), 4% SDS (w/v), 10% β-mercaptoethanol (v/v), and 0.1% bromophenol blue (w/v)) and heated (100 °C, 6 min) prior to gel running. As to SDS-PAGE, a separating gel of 15% acrylamide and a stacking gel of 5% acrylamide were used. The amount of sample loaded per lane was 10 μL for each. Molecular weight standards (10–170 kDa) (Fermentas, Burlington, ON, CNA) were used in the gel. Electrophoresis was performed in a vertical unit (Mini-PROTEAN® Tetra System, BIO-RAD, Philadelphia, PA, USA) with running buffer (50 mM Tris-HCl, 384 mM glycine and 0.1% SDS, pH 8.3). Electrophoresis was conducted at 80 V for stacking gel and 120 V for separating gel. Protein bands were fixed and stained with Coomassie Brilliant Blue (R-250) for 45 min, then destained with a solution containing 100 mL/L methanol and 100 mL/L acetic acid.

Statistical analysis

Data are presented as the mean ± SD. SPSS software (version 25.0) was used to perform t-test and ANOVA test to analyse the experimental results. Statistical significance was determined at P < 0.05. Each experiment was repeated three times.

Results and discussion

Changes in the quality of minced shrimp after fermentation and concentration of lactic acid bacteria during fermentation

The composition of medium and the culture conditions were the same as those of comparative test of supernatant and precipitate allergen of fermented shrimp. Minced shrimp without Lactobacillus inoculation were selected as the control fermentation set-up. Samples were taken every 12 h to count lactic acid bacteria, and the results are given in Table 2.

Table 2. Concentration of lactic acid bacteria in minced shrimp at different fermentation times.

Samples	Lactic acid bacteria concentration/ log CFU/g
	0 h	12 h	24 h	36 h	48 h	60 h	72 h
Control	2.88 ± 0.03*^f	3.42 ± 0.09^e**	4.52 ± 0.04^c	4.82 ± 0.04^a	4.75 ± 0.04^b	3.63 ± 0.06^d	2.92 ± 0.05^f
L. helveticus TS6024	8.37 ± 0.09^c	8.63 ± 0.08^b	8.94 ± 0.04^a	8.98 ± 0.03^a	8.11 ± 0.08^d	7.72 ± 0.07^e	6.87 ± 0.13^f
L. acidophilus 6005	8.23 ± 0.06^b	8.73 ± 0.13^a	8.78 ± 0.11^a	8.20 ± 0.04^b	7.97 ± 0.09^c	6.78 ± 0.12^d	5.71 ± 0.07^e

*Values are presented as the mean ± SD.

**Different superscript letters in the same row show significant difference (P < 0.05).

After fermentation, the control shrimp changed from bluish-white to yellowish-white and was still relatively thick, without layering or giving off a bad smell. On the contrary, the Lactobacillus-inoculated shrimp changed from bluish-white to pinkish-white, close to the colour of cooked shrimp, and from thick to clear, thin, and separated layers. In addition, the typical flavour of minced shrimp was obviously reduced after fermentation, similar to that in minced fish fermentation (Hu et al., 2007), and presented the natural flavour of shrimp.

The concentration of lactic acid bacteria reached a maximum of 8.98 log CFU/g at 36 h and 8.78 log CFU/g at 24 h in the L. helveticus TS6024- and L. acidophilus 6005-inoculated shrimps, respectively. With the prolonged fermentation time, the concentration of lactic acid bacteria in both L. helveticus TS6024-inoculated and L. acidophilus 6005-inoculated shrimps gradually decreased. This may be due to Lactobacillus producing organic acids or other antibacterial substances, and is a widely accepted factor in the bacteriostasis of lactic acid bacteria (Alvarez-Sieiro et al., 2016; Reuben et al., 2020; Soemarie et al., 2021). These results showed that both the strains could grow in the minced shrimp as fermentation substrate, inhibit native bacterial communities associated with shrimp, and thus, become the dominant bacteria in fermented shrimp.

