**5. Effects of soy protein inclusion on gut microbiota**

Healthy gut microbiota is essential to promote host health and well-being. Before the 1970s, there were some controversies regarding the existence and role of an indigenous microbiota in fish. However, it is now well established that fish and other aquatic animals have a microbiota in the GI tract (for review, see; [21, 23, 82–92]). The intestinal microbiota of fish, as is the case of mammals, is classified as autochthonous (indigenous) or allochthonous bacteria [90, 93]. The autochthonous bacteria are those able to colonize the host's gut epithelial surface or are associated with the microvilli, while the allochthonous bacteria are incidental visitors in the GI tract and are expelled after some time without colonizing [90, 93]. Several factors affect the gut microbiota in fish including host factors, environmental factors, microbial factors, etc. However, until recently, among different influencing factors affecting the fish microbiota, water and diet (environmental factors) have been studied extensively [49]. In this section, we address the effect of dietary soybean products on intestinal bacterial community of finfish and crustaceans (**Table 1**).

#### **5.1. In salmonids**

Research conducted until recently on SBM inclusion effects on gut microbiota of fish indicated that SBM modulated the intestinal microbiota toward developing an undesirable microbial community that can induce mucosal inflammation [110, 111]. Heikkinen et al. [94] reported that rainbow trout fish fed FM- and SBM-based diets for 4 weeks showed decreased number


**Species/initial weight**

**Soy protein type and feeding duration**

SBM (246 g/kg) for 84 days

SBM (246 g/kg) for 84 days

kg) for 84 days

for 9 weeks

for 8 weeks

for 8 weeks

SBM (40 g/kg) for 8 weeks

SBM for 8 weeks

FM replaced with graded level of SBM and fed for 63 days

SBM (300 g/kg) for 8 weeks

~534 g BPSBM (214 g/

~24 g SBM (313 g/kg)

given) SBM (13 g/kg)

(15 g) SBM (355 g/kg)

*Ctenopharyngodon idella* (weight not

*Carassius auratus* ♀ × *Cyprinus carpio* ♂ (24.7 g ± 0.4 g)

*Carassius auratus*

Three cyprinid species

*Oreochromis niloticus* ♀ × *Oreochromis aureus*

♂ (~2 g)

Northern snakehead and HC

and *Psychrobacter*

**Effects on gut microbiota References**

↑ species richness, Shannon- Weaver index Dimitroglou et al. [104]

→ on gut microbiota determined by DGGE Raggi and Gatlin III [107]

↑ *Plesiomonas* sp. BTOK4 *Aeromonas aquarium* Zhang et al. [109]

Refstie et al. [47]

101

The Potential Impacts of Soy Protein on Fish Gut Health http://dx.doi.org/10.5772/intechopen.92695

Ringø et al., [22]

Ringø et al. [22]a

Huang [105]

Cai et al. [106]

Li et al. [108]

Miao et al. [80]

↑ allochthonous bacterial level in FG, HG → allochthonous bacterial level in HC ↓ autochthonous bacterial level in FG, HG

Modulated gut microbiota. ↑ *Chryseobacterium*

↑ *Pseudomonas putida* Aeromonas sp. DH69

→ total culturable aerobic and anaerobic bacteria, presumptive *E. coli*, *Aeromonas*, Bifidobacterium,

Modulation of the allochthonous gut microbiota. ↓ Proteobacterium clone (EF707282.1), *Cetobacterium somerae* (AB353124), *Bacillus subtilis*, *Anoxybacillus* 

At the phylum level, ↓ *Firmicutes* abundance was the lowest in the diet group having 75% defatted fishmeal replacement with SBM, ↑In contrast with *Proteobacteria, Bacteroidetes* and

↓At the genus level, significantly lower abundance of Lactococcus, *Geobacillus*, *Pseudomonas*, *Streptococcus*, *Bacillus* and *Acinetobacter* in diet group (75% defatted fishmeal replacement with

