Nutritive Values of Protein Hydrolysate Products from Shrimp Waste Utilization by Fermentation with Lactobacillus plantarum

doi:10.21203/rs.3.rs-2307507/v1

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Nutritive Values of Protein Hydrolysate Products from Shrimp Waste Utilization by Fermentation with Lactobacillus plantarum

https://doi.org/10.21203/rs.3.rs-2307507/v1

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Shrimp hydrolysate proteins are produced from inedible parts of shrimp, such as the head, shell and tail, called GSW (ground shrimp waste). By fermentations with a proteolytic Lactobacillus plantarum strain 541, the inoculants were cultivated with commercial MRS (De Man, Rogosa and Sharpe) broth and pineapple juice broth (PA). There were four fermentation treatments: raw GSW fermented with inborn microorganisms, sterilized GSW fermented with MRS inoculant, raw GSW fermented with MRS inoculant, and raw GSW fermented with PA inoculant. The protein hydrolysate products of those treatments were compared with those of a nonfermented GSW and a commercial fishmeal, CFM. The changes in pH and viable cell number density concentrations (VSNC) or viable cell counts (CFU/g) were studied during fermentations at 0, 4, 8, 12, and 16 h. The pH values were controlled to 5.0–5.5 within the first 4 h. The hydrolysate protein products contained 48.6–52.5% (w/w) proteins, 5.4–8.0% (w/w) total lipids, 0.4–0.7% (w/w) fiber, and 12.0–13.5% (w/w) ash. The protein content in raw GSW fermented with MRS inoculant was the highest (p ≤ 0.05), close to the values of 55.6 and 63% (w/w) in nonfermented GSW and CFM. Moreover, the sixteen types of total amino acids of those hydrolysate protein products, nonfermented GSW and CFM presented high values. For this reason, the shrimp hydrolysate protein products here contain large amounts of amino acids, similar to those of the high-quantity protein source CFM. Thus, protein products from the hydrolysis of GSW by fermentation can replace CFM for animal feed protein sources. The in vitro results also showed that the shrimp hydrolysate proteins contained highly digestible proteins. They also presented varying molecular weights of 26–77 kDa of short peptides on SDS-PAGE.

Nutrition & Dietetics

Applied & Industrial Microbiology

Environmental Economics

shrimp waste

protein hydrolysates

Lactobacillus plantarum

lactic bacteria

fermented

nutritive values

nutrients

amino acids

Crustaceans such as shrimps, crabs, and lobsters are rich in amino acids, peptides, proteins and other biomolecules that can be recovered for utilization as ingredients in various food applications [1]. Shrimp have been used as one of the most popular and important raw materials for many countries around the world. In Mexico, it has been reported that shrimp production is edible in only 55% of animals, and the rest are inedible cephalothorax and exoskeletons; for example, those of the genus Penaeus are approximately 40,300 tons [2]. The head, shell and tail portions of shrimp were removed during processing, accounting for approximately 50% of the catch [3].

Many methods have been used to hydrolyze or extract the remaining proteins from shrimp waste, such as using acid hydrolysis, digesting with commercial enzymes, and with some microorganisms to obtain protein hydrolysate products. It has been reported that enzymatic hydrolysis of proteins frequently results in a bitter taste due to the formation of low molecular weight peptides composed of mainly hydrophobic amino acids. Bio-based methods include further hydrolysis of bitter peptides with enzymes such as aminopeptidase, alkaline/neutral proteases, and carboxypeptidase, condensation reactions of bitter peptides using proteases, and use of Lactobacillus as a debittering starter adjunct [4]. Shrimp flavor powder contains 17 types of amino acids, consisting of 9 essential amino acids and 8 nonessential amino acids. The highest essential amino acid is leucine, which is equal to 0.362% (w/w), and the highest nonessential amino acid is glutamate acid, which is equal to 0.913% (w/w) [5]. Generally, shrimp waste hydrolysate has a high content of essential amino acids, indicating a high nutritional value used for food, feed or as a nitrogen source in growth media for microorganisms [6].

Lactic acid fermentation of shrimp waste has been reported as an effective and economical technique to protect decomposition and fermented waste from silage containing a protein-rich liquor, a lipid fraction and insoluble chitin [7–11]. Some fermentations of shrimp waste to produce hydrolysate products have been reported. Shrimp waste protein (120.56 mg protein/g) was produced from a two-state solid culture using a combination of Lactobacillus brevis and Rhizopus oligosporus [12]. Shrimp waste fermentations to recover proteins were performed using the bacteria Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus [13]. Shrimp head wastes were treated to produce animal proteins by autolysis and fermentation with Bacillus licheniformis [14].

Plant families, such as papain, have the most cysteine peptidase, including those of Bromeliaceae, the botanical family of pineapple [15]. In recent years, a number of proteases from species belonging to Bromeliaceae have been isolated and characterized: stem and fruit bromealain, ananain and comosain, obtained from Ananas comosus [16–19], as well as protease from fruits of Bromelia penguin [20], B. balansae [21], and B. hieronymi [22].

The most important animal protein sources in aquatic animal feed industries are fishmeal, high efficiency and quality of proteins, such as digestibility efficiency and balance of amino acids more than the plant proteins, so that animals can most efficiently utilize them. In addition, animal proteins have a particular preferable smell for attraction. As a result, fishmeal is increasing its value [23]. The search for alternative protein sources with high nutritional quality at a reasonable cost is a current concern among farmers. Locally available feedstuffs, such as fish or plant seed meals, are normally used for the formulation of low-cost feed. Fish meal naturally contains a well-balanced mixture of essential amino acids and other nutrients that are readily digested [24]. For this reason, this study researched shrimp hydrolysate protein products, which can be used as alternative protein sources from ground shrimp waste.

To study and develop new sources of proteins, the materials are used for replacement and/or reduction of the amounts of fishmeal for environmental reasons, decreasing the quantity of caught fish to produce fishmeal. Thus, the utilization of shrimp waste, for instance, the protein extracts from shrimp heads, can be used as a protein source for more nutritive supplements and replacement of fishmeal in aquatic animal feed. The proteins from shrimp heads have two types: powder proteins and concentrated aqueous proteins. Moreover, they can be utilized as food ingredients and flavors or sweet meals for more nutrients.

Shrimps from farm culture have been produced in the highest quantity since 1991 in Thailand and have been increasingly produced. In 2019, approximately 290,000 tons (8.53%) of shrimp were produced worldwide, accounting for 3,400,000 tons. These quantities are expected to increase by 20% each year [25]. Recently, with the rapid growth of the fast-food industry, the consumption of shrimp has increased. Consequently, the production of inedible parts of shrimp, such as heads, shells, and tails, is increasing by approximately 290 tons/day from 130 shrimp processing and frozen factories in Thailand. As a result, TISTR (Thailand Institute of Scientific and Technological Research) intends to make use of these by-products as much valuable as possible by researching new methods and technologies for protein hydrolysates from shrimp wastes [26]. The objectives were to (i) assess and evaluate the quality of shrimp waste hydrolysate protein products from four fermentation methods. The shrimp waste was inoculated with Lactobacillus plantarum strain 541 produced by commercial MRS broth and compared with those produced by pineapple juice. (ii) to compare the nutritive values of shrimp waste proteins using proteolytic enzymes from Lactobacillus plantarum strain 541 with commercial fishmeal proteins.