In the study, sterilisation was not performed before fermentation to maintain the natural texture of shrimp. Therefore, both Lactobacillus and native bacteria were present in the shrimp during fermentation. In the preliminary experiments, we found that the number of viable lactic acid bacteria in each gram of fermentation medium should be at least 7.0 log CFU/g after inoculation for the inhibition of native bacterial growth (including spoilage bacteria) in the shrimp to prevent its rotting. This is consistent with the findings of Hu et al. (2007) who used lactic acid bacteria to ferment silver carp muscle and effectively inhibit the growth of Pseudomonas and Enterobacteriaceae in silver carp pulp.

Comparative assessment of supernatant and precipitate allergen of fermented shrimp

1. L. helveticus TS6024 and L. acidophilus 6005 were inoculated into minced shrimp. After fermentation, antigens were extracted from the supernatant and precipitate, and detected using ELISA. The results are listed in Table 3.

Table 3. Comparative assessment of supernatant and precipitate allergens of fermented shrimp.

Samples	L. helveticus TS6024		L. acidophilus 6005
	24 h	48 h	24 h	48 h
Supernatant OD492nm	0.343 ± 0.009*^b,1	0.150 ± 0.007^b,2	0.365 ± 0.008^b,1	0.179 ± 0.013^b,2
Precipitate OD492nm	0.406 ± 0.005^a,1**	0.251 ± 0.012^a,2	0.468 ± 0.009^a,1	0.303 ± 0.011^a,2

Note: Positive control 0.585 ± 0.014; Negative control 0.038 ± 0.001 (interval 0.030–0.050).

*Values are presented as the mean ± SD.

** Different superscript letters in the same column show significant difference (P < 0.05). Different superscript numbers between 24 and 48 h for the same strain in the same row mean significant difference (P < 0.05).

ELISA showed that after 48 h of fermentation, OD_492nm of the precipitate and the supernatant in both L. helveticus TS6024- and L. acidophilus 6005-inoculated shrimp were significantly lower than that of the 24 h (P < 0.05). Therefore, L. helveticus TS6024 and L. acidophilus 6005 can be used as starters for reducing the allergenicity of shrimp. This may be explained by the fact that allergic protein in shrimp is water-soluble or salt-soluble (Leung et al., 2014). With the prolongation of fermentation time, most of the water-soluble and salt-soluble protein gradually dissolved and decomposed in the liquid phase, and then served as nitrogen sources for Lactobacillus. This was also the original intent of adding water with an appropriate amount of salt to the minced shrimp before fermentation. Hu et al. (2007) confirmed that when silver carp sausages were inoculated with combinations of Staphylococcus xylosus-12 and L. plantarum-15, Pediococcus pentosaceus-ATCC33316, or L. casei subsp. casei-1.001, salt-soluble protein showed obvious decomposition with increase in fermentation time, and degradation of water-soluble protein was more apparent than that of salt-soluble protein. After 48 h of fermentation, OD_492nm of the precipitate in both L. helveticus TS6024- and L. acidophilus 6005-inoculated shrimp was still significantly higher than that of supernatant (P < 0.05). Hence, the level of allergen reduction in the fermented minced shrimp was determined by testing OD_492nm of the precipitate in subsequent experiments.

Fermentation of minced shrimp using L. helveticus TS6024

Optimal conditions for L. helveticus TS6024 Fermentation

Fermentation time of 48 h was chosen on the basis of results presented in Table 2 and to maintain the appropriate concentration of lactic acid bacteria during fermentation (to prevent rotting of shrimp). The results are presented in Table 4.

Table 4. Orthogonal test results of L. helveticus TS6024 fermentation.