↑ but higher abundance of *Cetobacterium*, *Planctomyces*, *Shewanella*, *Thermomonas*, *Rubrivivax* and *Carnobacterium* was observed in fish fed the same diet group (75% defatted fishmeal

*Actinobacterium bacilli* bacterium

*Clostridium perfringens*

*flavithermus*

*Planctomycetes*

replacement with SBM)

SBM)

**Table 1.** Effects of soy protein inclusion on gut microbiota of fish.

→ population levels of adherent and allochthonous bacteria in FG, HG and HC Modulated gut microbiota. ↑ *Psychrobacter*


**Table 1.** Effects of soy protein inclusion on gut microbiota of fish.

of cultivable intestinal bacteria (aerobic and anerobic). Afterward, by the 8 weeks of feeding trial, the bacterial numbers increased in the FM group, but not in the SBM group. Length heterogeneity analysis of PCR amplified 16S rDNA (LH-PCR) data also suggested a diet-related qualitative change in the intestinal microbiota of fish. The dominant identified genera were among aerobic species *Aeromonas*, *Sphingomonas*, and *Chryseomonas* and among the lactic acid bacteria, the genera *Lactococcus* and *Lactobacillus*. Rainbow trout fed SBM (450 g/kg) for 16 weeks showed decrease in total culturable species of *Aeromonas* spp., *Vibrio* spp., but the species *Actinomycetales*, *Psychrobacter* spp., *Saccharomyces* spp. were found as increased number. Total culturable aerobic levels, *Micrococcus* spp., were found unchanged in numbers. Mansfield et al. [96] evaluated the effect of FM and SBM (300 g/kg) on the allochthonous distal intestinal microbiota of triploid female rainbow trout by three cpn60 universal clone libraries, resulting in 1000 and 1181 sequences from FM and SBM, respectively. There were total 32 different sequences were noticed. The most frequently observed sequences were identical to *Carnobacterium (piscicola) maltaromaticum* and accounted for 55 and 97.2% of the clones from the FM and SBM group, respectively. Overall, fish fed FM showed highest diversity (14 different sequences) and only four different sequences observed in the SBM library. In another study, Desai et al. [97] observed that 30% SBM inclusion in rainbow trout diets led to a reduction in *Proteobacteria* and increase in *Firmicutes*. Recently, Bruce et al. [29] evaluated different processed soybean products as a replacement of fishmeal on gut microiota of rainbow trout and observed that the incorporation of processed soy-based proteins alters the microbial community composition within the distal intestine. Species diversity based on abundance and evenness were lowest in the defatted soybean meal group and were significantly less than the bioprocessed soybean meal in low concentration (p = 0.003) and commercial soy protein concentrate (p = 0.003) treatments.

*Streptococcaceae* in PI, and Bacilli-like and *Streptococcaceae* in DI by SPC feeding. In contrast, a

The Potential Impacts of Soy Protein on Fish Gut Health http://dx.doi.org/10.5772/intechopen.92695

Most of the literature available on the effects of different soy products on the gut microbiota are on salmonid fish, and less is known for other species. The possible reasons behind this might be due to the less susceptibility of non-salmonid fish to SBMIE and histological damage [39]. In cyprinid fish, like in grass carp, the effects of dietary SBM inclusion (1.3% by dry weight) were compared with the inclusion of casein meal (CM; 1.0% by dry weight) on the autochthonous gut microbiota [105]. After 8 weeks of feeding, 16S rRNA PCR-DGGE analysis revealed a clear difference between the microbiota of the SBM group and the CM group with similarity between the groups of only 26% (p < 0.05). Unique bacteria isolated from the CM group were identified as follows: uncultured *Lachnospiraceae bacterium*, uncultured *Lactobacillus*, uncultured *Clostridium* spp., and uncultured *Proteobacterium*, while bacteria isolated from the SBM group were identified as *Pseudomonas* sp., *Aeromonas* sp., uncultured