The lactic acid bacterium Lactobacillus plantarum strain 541 can be used for digestion of residual proteins from shrimp waste to produce protein hydrolysates. This will be proven through the in vitro method by using commercial enzymes, trypsin, chymotrypsin, and bacterial proteases (Streptomyces griseus). L. plantarum strain 541 can grow in acidic conditions at low pH. Pineapple also has acidic juice, which can be used to replace the commercial MRS medium for the production of the L. plantarum strain 541 inoculant. The nutritive values of the protein hydrolysates from shrimp waste will be analyzed and then compared with the nonhydrolyzed shrimp waste and with the commercial fishmeal protein. The protein hydrolysates promise to have higher nutritive values than those of shrimp waste without hydrolysis. The protein hydrolysates produced from shrimp waste increase the nutritive supplement values for animal feed. In addition, they can be used for the replacement of fishmeal. This can efficiently be developed to the industrial production scale. Moreover, for environmental conservation, we expect that it is important to reduce the large quantities of caught fish to produce fishmeal.

Scopes of the Study: (i) The shrimp wastes were obtained from the seafood frozen factory in Chon Buri Province, Thailand. The fresh ground shrimp wastes (GSW) were split into four treatments and with three replicates in each for further fermentations as described in treatments 1–4 as follows: Treatment 1: the raw GSW fermented with inborn microorganisms (without the addition of L. plantarum strain 541 starter seed, used as the Control). Treatment 2: Sterilized GSW was fermented with L. plantarum strain 541 inoculant from cultivation in sterilized MRS broth (sterilized). Treatment 3: Raw GSW was fermented with L. plantarum strain 541 inoculant from cultivation in sterilized MRS broth (MRS). Treatment 4: Raw GSW was fermented with L. plantarum strain 541 inoculant from cultivation in sterilized pineapple juice (PA). The experiments were conducted at “Coastal Aquatic Feed Research Institute, (CAFRI)”, Sriracha, and at Burapha University, both in Chon Buri. (ii). The nutritional compositions, such as moisture, ash, fiber, crude proteins, crude lipid, and amino acid profiles, of all the hydrolysate shrimp protein products were analyzed. (iii). The in vitro method was applied for the assessment of digestion with commercial enzymes (trypsin, chymotrypsin and bacterial protease (Streptomyces griseus)) similar to in vivo digestive systems to determine the digestibility of the hydrolysate shrimp protein products. (iv). The molecular weights of the hydrolysate shrimp protein products were determined using SDS-PAGE.

2.1. Materials

The lactic bacterium Lactobacillus plantarum strain 541 was derived from the Thailand Institute of Scientific and Technological Research (TISTR). The shrimp waste of white shrimp (Penaeus vannamei) was from a seafood frozen factory, Tipwanchai Seafoods Co. Ltd., in Chonburi Province, Thailand.

Culture Media: (i) MRS broth of Difco™ (formula per L: protease peptone No. 3 10 g, beef extract 10 g, yeast extract 5 g, dextrose 20 g, Tween-80 1 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, manganese sulfate 0.05 g, dipotassium phosphate 2 g). Preparation: A total of 55 g of the powder was suspended in 1 L of purified water, mixed thoroughly, adjusted to pH 6.5, heated and boiled for 1 min to completely dissolve the powder, and autoclaved at 121^°C for 15 min. (ii) For MRS agar, 15 g of agar powder was added. (iii) Nutrient agar, NA of Difco™ (per L: beef extract 3 g, peptone 5 g, agar 15 g. Preparation: A total of 23 g of the powder was suspended in 1 L of purified water, mixed thoroughly, adjusted pH to 6.8, heated and boiled for 1 min, and then autoclaved and (iv) pineapple juice medium, PA (see Topic 2.2.4).

2.2. Methods

2.2.1. Raw Material Preparation

Shrimp waste (SW) used as the raw material obtained was transported to the Laboratory of Coastal Aquatic Feed Research Institute (CAFRI). The SW was ground and homogenized immediately with an industrial blender to obtain ground shrimp waste (GSW), collected in plastic bags and kept frozen at yeast extract 5 g, dextrose 20 g, Tween-80 1 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate − 80°C for further experiments.

2.2.2. First Inoculum Preparation

The stock cultures of Lactobacillus plantarum strain 54 were kept at − 80°C in 2-mL sterilized cryotubes supplemented with 20% (v/v) glycerol. To activate the cells for culture, the stock culture was transferred into 250 mL MRS broth contained in a 1-L shake flask, cultivated overnight at room temperature of 30°C and shaken at 180 rpm on a rotary shaker. One loop full of the cultured broth was streaked on a sterilized MRS agar plate and incubated at 35–37°C for 24–48 h in an incubator. When they grew, the characteristics of L. plantarum strain 541 colonies were convex, white-cream in color, and small in size of ~ 1–2 mm (Fig. 1). One colony was picked up and put into a 10-mL sterilized MRS broth in a 20-mL vial and then incubated at 35–37°C for 24 h.

2.2.3. MRS Broth Inoculant Preparation

The first inoculum culture from Topic 2.2.2. of 2.5 mL volume was inoculated into the 500-mL sterilized MRS broth contained in a 1-L flask and incubated at 35–37°C in an incubator shaker at 50 rpm for 24 h. This cultivation generated a viable cell number density concentration (VCNC) of approximately 1×10⁹ CFU/mL, checked by the total viable plate count method. It was further used as the inoculant in experimental Treatments 2 and 3: raw GSW and sterilized GSW fermentations (also see Topic 2.2.5.).

2.2.4. Pineapple Juice Inoculant Preparation

This was to compare the utilization of L. plantarum strain 541 inoculants produced from preparations with the MRS broth above and from the PA. Fresh pineapple fruits (Ananas comosus) were purchased from a local market. The skins were peeled after the crowns and stalk portions were removed. After that, the fruit fleshes were sliced using a knife and crushed in a fruit juicer for one to two minutes. Then, the pineapple juices were filtrated by a filter cloth to obtain the pineapple juice. Its 500 mL contained in a 1-L flask was adjusted to pH 6.5 ± 0.2 with 0.1 N NaOH solution and then supplanted with sucrose sugar at a concentration of 5% Brix. It was then autoclaved at 110°C under 15 lb/in² for 15 min. After that, 2.5 mL of the first inoculant from MRS broth (Topic 2.2.2) was inoculated into it and incubated at 35–37°C for 24 h. This cultivation generated a VCNC of approximately 1×10⁹ CFU/mL. (confirmed by total viable plate count). It was further used as the inoculant of experimental treatment 4: raw GSW fermentation (see Topic 2.2.5.).