Test No.	A-glucose (g/100 g)	B-minced shrimp (g/100 g)	C-inoculum level (mL/100 g)	D-NaCl (g/100 g)	Average OD492nm
1	1 (1)	1 (20)	1 (2.5)	1 (1)	0.279 ± 0.007*
2	1	2 (30)	2 (15)	2 (2)	0.227 ± 0.013
3	1	3 (40)	3 (25)	3 (3)	0.174 ± 0.009
4	2 (2)	1	2	3	0.280 ± 0.011
5	2	2	3	1	0.279 ± 0.007
6	2	3	1	2	0.211 ± 0.010
7	3 (3)	1	3	2	0.249 ± 0.006
8	3	2	1	3	0.139 ± 0.011
9	3	3	2	1	0.317 ± 0.008
M1	0.680	0.808	0.629	0.875
M2	0.770	0.645	0.824	0.687
M3	0.705	0.702	0.702	0.593
m1	0.227	0.269	0.210	0.292
m2	0.257	0.215	0.275	0.229
m3	0.235	0.234	0.234	0.198
Sj	0.0204	0.0236	0.0255	0.0327
Optimal level	A1	B2	C1	D3

*Values are presented as the mean ± SD.

Orthogonal test analysis demonstrated that the effect of glucose concentration on the OD_492nm was the lowest among all influencing factors analysed, and NaCl concentration, inoculum level, and minced shrimp concentration were ranked first, second, and third, respectively. Based on the statistical results of the orthogonal test, 1% glucose, 30% minced shrimp, 2.5% inoculum level, and 3% NaCl were identified as the optimum conditions for attaining high efficacy of L. helveticus TS6024 fermentation. Here, 3% NaCl was the optimal concentration, the reason for which may be that the inhibition of miscellaneous bacteria promotes the growth of L. helveticus TS6024. In addition, an appropriate amount of salt promoted the dissolution of allergenic proteins.

L. helveticus ts6024 Fermentation temperature and time

Based on the obtained orthogonal test results (Table 4), a validation and supplementary test was performed. Since the temperature and time of fermentation would directly affect the final OD_492nm, we examined different fermentation temperatures and adjusted the fermentation time appropriately during the tests. The results are shown in Table 5.

Table 5. L. helveticus TS6024 fermentation temperature and time.

TEMP (°C)	Time (h)	Average OD492nm
37	48	0.194 ± 0.005*^b
37	60	0.123 ± 0.009^c**
42	36	0.296 ± 0.012^a
42	48	0.281 ± 0.010^a

*Values are presented as the mean ± SD.

**Different superscript letters in the same column show significant difference (P < 0.05).

Statistical results showed that at 37°C, the OD_492nm significantly decreased with prolonged fermentation time (P < 0.05), whereas at 42°C, the decrease in OD_492nm was not significant (P > 0.05). In the medium supplemented with glucose and NaCl, the optimum fermentation temperature of L. helveticus TS6024 was 37°C. After 60 h of fermentation, the OD_492nm significantly decreased from 0.585–0.123, and the allergenicity was reduced by 78.97%. These results indicated that L. helveticus TS6024 could significantly reduce shrimp allergens and may be used to develop low-risk or risk-free products for consumption by people diagnosed with shrimp allergy. Many studies have shown that L. helveticus has high proteolytic activity due to the presence of CEPs (Skrzypczak et al., 2018; Raveschot et al., 2020). In the studies by Meiyan and Qingsen (Meiyan & Qingsen, 2009), L. helveticus TS6024 was used to produce angiotensin converting enzyme inhibitory peptide, and was proven to have high CEP activity. This may be one of the factors that aid L. helveticus TS6024 in reducing shrimp allergenicity.

Fermentation of minced shrimp using L. acidophilus 6005

Optimal conditions for L. acidophilus 6005 fermentation

Fermentation time of 48 h was chosen on the basis of results presented in Table 2 and to maintain appropriate concentration of lactic acid bacteria during fermentation (to prevent rotting of shrimp). The results are presented in Table 6.

Table 6. Orthogonal test results of L. acidophilus 6005 fermentation.