Raggi and Gatlin [107] evaluated four probiotics diets based on FM and SBM on gut microbiota of goldfish (*Carassius auratus*). After 8 weeks of feeding, denaturing gradient gel electrophoresis (DGGE) analysis results revealed no difference in gut microbiota. The probable reason explained

have reduced the quantity and complexity of the bacterial community as reported by Ringø [112] for Arctic charr (*Salvelinus alpinus* L.). Cai et al. [106] also reported no significant effects of fishmeal replacement by SBM (30%) on the levels of total aerobic bacteria, total anaerobic bacteria, presumptive *E. coli*, *Aeromonas*, *Bifidobacterium*, or *Clostridium* in the intestine of silver crucian carp (*Carassius auratus gibelio × Cyprinus carpio*). Recently, the effect of partial replacement of SBM (4%) by intestinal casing meal (ICM), prepared from the wastewater of enteric coating and heparin processing, was used to evaluate the effect on the allochthonous gut microbiota of three cage-cultured cyprinid species [108]. Results indicated that the allochthonous bacterial diversity was altered by ICM substitution; however, by feeding ICM, some bacterial species were significantly stimulated, *E. coli*, and *Exiguobacterium* in black carp (*Mylopharyngodon piceus*) and species

) which may

103

for this observation is due to the incorporation of dietary chromic oxide (10 g kg−<sup>1</sup>

belonging to *Firmicutes*, Fusobacteria, and *Proteobacteria* in gibel carp (*Carassius gebelio*).

The effects of replacing dietary SBM or cottonseed meal (CSM) by completely hydrolyzed feather meal (CHFM) on the composition of gut microbiota was investigated by Zhang et al. [109] for hybrid tilapia. After 8 weeks of feeding, 16S rRNA PCR-DGGE analysis results revealed that CHFM induced modulation of the whole intestinal microbiota in hybrid tilapia and prevented colonization of potentially harmful species in the intestinal tract. *Plesiomonas* sp. BTOK4 and *Aeromonas aquarium* were found in decreased level in diet group where

of dietary SBM on the intestinal microbial community of northern snakehead. After 63-day feeding, trial results indicated that dietary soybean meal substitutions significantly affected

CSM was replaced with CHFM. Miao et al. [80] reported the substitution effects

significant (p = 0.05) decrease was revealed in *Vibrionaceae* in PI.

bacteria, uncultured *Actinobacterium*, and uncultured *Bacillus* spp.

**5.2. In cyprinid fish**

**5.3. Cichlids and others**

120 g kg−<sup>1</sup>

In Atlantic salmon, fish fed the SBM (250 g/kg) diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine observed by Bakke-McKellep et al. [98]. Green et al. [100] investigated the influence of FM and soybean protein concentrate (SPC; 50 g/kg) on intestinal microbiota of Atlantic salmon. Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA clone library analysis revealed that the SPC diet modulated the intestinal microbiome by increasing the bacterial diversity, and a *Pseudomonadales* was more frequently revealed species. In addition, increased *Escherichia coli* also observed in SPC-based diet, but it was absent in FM-based diet. In another study, Navarrete et al. [101] reported SBM supplementation (378 g/kg) effects on distal intestine microbial community of Atlantic salmon. Principal component analysis (PCA) revealed correlations that fish fed SBM diet was correlated with *Aeromonas* VIb and *Sporosarcina aquimarina*, while *Microbacterium*, *Pseudomonas*, *Lactococcus lactis sp*. cremoris, and *Aeromonas* VIa were correlated with the FM-based diet. Reveco et al. [103] investigated the microbiota in the mid and distal intestine of Atlantic salmon fed FM and solvent extracted SBM (200 g/kg) by DGGE analysis. Results showed increased *Lactococcus lactis* subsp. lactis in the mid-intestine, while a reduction in *Weissella confusa* in the distal intestine of Atlantic salmon fed 20% solvent extracted SBM-contained diet. Hartviksen et al. [102] revealed no dietary effect of soy protein concentrate (SPC) on total autochthonous bacteria isolated from PI and total allochthonous and total autochthonous bacteria isolated from DI of Atlantic salmon by qPCR analysis. However, significant (p = 0.05) effect was observed regarding community composition. An increase was noticed in autochthonous *Enterobacteriaceae*, Bacilli-like, *Lactobacillaceae*, and *Streptococcaceae* in PI, and Bacilli-like and *Streptococcaceae* in DI by SPC feeding. In contrast, a significant (p = 0.05) decrease was revealed in *Vibrionaceae* in PI.