2.2.5. Viable Cell Number Density Concentration during Fermentation (CFU/g)

The viable cell number density concentration (VCNC) was measured as the viable cell count, determined during fermentations at 0, 4, 8, 12, and 16 h by the standard plate count method [27]. A 1.0 g sample was diluted 10 times in 9 mL of 0.85% (w/v) sterilized normal saline. The suspension of 0.1 mL was spread on MRS agar plates for L. plantarum 541 and other lactic bacteria cell counts and on NA for total bacteria count. They were incubated at 37°C for 48 h, and all numbers of colonies were counted and calculated as VCNC in CFU/g. As in the control treatment, the GSW was not inoculated with the L. plantarum inoculant, and fermentation was performed by natural inborn microorganisms. The total viable cell count of the control treatment was determined by plating on MRS agar for lactic acid bacteria and on NA agar for total bacteria counts. As their culture samples were both plated on MRS agar and NA, natural lactic bacteria in the control treatment and L. plantarum 541 in other treatments can grow on both media, but other bacteria cannot grow on MRS agar because ammonium citrate and sodium acetate inhibit their growth and can grow only on NA. When the numbers of both bacterial types grew on both media with the same amounts, they were all lactic bacteria. For the control treatment, there were both natural lactic acid and other bacteria; the differences in cell numbers between the same samples plated on both MRS agar and NA were the other bacteria.

2.2.6. Protein Hydrolysis by Fermentation

To hydrolyze proteins in GSW, it was thawed at a room temperature of ~ 30°C overnight and then fermented by inoculation with L. plantarum inoculum from different sources, i.e., from cultivations with MRS (Topic 2.2.2.) and PA (Topic 2.2.3.). They were compared with the control treatment of GSW fermented with inborn microorganisms. There were four treatments performed in three replicates as follows: Treatment 1 (control treatment): raw GSW fermented with inborn microorganisms without the addition of starter seed, Treatment 2 (sterilized MRS): sterilized GSW fermented with L. plantarum strain 541 from MRS broth inoculant, Treatment 3 (MRS): raw GSW fermented with L. plantarum strain 541 from MRS broth inoculant, and Treatment 4 (PA): raw GSW fermented with L. plantarum strain 541 from pineapple juice inoculant.

The GSW samples of each treatment were fermented in stainless open stirred reactors (40 cm diameter and 60 cm height) with L. plantarum 541 from MRS inoculant, PA inoculant, or none at a ratio of 2.5 kg of GSW per 0.5 litres of inoculant broth. The GSW fermentations were mixed by two-arm stirrers at all times. The initial pH of GSW fermentations in all treatments was adjusted to ~ 5.0 with a concentrated acetic acid solution before the addition of inoculants, while the Control treatment had no inoculant added. Their fermentation pH was controlled between 5.0–5.5 with the same acid solution only during the first 4 h of fermentation. They were done for 16 h at room temperature (25–30°C) for good quality of protein hydrolysate products [28]. During fermentation, microorganisms enzymatically hydrolyzed protein contents in the GSW. Samples were withdrawn at 0, 4, 8, 12, and 16 h to measure pH change and microorganism growth. Then, crude liquid hydrolysates were separated from solid GSW (solid portion) by filtration with a filter cloth. They were kept at − 80°C before drying using a freeze dryer. The dried hydrolysate samples were analyzed for proximate compositions of moisture, protein, lipid, fiber, and ash. More profound analyses of amino acid compositions, in vitro protein digestion, and molecular sizes of proteins on SDS-PAGE were carefully undertaken. They were all performed in triplicate.

2.2.7. Proximate Analysis of the Compositions

According to the method of AOAC [29], the moisture was calculated by freeze dryer method, the crude ash was derived from incineration in a muffle furnace at 550°C and the crude fiber was from boiling with 1.25% (v/v) HCl and 1.25% (w/v) KOH and then incinerated in a muffle furnace at 500°C. The total nitrogen was analyzed using a TruSpec CN Carbon/Nitrogen Determinator (LECO Instruments St. Joseph, MI, USA). The total lipids were analyzed using a TFE 2000 Fat Extractor (LECO). Proximate analyses of the compositions were performed at the laboratory of the Coastal Aquatic Feed Research Institute (CAFRI), Department of Fisheries, Ministry of Agriculture and Cooperatives, Sriracha, Chonburi Province.

(i) Carbohydrate contents in the commercial fishmeal (CFM), C_CFM was calculated by:

C_CFM (% w/w) = 100–(m + p + l + f + a)

where m, p, l, f, and a are the concentrations (% w/w) of moisture, protein, lipid, fiber, and ash, respectively.

(ii) Carbohydrate contents in hydrolysate protein products (HPP), C_HPP (dry powder by freeze dryer) was calculated by:

C_HPP (% w/w) = 100–(p + l + f + a)

2.2.8 Amino Acid Composition Analysis

Amino acid compositions were analyzed using an amino acid analyzer (1100 series HPLC system, Agilent, USA) with postcolumn derivatization instrument and detected by a photodiode array detector at 405 nm (primary amino acids), 440 nm (secondary amino acids) or 570 nm (tertiary amino acids). A 0.5 g sample was dissolved in 5 mL of 6 M HCl and then incubated in an oven at 100°C for 24 h. After cooling at room temperature, the sample was filtered through a 0.45 µm filter to remove the sediments. The supernatant was diluted 1:50 and then injected into the amino acid analyzer (20 µL) to estimate the amino acid profiles. The standard sets of 16-component amino acids were used to create standard curves for calculations of the amounts of each amino acid in the samples.

2.2.9. In vitro Protein Digestion Analysis

The in vitro protein digestion method was used for estimation of protein digestibility using a multienzyme system developed by [30–32]. The multienzyme system was consisted of trypsin from porcine pancreas type IX-S (13,000–20,000 BAEE units/mg protein), chymotrypsin from bovine pancreas type II (≥ 40 units/mg protein) and protease type XIV (≥ 3.5 units/mg solid, powder) from Streptomyces griseus. Reportedly, an enzyme peptidase preparation from Streptomyces griseus was also found to be a good predictor of protein digestibility [33]. All enzymes were obtained from Sigma-Aldrich, USA. Albumin powder from bovine serum (≥ 97% w/w) was used as the reference protein.

The Lowry method [34] was used to quantify soluble proteins. The samples were ground to a fine powder and made into solutions with distilled water. The solutions were centrifuged at 8000 rpm for 15 minutes to obtain clear supernatants for protein quantity analyses after overnight incubation at 4°C. The solutions of 25 mL of proteins (~ 6.25 mg proteins/mL) were adjusted to pH 8.0 with 0.1 N HCl and/or 0.1 N NaOH during stirring at 37°C in a waterbath (Fig. 8). A solution of 2.5 mL of multienzymes (1.6 mg trypsin, 3.1 mg chymotrypsin, and 1.3 mg peptidase/mL, pH 8.0) was freshly prepared and kept on ice. Then, the multienzyme solution was added to the protein solutions, and the mixtures were stirred at 37°C. After measuring changes in pH over 10 min, rapid declines in pH occurred immediately due to freeing amino acid carboxyl groups from the protein chains because of proteolytic enzyme activities.