Test No.	A-glucose (g/100 g)	B-minced shrimp (g/100 g)	C-inoculum level (mL/100 g)	D- NaCl (g/100 g)	Average OD492nm
1	1 (1)	1 (20)	1 (2.5)	1 (1)	0.302 ± 0.012*
2	1	2 (30)	2 (15)	2 (2)	0.326 ± 0.010
3	1	3 (40)	3 (25)	3 (3)	0.319 ± 0.008
4	2 (2)	1	2	3	0.277 ± 0.005
5	2	2	3	1	0.330 ± 0.015
6	2	3	1	2	0.306 ± 0.007
7	3 (3)	1	3	2	0.318 ± 0.011
8	3	2	1	3	0.434 ± 0.012
9	3	3	2	1	0.344 ± 0.013
M1	0.947	0.897	1.042	0.976
M2	0.913	1.090	0.947	0.950
M3	1.096	0.969	0.967	1.030
m1	0.316	0.299	0.347	0.325
m2	0.304	0.363	0.316	0.317
m3	0.365	0.323	0.322	0.343
Sj	0.00632	0.00634	0.00167	0.00111
Optimal level	A2	B1	C2	D2

*Values are presented as the mean ± SD.

Orthogonal test analysis demonstrated that the effect of NaCl on the OD_492nm was the lowest among all influencing factors, while minced shrimp concentration, glucose concentration, and inoculum level were ranked first, second, and third, respectively. Based on the statistical results of the orthogonal test, 2% glucose, 20% minced shrimp, 15% inoculum level, and 2% NaCl were identified as the optimum conditions for achieving high efficacy of L. acidophilus 6005 fermentation.

L. acidophilus 6005 fermentation temperature and time

Based on the orthogonal test results (Table 6), a validation and supplementary test was performed. Different fermentation temperatures and time were examined during the tests. The results are presented in Table 7.

Table 7. L. acidophilus 6005 fermentation temperature and time.

TEMP (°C)	Time (h)	Average OD492nm
37	48	0.274 ± 0.011*^a
37	60	0.210 ± 0.009^b**
42	36	0.211 ± 0.010^b
42	48	0.175 ± 0.006^c

*Values are presented as the mean ± SD.

**Different superscript letters in the same column show significant difference (P < 0.05).

The statistical results revealed that at 37°C and 42°C, the OD_492nm significantly decreased with prolonged fermentation time (P < 0.05). These results suggest that the optimum temperature for L. acidophilus 6005 fermentation was 42°C. After 48 h of fermentation, the OD_492nm significantly decreased from 0.585 to 0.175, and the allergenicity was reduced by 70.09%. These results, in addition to those obtained previously from L. helveticus TS6024, suggest that a reduction in allergenicity may be due to the ability of Lactobacillus to produce CEPs and peptidases (Raveschot et al., 2020; Skrzypczak et al., 2018; Zhong et al., 2021).

In recent years, many studies have shown that lactic acid bacteria can hydrolyse muscle protein. Fadda et al. (2002) studied the proteolytic activity of a starter culture involving L. plantarum and L. casei on meat sarcoplasmic and myofibrillar proteins during the fermentation of a sausage-like system. After 96 h of incubation, the proteolytic system of L. plantarum CRL681 caused a degradation of both the sarcoplasmic and myofibrillar proteins. Wang et al. (2017) found that proteolysis of fish sarcoplasmic proteins by L. plantarum 120, which is isolated from Suanyu (a traditional Chinese low salt fermented fish), occurred during fermentation, resulting in an increase in peptides and free amino acids.

However, in fermented meat products, pronounced hydrolysis of muscle proteins requires enzymatic activity of endogenous cathepsin in addition to those of the microorganism. In meat products, such as dry sausages (Fadda et al., 1999), proteolysis results from the combined action of enzymes of both endogenous and microbial origins. Therefore, the initial degradation of myosin and actin into peptides was accomplished by cathepsin D, whereas the later decomposition of peptides into free amino acids was performed by the bacteria. Hughes et al. (2002) confirmed that the initial degradation of sarcoplasmic proteins was due to the activity of endogenous proteinases, while both endogenous and bacterial enzymes from Staphylococcus carnosus MC 1 contributed to the initial degradation of myofibrillar proteins in fermented sausages. Furthermore, endogenous enzymes were responsible for the release of trichloroacetic acid-soluble peptides, which were further hydrolysed by bacterial enzymes.