#### **5.2. In cyprinid fish**

Most of the literature available on the effects of different soy products on the gut microbiota are on salmonid fish, and less is known for other species. The possible reasons behind this might be due to the less susceptibility of non-salmonid fish to SBMIE and histological damage [39]. In cyprinid fish, like in grass carp, the effects of dietary SBM inclusion (1.3% by dry weight) were compared with the inclusion of casein meal (CM; 1.0% by dry weight) on the autochthonous gut microbiota [105]. After 8 weeks of feeding, 16S rRNA PCR-DGGE analysis revealed a clear difference between the microbiota of the SBM group and the CM group with similarity between the groups of only 26% (p < 0.05). Unique bacteria isolated from the CM group were identified as follows: uncultured *Lachnospiraceae bacterium*, uncultured *Lactobacillus*, uncultured *Clostridium* spp., and uncultured *Proteobacterium*, while bacteria isolated from the SBM group were identified as *Pseudomonas* sp., *Aeromonas* sp., uncultured bacteria, uncultured *Actinobacterium*, and uncultured *Bacillus* spp.

Raggi and Gatlin [107] evaluated four probiotics diets based on FM and SBM on gut microbiota of goldfish (*Carassius auratus*). After 8 weeks of feeding, denaturing gradient gel electrophoresis (DGGE) analysis results revealed no difference in gut microbiota. The probable reason explained for this observation is due to the incorporation of dietary chromic oxide (10 g kg−<sup>1</sup> ) which may have reduced the quantity and complexity of the bacterial community as reported by Ringø [112] for Arctic charr (*Salvelinus alpinus* L.). Cai et al. [106] also reported no significant effects of fishmeal replacement by SBM (30%) on the levels of total aerobic bacteria, total anaerobic bacteria, presumptive *E. coli*, *Aeromonas*, *Bifidobacterium*, or *Clostridium* in the intestine of silver crucian carp (*Carassius auratus gibelio × Cyprinus carpio*). Recently, the effect of partial replacement of SBM (4%) by intestinal casing meal (ICM), prepared from the wastewater of enteric coating and heparin processing, was used to evaluate the effect on the allochthonous gut microbiota of three cage-cultured cyprinid species [108]. Results indicated that the allochthonous bacterial diversity was altered by ICM substitution; however, by feeding ICM, some bacterial species were significantly stimulated, *E. coli*, and *Exiguobacterium* in black carp (*Mylopharyngodon piceus*) and species belonging to *Firmicutes*, Fusobacteria, and *Proteobacteria* in gibel carp (*Carassius gebelio*).

#### **5.3. Cichlids and others**

The effects of replacing dietary SBM or cottonseed meal (CSM) by completely hydrolyzed feather meal (CHFM) on the composition of gut microbiota was investigated by Zhang et al. [109] for hybrid tilapia. After 8 weeks of feeding, 16S rRNA PCR-DGGE analysis results revealed that CHFM induced modulation of the whole intestinal microbiota in hybrid tilapia and prevented colonization of potentially harmful species in the intestinal tract. *Plesiomonas* sp. BTOK4 and *Aeromonas aquarium* were found in decreased level in diet group where 120 g kg−<sup>1</sup> CSM was replaced with CHFM. Miao et al. [80] reported the substitution effects of dietary SBM on the intestinal microbial community of northern snakehead. After 63-day feeding, trial results indicated that dietary soybean meal substitutions significantly affected the intestinal microbiota composition of fish. At the phylum level, *Firmicutes* abundance was the lowest in the diet group having 75% defatted fishmeal replacement with SBM, in contrast with *Proteobacteria*, *Bacteroidetes*, and *Planctomycetes*. At the genus level, significantly lower abundance of *Lactococcus*, *Geobacillus*, *Pseudomonas*, *Streptococcus*, *Bacillus*, and *Acinetobacter*, but higher abundance of *Cetobacterium*, *Planctomyces*, *Shewanella*, *Thermomonas*, *Rubrivivax*, and *Carnobacterium* was observed in fish fed the same diet group (75% defatted fishmeal replacement with SBM).