2.2.10. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was prepared by the method of [35]. The gel system consisted of a 12% (w/v) polyacrylamide-resolving gel (pH 8.8) and a 4% (w/v) stacking gel (pH 6.8). The lengths of the resolving and stacking gels were 6 and 1 cm, respectively, with a gel thickness of 0.75 mm. Soluble protein samples were quantified by the Lowry method [34]. The solutions of 100-µL protein samples at a concentration of 8 µg protein/µL of 0.01 M PBS (phosphate-buffered saline) was added to 100 µL of a buffer solution [20% (w/v) of 0.5 M Tris–HCl, pH 6.8, 30% (v/v) of 10% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 15% (v/v) glycerol, and 30% (w/v) bromophenol blue]. Then, the solutions were heated in a boiling water bath for 3 minutes prior to loading into the slab gel wells. The solutions (80 µg of proteins/well) of 20 µL were loaded in each lane well. Electrophoresis was carried out at a constant current of 120 volts. Protein bands were stained by immersion of the gels in a 0.1% (w/v) Coomassie Brilliant Blue R-250 solution in 50% (v/v) methanol and 10% (v/v) acetic acid. The gels were destained with 10% (v/v) acetic acid and 30% (v/v) methanol. The molecular weights of proteins were calculated from the standard curve of known molecular weights of protein markers versus their relative fractions (RF) of their molecular weight relative migrations on SDS-PAGE.

2.2.11. Statistical Analysis

The quantitative performance of proximate compositions of moisture, protein, lipid, fiber, and ash of all sample groups were statistically analyzed by one-way ANOVA, and the mean values of each treatment were compared by Tukey’s multiple comparison test. Differences were considered significant at p-value ≤ 0.05 [36].

3.1 Growth of Lactobacillus plantarum 541 and Total Bacteria

The raw GSW samples were fermented with L. plantarum inoculants from different cultured sources, i.e., from cultivations with MRS broth and pineapple juice compared with the control treatment fermented with inborn microorganisms and the sterilized ground shrimp waste fermented with L. plantarum 541 inoculant from MRS broth. The inoculum cultures had amounts of L. plantarum 541 inoculants of 1.3–1.8×10⁹ CFU/mL (Table 1).

The growth of Lactobacillus plantarum strain 541 and total bacterial counts are shown in Fig. 1. PA treatment showed the highest VCNC of L. plantarum 541 on MRS agar and the height total bacteria on NA agar. The MRS treatment showed lower VCNC of both bacterial types than that of the PA treatment, but it was higher than those of the Sterilized treatment. The control treatment, without the addition of starter seed, showed the lowest growth of microorganisms. The growths of L. plantarum 541 and of total bacteria in MRS, Sterilized, and PA treatments gradually grew until reaching the highest growths at the end of fermentation (16 h). Figure 1 compares the cell counts on MRS agar and NA agar. The different results were found in the Control treatment, in which the total bacteria amount slightly declined within the first 4 h, after which they gradually grew until the end at 16 h while lactic acid bacteria on MRS agar grew slowly until they reached the highest growth at the end of fermentation.

Table 1

Growth of the lactic acid bacterium *L. plantarum* 541 on MRS agar and total bacteria on NA of GSW fermented with inoculants from different sources in the Control, Sterilized-GSW, MRS, and PA treatments (n = 6)
GSW fermented without inoculant, and with MRS and PA inoculants	Inoculants cultured*	×10⁶ (CFU/g)
		0 h		4 h		8 h		12 h		16 h
		MRS^**	NA^**	MRS	NA	MRS	NA	MRS	NA	MRS	NA
Control GSW without inoculant	No inoculant	0.17 ± 0.1	5.2 ± 0.7	0.18 ± 0.1	3.7 ± 0.7	0.29 ± 0.1	3.8 ± 1.0	0.28 ± 0.1	3.9 ± 1.0	0.39 ± 1.1	4.8 ± 1.1
Sterilized GSW with MRS inoculant	1.6 ± 0.1×10⁹	270 ± 0.1	250 ± 0.1	650 ± 0.8	530 ± 1.1	930 ± 0.1	1000 ± 0.1	1200 ± 0.1	1100 ± 0.1	1600 ± 0.1	1400 ± 0.1
Raw GSW with MRS inoculant	1.8 ± 0.1×10⁹	430 ± 0.8	410 ± 0.9	450 ± 0.9	403 ± 0.8	1500 ± 0.1	1400 ± 0.1	1900 ± 0.1	1900 ± 0.2	2700 ± 0.1	2700 ± 0.2
Raw GSW with PA Inoculant	1.3 ± 0.1×10⁹	110 ± 0.1	110 ± 0.1	160 ± 0.1	170 ± 0.2	280 ± 0.1	270 ± 0.1	2600 ± 0.1	2600 ± 0.1	4500 ± 0.9	5800 ± 1.0
* CFU/mL of L. plantarum 541 in MRS and pineapple juice inoculants

** MRS: Cell numbers of only L. plantarum 541 and lactic acid bacteria (in Control) can grow on MRS agar.

** NA: Cell numbers of total bacteria (both lactic and other bacteria) that can grow on NA.

The results of this study show that the GSW fermented with L. plantarum 541 inoculant from pineapple juice (PA inoculant) grew rapidly with a VCNC of 4500×10⁶ CFU/g (on MRS agar) after 16 h of fermentation at room temperature (~ 30°C). Using inoculants from MRS broth (MRS inoculant) resulted in less growth of L. plantarum 541 (2700×10⁶ CFU/g) than that of the PA inoculant (4500×10⁶ CFU/g) at 16 h the end of fermentation. The Sterilized GSW treatment inoculated with inoculants from MRS broth (MRS inoculant) obtained the lowest growth with a VCNC of 1600×10⁶ CFU/g. This may be due to the destruction of nutrients by sterilization at 121°C for 15 minutes. In the GSW control treatment without the addition of L. plantarum 541 inoculant, the hydrolysate was from inborn microorganism activities. The VCNC of total bacteria was 4.8×10⁶ CFU/g on NA. The VCNC of L. plantarum 541 and the VCNC of total bacteria from enzymatic hydrolyses of GSW by inoculants of all treatments (MRS, sterilized GSW with MRS, and PA) were similar. This indicated that L. plantarum was the main lactic bacterial species grown. It produced lactic acid, which inhibited the growth of other contaminating bacteria. This result agrees well with the results reported by [37].

However, we expect that pineapple juice could be used as a basal medium for the cultivation of L. plantarum 541 because of its suitable acid and other contents, as in the tomato juice research work of [33]. They also used tomato juice for the production of mainly probiotic lactic acid bacteria. They still reported that L. plantarum was capable of rapidly utilizing tomato juice for cell synthesis and produced lactic acid without nutrient supplementation and pH adjustment. Most of the lactic acid bacteria survived under low pH or high acidity conditions during four weeks of cold storage at 4^°C. They also found that lactic acid starters produced bacteriocin against other bacteria and vice versa.

In addition, [38] reported that 100 g of pineapple contains 47–52 calories, 85.3–87.0 g water, 0.4–0.7 g proteins, 0.2–0.3 g fat, 11.6–13.7 g total carbohydrate, 0.4–0.5 g fiber, 0.3–0.4 g ash, 17–18 mg calcium, 8–12 mg phosphorus, 0.5 mg iron, 1–2 mg sodium and 125–146 mg potassium. Pineapple also contains 12–15% (w/w) sugars, of which two-thirds are in the form of sucrose and the rest are glucose and fructose. The pH 3.71 of pineapple is acidic, which was 53.5% (w/w) of the acidity percentage. The composition of the juice could vary depending on geographical, cultural, and seasonal harvesting and processing [39, 40]. As a result, pineapple juice sugar at low pH is suitable for the growth of the lactic acid bacterium L. plantarum 541.