Therefore, it can be preliminarily inferred that shrimp proteins can be hydrolysed by enzymes from endogenous as well as microbial sources in this study. As confirmed by previous studies (Azcarate-Peril et al., 2005, 2009; Rodríguez-Serrano et al., 2018), small molecular peptide chains are transported to cells as nitrogen sources by oligopeptide, tripeptide, and dipeptide transport systems, and further hydrolysed into smaller peptides or amino acids by intracellular peptidases to exert physiological functions. This can be validated in future experiments.

SDS-PAGE analysis on the degradation effects of L. helveticus TS6024- and L. acidophilus 6005-fermentation on TM molecules

The optimal fermentation results of L. helveticus TS6024 and L. acidophilus 6005 were analysed using SDS-PAGE. As shown in Figure 1, after fermentation by the selected Lactobacillus species, the band intensity of various proteins weakened to varying degrees between 10 and 170 KDa in both the supernatant and precipitate, among which 36 KDa (TM) weakened more evidently. This preliminary analysis indicated that fermentation by the selected Lactobacillus species had the effect of protein degradation. Previous research (Ruethers et al., 2018) has confirmed that Lit v 1, a 36 kDa TM, is the major potent allergen in Litopenaeus vannamei, and its reduction is one of the main factors that results in the decrease in shrimp allergenicity. These, combined with the results of ELISA, show that the selected Lactobacillus species can effectively reduce the allergenicity of shrimp.

Figure 1. SDS-PAGE analysis on the degradation effects of L. helveticus TS6024- and L. acidophilus 6005-fermentation on TM molecules. M, marker; lane 1, protein extracts from unfermented shrimp; lane 2, protein extracts from the precipitate of L. acidophilus 6005-fermentation, OD_492nm: 0.175; lane 3, protein extracts from the supernatant of L. acidophilus 6005-fermentation, OD_492nm: 0.145; lane 4, protein extracts from the precipitate of L. helveticus TS6024-fermentation, OD_492nm: 0.123; lane 5, protein extracts from the supernatant of L. helveticus TS6024-fermentation, OD_492nm: 0.107.

Conclusions

In this study, the optimal fermentation conditions for minced shrimp using Lactobacillus strains were determined. Results showed that the optimal fermentation reduced the antigen concentration by 78.97% and 70.09% in L. helveticus TS6024 and L. acidophilus 6005 fermentation, respectively. Our results revealed that fermentation using Lactobacillus, especially L. helveticus TS6024, effectively reduced the allergenic potential of Litopenaeus vannamei. However, this is still not enough for patients with shrimp allergy. In this study, we found that the key to successful fermentation may be the maintenance of high lactic acid bacteria concentration for the prolonged duration in the fermentation process. However, from a health perspective, only glucose and NaCl were added to the minced shrimp, resulting in weaker growth of Lactobacillus as well as a consequently diminished reduction in the allergens in minced shrimp. In future studies, fermentation medium could be supplemented with additional nutrients to promote the growth of Lactobacillus, which might be more effective in reducing the allergens in minced shrimp. Overall, this study presented a great value for the processing of the hypoallergenic shrimp/shrimp products, which will provide optimal nutrition with a reduced risk of adverse reactions in hypersensitive individuals.

Author contribution

Conceptualisation: HL, ZH and YY; formal analysis: HL; methodology: HL, ZH and YY; data curation: HL and ZH; original draft preparation: HL; review and editing: HL, ZH and YY.

Acknowledgements

The authors acknowledge Professor Qingsen Chen (School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin, China) for his advice regarding development of the fermentation process and for providing the Lactobacillus strains.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data can be made available upon reasonable request.

Additional information

Funding

This work was supported by a project funded by National Natural Science Foundation of China (project number 31271841).