**Acknowledgements**

**Conflict of interest**

**Author details**

Bangladesh

**References**

of Idaho, Moscow, ID, USA

The authors declare no conflict of interest.

Vikas Kumar1,2\*, Md. Sakhawat Hossain2,3, Janice A. Ragaza<sup>4</sup>

\*Address all correspondence to: vikaskumar@uidaho.edu

We gratefully thank Dr. Nicholas Romano (University of Arkansas at Pine Bluff, Pine Bluff, AR, USA) for his help to make figure 3. The authors wish to acknowledge Ajinomoto Co. Inc. (Kanagawa, Japan) for their technical support to make histological slides for Amberjack.

1 Department of Animal and Veterinary Science, Aquaculture Research Institute, University

2 Hagerman Fish Culture Experiment Station, University of Idaho, Hagerman, ID, USA

4 Department of Biology, Ateneo de Manila University, Quezon City, Philippines

Trout Fed Soy Protein Based Diets. Unpublished; 2020a

Atlantic salmon (*Salmo salar*). Aquaculture. 2000;**190**:49-63

Nutrition. 2019;**25**(4):917-931

3 Department of Aquaculture, Faculty of Fisheries, Sylhet Agricultural University, Sylhet,

[1] Kumar V, Bledsoe J, Lee S, Romano N, Small BC, Lalgudi R, et al. Gut Microbiota Homoeostasis Maintains Via Changing the Distal Intestinal Morphology in Rainbow

[2] Kumar V, Lee S, Cleveland B, Romano N, Lalgudi R, Rubio M, et al. Comparative evaluation of processed soybean meal (EnzoMealTM) vs. regular soybean meal as a fishmeal replacement in diets of rainbow trout (*Oncorhynchus mykiss*): Effects on growth performance and growth-related genes. Aquaculture. 2020b:**516**. DOI: 10.1016/j.aquaculture.2019.734652 [3] Kumar V, Wang H-P, Lalgudi R, Cain R, McGraw B, Rosentrater KA. Processed soybean meal as an alternative protein source for yellow perch (*Perca flavescens*) feed. Aquaculture

[4] Refstie S, Korsøen ØJ, Storebakken T, Baeverfjord G, Lein I, Roem AJ. Differing nutritional responses to dietary soybean meal in rainbow trout (*Oncorhynchus mykiss*) and

and Marina Rubio Benito1,2

The Potential Impacts of Soy Protein on Fish Gut Health http://dx.doi.org/10.5772/intechopen.92695 105

From previous research, it is established that gut microbiota influences several physiological and immunological aspects of aquatic animals like development, digestion, nutrition, immunological functions, and disease resistance [113, 114]. The gut microbiota together with digestive enzymes, mucins, peristalsis, and epithelial barrier with tight junctions belongs to the so-called non-immune component of mucosal immunity [115]. Moreover, several previous research findings indicated that intestinal microbiota is required for full immune maturation [116, 117], inflammatory diseases [117, 118], and to increase the host's resistance toward pathogenic invasion and infection [119]. However, until recently, research relating on the effects of soy protein inclusion in fish feed and their interaction among gut microbiota and immune responses is scarce. So, further research on the interaction effect on gut microbiota and innate immune system due to soy protein utilization are required for further confirmation of the usability of SBM.