3.2. The pH Change during Fermentation

The results of the pH change during fermentation are shown in Fig. 3. The initial pH of the GSW in the four treatments fermented with inoculants from MRS, PA, and none are shown in Table 2. During the first 4 h, the pH values of all treatments ranged from 5.0 to 5.5. As the control batch, no inoculation of L. plantarum 541, fermentation performed by natural inborn microorganisms in the raw GSW. Of this treatment, pH increased and was in accordance with the lowest VCNC of lactic acid bacteria and total bacteria (Fig. 2). The GSW fermented with the inoculants from MRS and PA and the sterilized GSW fermented with the inoculants from MRS showed pH values of approximately 5.0 to 5.8 throughout the fermentation period. However, the GSW fermented with the L. plantarum 541 inoculant from PA generated the highest VCNC (Fig. 2). The decrease in the pH value of GSW fermented with L. plantarum 541 from the PA inoculant agrees well with the increase in VCNC. The pH profile of GSW fermented with the inoculant from MRS increased (5.1–5.5), and then after 8 h, it was constant with a value of 5.7 because an accumulation of lactic acid during fermentation, produced from L. plantarum 541, occurred with a small amount after that time. However, VCNC gradually increased until it reached the maximum density. The sterilized GSW fermented with inoculant from MRS was similar to that of GSW fermented with inoculant from MRS, but it showed lower values of both pH and VCNC of L. plantarum 541, and the pH was constant after 4 h with a value of 5.3. In addition, the protein hydrolysate products of the GSW fermented with L. plantarum 541 inoculants from MRS, and PA and of the sterilized GSW fermented with inoculants from MRS had a better smell and color (Fig. 4) than the product from the Control GSW without the addition of L. plantarum 541 inoculant. This result also agrees with the report of [28].

Table 2

The pH change profile during fermentations of GSW fermented without inoculant and with inoculants from different cultures (n = 3)
GSW fermented without inoculant, and with MRS and PA inoculants	pH
	Initial pH ^a	4 h ^b	8 h	12h	16 h
Control GSW without inoculant (with inborn microorganisms)	7.3 ± 0.1	5.0 ± 0.0	5.4 ± 0.1	6.2 ± 0.1	6.2 ± 0.1
Sterilized GSW with MRS inoculant	7.5 ± 0.0	5.1 ± 0.1	5.3 ± 0.1	5.3 ± 0.1	5.3 ± 0.1
Raw GSW with MRS inoculant	7.2 ± 0.1	5.1 ± 0.0	5.5 ± 0.0	5.7 ± 0.1	5.7 ± 0.1
Raw GSW with PA Inoculant	7.4 ± 0.0	5.2 ± 0.0	5.4 ± 0.0	5.8 ± 0.1	5.4 ± 0.1
^a Initial pH values of GSW before fermentations and ^b controlled pH of 5 ± 0.2 during 0–4 h

The pH values of all treatments were controlled at 5.0–5.5 for the first 4 h using concentrated acetic acid added into the GSW to adjust the pH before adding and mixing with each designed inoculant culture. A pH of 6.5 was obtained in the fermentation of the inedible portions of shrimp without pH control by lactic acid bacteria [41]. In addition, it was found that the hydrolysate product had a tendency to spoil when the pH increased. It was reported that L. plantarum showed a more rapid drop in pH than the other cultures examined and found acid production ability by lactic acid bacteria [38]. Additionally, pH values were maintained ranging from 5.0 to 5.5 during the first 4 h for optimum growth of L. plantarum to enhance the quality of appearance and the good smell of hydrolysate protein products [28]. They mentioned that the controlled pH at 5.0 to 5.5 during the first 0–4 h was effective inhibiting other bacterial cell growth.

3.3. Proximate Compositions of Moisture, Protein, Carbohydrate, Lipid, Fiber, and Ash

The hydrolysate protein products are shown in Figure. The careful proximate analyses of the compositions of moisture, protein, carbohydrate, lipid, fiber, and ash are revealed in Table 3. The moisture content of the products was rather high (~ 80% w/w) while that of the commercial fish meal (CFM) was only 8.6% (w/w). The moisture content of the dry powder hydrolysates was ~ 80%, which depended on the evaporation process of freeze drying.

3.3.1. Protein Contents

The highest protein content of 52.5% (w/w) (83.33% of CFM) was from the hydrolysate product of GSW fermented with the inoculants from MRS, which was significantly higher than those of the other treatments, which value between 48.6–49.6% (w/w) (77.14–78.73% of CFM). As the standard commercial product, the protein content of CFM (commercial fish meal) was 63%, which was higher than those of protein products from shrimp waste hydrolysates here. Not surprisingly, because of the lack of processing of GSW by either lactic bacteria fermentation or physical treatment, the nonfermented GSW has a protein content of 55.6% (w/w), which is higher than those of all the hydrolyzed shrimp protein products from fermentations of GSW (Table 3). However, the protein contents in the hydrolysate products from those GSW fermented in this study of 48.6–52.5% (w/w) were higher than those of the shrimp head silage with values of 39.9% (w/w) [42] and 46.73% (w/w) prepared by lactic acid fermentation of inedible portions of shrimp [41]. On the other hand, the protein content of the nonfermented GSW product from this study was 55.6% (w/w), which was higher than those of the fermented GSW hydrolysate products (Table 2).

3.3.2. Carbohydrate Contents

Carbohydrate contents in the hydrolysate products were 31.7% (w/w) from GSW fermented without the addition of L. plantarum 541 inoculant but by inborn microorganisms, 31.6% (w/w) from GSW fermented with L. plantarum 541 inoculant from PA culture, and 30.6% (w/w) from the sterilized GSW fermented with L. plantarum 541 inoculant from MRS culture. These values showed no significant difference (p ≥ 0.05) from each other. The carbohydrate content of nonfermented GSW was 8.3% (w/w), which was lower than all the hydrolyzed products of all treatments (Table 3).

3.3.3. Lipid Contents

The high lipid contents of hydrolysate protein products were 8.0% and 7.0% (w/w) from raw GSW and sterilized GSW fermented with L. plantarum 541 inoculant from MRS culture, respectively. They were not significantly different (p ≥ 0.05). These values were 5.4% (w/w) from GSW fermented with L. plantarum 541 inoculant from PA culture and 5.9% (w/w) from GSW fermented with inborn microorganisms. They were significantly less than the former (p ≤ 0.05). However, the lipid contents of the CFM and the nonfermented GSW were higher than those of other treatments, with values of 9.4% and 8.7% (w/w), respectively. (Table 3). The overall lipid contents of the hydrolysate protein products in this study were between 5.4% and 8% (w/w), which are lower than the values of 13.6% (w/w) of fermented shrimp head silage using molasses as a carbohydrate source [43] and 12.5% (w/w) [42].

3.3.4. Fiber Contents

The highest fiber content of the hydrolysate protein product was 0.7% (w/w) from the control treatment of GSW fermented with inborn microorganisms, which was significantly higher than those of the other treatments (p ≤ 0.05). The lower fiber contents were 0.4% and 0.5% from GSW fermented with L. plantarum 541 inoculant from PA culture and from sterilized GSW fermented with L. plantarum 541 inoculant from MRS. Again, the nonfermented GSW still had a fiber content of 13.3% (w/w), which was higher than those of all the hydrolyzed GSW treatments (Table 3).

The fiber and carbohydrate contents in all hydrolysate protein products were higher than those of the CFM. There have been a few reports related to the fiber and carbohydrate contents in hydrolysate protein from shrimp waste. In addition, chitin, which is similar to cellulose fiber both in chemical structure and in biological function [43], was found in shrimp shells of the nonhydrolyzed shrimp waste. The quantity of chitin showed a higher fiber content than in all hydrolysate protein products.

3.3.5. Ash Contents

The ash contents of hydrolysate protein products from GSW fermented with inoculants from MRS and PA cultures and from sterilized GSW fermented with inoculants from MRS culture were 12.9–13.5% (w/w), which were not significantly different from each other (p ≥ 0.05), but significantly different from 12% (w/w) (p ≤ 0.05) of the control treatment of GSW fermented with inborn microorganisms. The ash contents of all the hydrolysate protein products from all treatments were between 12.0% and 13.5% (w/w), while those of the CFM and the nonfermented GSW were 21.1% and 14.1% (w/w), respectively (Table 3). In this study, the overall ash content of all hydrolysate products was 12.0–13.5% (w/w). Compared with the values of [42–44] and [41], the ash contents of shrimp waste hydrolysate products were reported to be 12.3, 12.2, 10.2 and 8.25% (w/w), respectively.

Table 3

Proximate compositions (% w/w) of hydrolysate protein products from GSW fermented with different inoculants compared with nonfermented GSW and commercial fish meal (CFM)
GSW fermented without inoculant, and with MRS and PA inoculants; Non-hydrolyzed GSW, CFM	Dry matter basis (g/100 g) or % (w/w)
	Moisture	Protein	Carbohydrate	Lipid	Fiber	Ash
Control GSW without inoculant	80.2 ± 1.4	49.6 ± 1.0^c	31.7 ± 1.6^a	5.9 ± 0.6^c	0.7 ± 0.1^b	12.0 ± 0.2^b
Sterilized GSW with MRS inoculant	83.5 ± 1.9	48.6 ± 1.6^c	30.6 ± 0.9^a	7.0 ± 0.9^bc	0.4 ± 0.0^b	13.4 ± 0.4^b
Raw GSW with MRS inoculant	83.1 ± 1.3	52.5 ± 0.8^bc	26.1 ± 1.4^b	8.0 ± 0.8^b	0.6 ± 0.1^b	12.9 ± 0.5^b
Raw GSW with PA Inoculant	82.1 ± 0.9	49.1 ± 2.1^c	31.6 ± 2.6^a	5.4 ± 0.6^c	0.5 ± 0.1^b	13.5 ± 0.5^b
Non-fermented GSW	76.4 ± 1.7	55.6 ± 1.1^b	8.3 ± 1.0^c	8.7 ± 0.7^a	13.3 ± 0.2^a	14.1 ± 0.4^b
Commercial fish meal (CFM)	8.6 ± 0.2	63.0 ± 0.6^a	6.2 ± 0.4^c	9.4 ± 0.3^a	0.3 ± 0.0^b	21.1 ± 0.1^a

Statistical comparisons of those mean values within their own columns (among treatments and two comparators) at p-values of ≤ 0.05 show different characters, a, b, c, and d, which indicate statically significant differences.

3.4. Quantitative Analysis of Amino Acid Components

The nutritional value of a protein depends on the types and amounts of amino acids available in its sources. The amino acid compositions of the hydrolysate protein products from GSW fermented with different inoculant cultures compared with a nonfermented GSW and a commercial fish meal (CFM) are presented in Table 4 and Fig. 4. Of all the treatments and the controls, our results show the contents of sixteen known amino acids, i.e., The eight essential amino acids (EAAs) and the eight nonessential amino acids (NEAAs) included S-amino acids methionine and cysteine. From all the samples, the overall amounts of EAA in descending order are Leu > Lys > Val ≈ Phe > Thr > Iso > His > Met, and the overall amounts of NEAA in descending order are Glu > Asp > Gly ≈ Ala > Arg > Pro > Ser > Tyr. Because of the large amount of data in Table 4, there are two suggestions to study the amino acid components here: (Topic 3.4.1.) the comparative study of each 16 amino acids among those four fermentation treatments plus the two comparators, see Table 4 and Fig. 4. (Topic 3.4.2.) the studies of total amounts and their total fractions (Tables 5 and 6) which we can make the decision that which treatments were good using statistic comparative results provided.

3.4.1. Comparative Study of Each 16 Amino Acid among the Four Treatments and Comparators

(i) Example Studies of Some Essential Amino Acids

The lysine contents of hydrolysate products of the control treatment of raw GSW fermented with inborn microorganisms, sterilized GSW fermented with MRS inoculant, raw GSW fermented with MRS inoculant, and raw GSW fermented with PA inoculant were 6.7, 6.2, 5.2, and 6.1% (w/w) of total amino acids, respectively (6.1% (w/w) in CFM). Similar to [44], autolysis hydrolysate from shrimp head contained a very high level of both lysine at approximately 7.42% (w/w) and arginine approximately 8.63% (w/w) of total amino acids. As raw shrimp wastes of different nutritional values were used, their values were higher than the results in this study. Lysine benefits were referred to, which are usually deficient in cereals. It is an essential amino acid required for proper development and is also a precursor in the production of carnitine, a nutrient with roles in converting fatty acids into energy and in regulating cholesterol levels [45].

The methionine contents were 2.9% and 1.8% (w/w) in CFM and nonfermented GSW, respectively. They were 2.5, 1.8, 1.9, and 1.7% (w/w) in raw GSW fermented with inborn microorganisms, sterilized GSW fermented with MRS inoculant, raw GSW fermented with MRS inoculant, and raw GSW fermented with PA inoculant, respectively. Essentially, methionine was the limiting EAA in all hydrolysate products of ground shrimp waste. The highest EAA concentration in all hydrolysate products was leucine, including in the comparators of control groups.

(ii) Example Studies of Some Nonessential Amino Acids

Arginine is sometimes classified as a semiessential or conditionally essential amino acid and participates in protein synthesis and other physiological functions, such as detoxification and energy conversion. The arginine contents were 6.3, 6.1, 5.9, and 6.2 (w/w) in raw GSW fermented with inborn microorganisms, sterilized GSW fermented with MRS inoculant, raw GSW fermented with MRS inoculant, and raw GSW fermented with PA inoculant, respectively. They were 6.10 and 5.0% (w/w) in nonfermented GSW and CFM, respectively.

Glutamic acid is a nonessential amino acid (NEAA) found in the major high quantity of ~ 14% (w/w) of total amino acids of all the treatments, including the nonfermented GSW and CFM. This result agrees well with the result of [44], who used the autolysis method for protein recovery from white shrimp head (Penaeus vannamei). Here, the hydrolysate protein products from raw GSW fermented with inborn microorganisms, sterilized GSW fermented with MRS inoculant, raw GSW fermented with MRS inoculant, and raw GSW fermented with PA inoculant are composed of glutamic acids at 14.17, 13.74, 13.86, 13.68, and 13.92% (w/w) of total amino acids, respectively. The highest concentration of NEAA in the dry hydrolysate powder from lactic bacteria fermentation was glutamic acid at 38.8 mg/g dry powder, but it was lower than the 47–54 mg/g dry powder in this study [41]. An agreement with this study was that the highest concentration of amino acids was glutamic acid, and the limiting amino acid was EAA methionine [46].

In addition, the shrimp hydrolysate protein products from this study had extremely high contents of 38.4–39.3% (w/w) of the total amino acids of the flavor enhancers glutamic acid, aspartic acid, glycine, and alanine, while the nonfermented GSW and CFM were found at 34.5 and 42.1% (w/w), respectively.

Table 4

Amino acid compositions in hydrolysate protein products from GSW fermented with different inoculant cultures compared with the nonfermented GSW and a commercial fishmeal (CFM)
Amino acids, (Italic is essential amino acids)	Control 1 : GSW fermented inborn microorganisms		Sterilized GSW fermented with MRS inoculant		Raw GSW fermented with MRS inoculant		Raw GSW fermented with PA inoculant		Control 2 : Non-fermented GSW		Control 3 : Commercial fish meal (CFM)
Amino acids, (Italic is essential amino acids)	/p	/ds	/p	/ds	/p	/ds	/p	/ds	/p	/ds	/p	/ds
Histidine	27.0 ± 7.7	13.4 ± 3.71	23.2 ± 4.1	11.3 ± 2.37	24.4 ± 0.9	12.8 ± 0.70	24.3 ± 0.8	11.9 ± 0.93	22.5 ± 4.6	12.5 ± 2.47	21.7 ± 5.6	13.6 ± 3.47
Isoleucine	33.2 ± 0.2	16.5 ± 0.33	29.3 ± 0.9	14.2 ± 0.51	30.7 ± 0.6	16.1 ± 0.56	30.8 ± 1.2	15.1 ± 1.31	40.1 ± 1.2	22.3 ± 0.92	21.7 ± 1.5	13.7 ± 0.99
Leucine	62.2 ± 1.0	30.9 ± 0.51	54.5 ± 1.4	26.5 ± 0.77	56.9 ± 2.0	29.9 ± 1.50	58.1 ± 2.0	28.6 ± 2.34	114.4 ± 2.1	35.5 ± 2.01	47.0 ± 3.1	29.6 ± 2.11
Lysine	51.7 ± 2.0	25.7 ± 1.48	47.6 ± 0.4	23.1 ± 0.68	40.0 ± 17.6	21.1 ± 2.47	47.4 ± 1.0	23.3 ± 1.58	47.2 ± 1.0	26.2 ± 0.61	47.4 ± 1.1	29.8 ± 0.71
Methionine	19.4 ± 8.3	9.6 ± 3.91	13.7 ± 0.6	6.7 ± 0.40	14.9 ± 0.9	7.9 ± 0.59	13.5 ± 1.2	6.6 ± 0.70	13.1 ± 5.0	7.3 ± 3.50	22.2 ± 6.0	14.0 ± 3.84
Phenylalanine	39.6 ± 1.2	19.6 ± 0.39	38.4 ± 1.6	18.7 ± 0.71	37.9 ± 1.6	19.9 ± 1.03	38.9 ± 0.7	19.1 ± 1.25	35.7 ± 1.5	19.8 ± 1.01	25.2 ± 1.7	15.9 ± 1.12
Threonine	37.0 ± 5.8	18.3 ± 2.51	29.1 ± 2.6	14.1 ± 0.82	28.3 ± 1.6	14.9 ± 1.02	30.4 ± 0.2	14.9 ± 0.69	26.2 ± 5.0	14.5 ± 2.82	28.3 ± 6.0	17.8 ± 3.82
Valine	40.1 ± 0.5	19.9 ± 0.33	37.4 ± 1.2	18.2 ± 0.56	38.3 ± 1.0	20.1 ± 0.70	38.0 ± 1.1	18.7 ± 1.38	32.9 ± 1.5	18.3 ± 1.10	26.9 ± 1.7	17.0 ± 1.14
Alanine	51.8 ± 1.8	25.7 ± 0.72	47.7 ± 1.3	23.2 ± 0.82	50.1 ± 2.5	26.3 ± 1.63	49.9 ± 2.1	24.5 ± 2.12	44.1 ± 2.5	24.5 ± 1.50	47.3 ± 2.8	29.8 ± 1.90
Arginine	48.3 ± 0.5	24.0 ± 0.48	45.7 ± 1.1	22.2 ± 1.22	48.1 ± 4.3	25.3 ± 2.54	47.0 ± 1.3	23.1 ± 1.61	44.0 ± 3.0	24.5 ± 2.14	38.7 ± 3.7	24.4 ± 2.44
Aspartic acid	91.8 ± 14.7	45.5 ± 4.51	72.7 ± 3.3	35.3 ± 0.84	71.89 ± 2.9	37.8 ± 1.89	72.3 ± 0.9	35.5 ± 1.98	71.3 ± 14.4	39.6 ± 8.49	72.5 ± 16.4	45.7 ± 10.49
Glutamic acid	109.4 ± 9.2	54.3 ± 3.43	95.7 ± 2.3	46.5 ± 0.83	94.3 ± 4.9	49.5 ± 3.30	95.0 ± 4.1	46.7 ± 4.20	88.9 ± 10.2	49.4 ± 5.88	86.9 ± 12.2	54.8 ± 7.88
Glycine	50.7 ± 1.5	25.2 ± 0.29	49.2 ± 1.5	23.9 ± 0.29	50.3 ± 1.5	26.4 ± 0.95	49.2 ± 0.8	24.1 ± 1.53	46.1 ± 2.0	25.6 ± 1.55	59.5 ± 2.4	37.5 ± 1.67
Proline	46.1 ± 1.8	22.9 ± 0.79	44.0 ± 0.9	21.4 ± 0.59	46.2 ± 2.5	24.2 ± 1.42	44.5 ± 2.0	21.8 ± 0.68	43.0 ± 4.3	23.9 ± 2.44	39.0 ± 5.3	24.6 ± 3.44
Serine	40.1 ± 5.7	19.8 ± 2.44	32.5 ± 1.9	15.8 ± 0.36	23.9 ± 0.6	12.6 ± 0.43	30.7 ± 0.8	15.1 ± 1.12	32.2 ± 4.4	17.9 ± 2.45	30.9 ± 5.4	19.5 ± 3.45
Tyrosine	23.5 ± 1.1	11.7 ± 0.30	27.2 ± 1.7	13.2 ± 0.56	24.4 ± 0.1	12.8 ± 0.22	24.5 ± 0.8	12.1 ± 0.95	24.4 ± 1.2	13.6 ± 0.75	17.2 ± 1.4	10.8 ± 0.93
ΣAA	771.88	382.85	687.69	334.11	680.49	357.54	694.61	341.17	725.87	403.20	632.23	398.32
ΣEAA(italics)	310.22	153.89	273.16	132.75	271.29	142.59	281.46	138.25	331.92	184.38	240.34	151.41
ΣNEAA	461.65	228.96	414.54	201.36	409.19	214.95	413.15	202.91	393.92	218.82	391.88	246.91
ΣEAA/ΣAA	0.40	-	0.40	-	0.40	-	0.41	-	0.46	-	0.38	-
ΣEAA/ΣNEAA	0.67	-	0.66	-	0.66	-	0.68	-	0.84	-	0.61	-
/p: amino acids (mg/g protein); /ds: amino acids (mg/g dry sample)

AA (amino acids), EAA (essential amino acids), and NEAA (non-essential amino acids).

Values in the table are shown as the mean ± SD of three replicates.

Overall EAA amounts in descending order: Leu > Lys > Val ≈ Phe > Thr > Iso > His > Met

Overall NEAA amounts in descending order: Glu > Asp > Gly ≈ Ala > Arg > Pro > Ser > Tyr

3.4.3. Studies of Total Amounts and their Total Fractions

The results showed that the seven EAAs (Table 4) and the sum of essential amino acids, ΣEAA (Tables 5 and 6), were most present in the nonhydrolyzed GSW, and second in the control treatment of GSP fermented without the addition of L. plantarum 541 (but with inborn microorganisms). Additionally, the sums of total amino acids and ΣAA of both cases were higher than those of other treatments, as they were not used by microorganisms for nonhydrolyzed GSW and less used for the control treatment because of the least bacterial cell growth (Table 1). The growth and concentration of microorganisms in the GSW fermented with the inoculants from MRS and PA cultures were higher than those of the control treatment and the nonhydrolyzed GSW. Therefore, nitrogen sources from amino acids were used for their growth. Reported the result of protein hydrolysates from the head waste of Penaeus vannamei by autolysis conditions (at 50°C, pH at 7.85, and 23% (w/v) of substrate concentration) [47]. They found that endogenous enzymes in the shrimp heads had a strong autolysis capacity (AC) for releasing almost all amino acids and reached a plateau at 3 h. This indicated that the endogenous enzymes in the shrimp head were compounded by several or dozens of enzymes due to their broad specificities. For this reason, the hydrolysate protein product from the control and from the nonhydrolyzed GSW presented higher contents of almost all amino acids, EAA and ΣEAA than the others.

The ratios of ΣEAA/ΣAA were 0.40, 0.40, 0.40, 0.44, 0.46 and 0.38 for the hydrolysate protein products from GSW fermented with inborn microorganisms, sterilized GSW fermented with MRS inoculant, raw GSW fermented with MRS inoculant, raw GSW fermented with PA inoculant, compared with nonfermented GSW and from CFM, respectively. Again, the ratio of ΣEAA/ΣAA was 0.40 and that of ΣNEAA/ΣAA was 0.60 for all the hydrolysate protein products of all the treatments of fermented GSW. Those ratios of 0.46 and 0.54 and 0.38 and 0.62 belonged to the nonfermented GSW and CFM, respectively. The ratios of ΣEAA/ΣNEAA were between 0.66 and 0.68 for all the hydrolysate protein products. Surprisingly, these values are definitely the most similar to 0.70 and 0.67 obtained from [48], who reported the ratios of EAA/NEAA in black tiger shrimp and white shrimp, respectively. In addition, [49] explained that the EAA/NEAA ratio of many fish species was 0.70 on average, but the ratio of 0.59 was reported to be in crab (Portunus trituberculatus) and squid (Doryteusthis bleekeri), and in this study, the ratio of 0.61 belonged to the CFM. Moreover, the ratio of 0.84 was of the nonfermented GSW. Thus, these results indicated that the EAA from all the fermented GSW was approximately 10% higher, and the EAA from nonfermented GSW was 27% higher than that of the CFM. This essentially indicates that the GSW can be used to substitute the CFM in the composition of animal feed. Consequently, shrimp hydrolysate protein products with a 0.7 ratio of EAA/NEAA can be a good alternative protein source in animal feed. In addition, the percentage of EAA in total amino acids and the ratios of EAA/NEAA of the shrimp hydrolysate protein product obtained from this study were 40% (w/w) and 0.7, respectively. These values meet and exceed the reference values of 40% (w/w) and 0.6, which were recommended by the World Health Organization (WHO)/Food and Agriculture Organization (FAO) (FAO/WHO, 1991) [50].

Table 5

Total amino acids, total essential and total nonessential amino acids per g protein, percentages and their fractions in hydrolysate protein products from GSW fermented with different inoculant cultures compared with nonfermented GSW and a commercial fishmeal (CFM)
GSW fermented without inoculant, and with MRS and PA inoculants; Non-fermented GSW, CFM	Amino acids (mg/g protein) and % of EEA, %NEAA and their fractions
	ΣAA	ΣEAA	ΣNEAA	EAA % (w/w)	NEAA % (w/w)	ΣEAA/ΣNEAA
Control GSW without inoculant	771.88^a	310.22^b	461.65^a	40^b	60^a	0.67^b
Sterilized GSW with MRS inoculant	687.69^c	273.16^c	414.54^b	40^b	60^a	0.66^b
Raw GSW with MRS inoculant	680.49^c	271.29^c	409.19^b	40^b	60^a	0.66^b
Raw GSW with PA Inoculant	694.61^c	281.46^c	413.15^b	41^b	59^a	0.68^b
Non-fermented GSW	725.87^b	331.92^a	393.92^c	46^a	54^b	0.84^a
Commercial fish meal (CFM)	632.23^d	240.34^d	391.88^c	38^c	62^a	0.61^c

Statistical comparisons of those mean values within their own columns (among treatments and two comparators) at p-values of ≤ 0.05 show different characters, a, b, c and d, which indicate statistically significant differences.

Table 6

Total amino acids, total essential and total nonessential amino acids per g dry sample matter, in hydrolysate protein products from GSW fermented with different inoculant cultures compared with nonfermented GSW and a commercial fish meal (CFM)
GSW fermented without inoculant, and with MRS and PA inoculants; Non-hydrolyzed GSW, CFM	Amino acids (mg/g dry sample)
	ΣAA	ΣEAA	ΣNEAA
Control GSW without inoculant	382.85^ab	153.89^b	228.96^b
Sterilized GSW with MRS inoculant	334.11^e	132.75^cd	201.36^d
Raw GSW with MRS inoculant	357.54^c	142.59^c	214.95^c
Raw GSW with PA Inoculant	341.17^cd	138.25^c	202.91^d
Non-hydrolyzed GSW	403.20^a	184.38^a	218.82^c
Commercial fish meal (CFM)	398.32^a	151.41^b	246.91^a

In addition, the shrimp hydrolysate protein products from this study were found to have extremely high contents of flavor enhancers, i.e., glutamic acid, aspartic acid, glycine, and alanine in between 38.4% and 39.3% (w/w) of the total amino acids. Similar values of 37.1–39.2% (w/w) were reported with our results [47]. As the hydrolysate protein products from fermented GSW, especially nonfermented GSW, were composed of high nutritional value when compared with commercial fish meal (CFM), they can essentially be utilized and substituted as an excellent alternative feedstuff or a good taste enhancer in animal feed. This is to save the environment in two ways: One is to reduce catching the world’s huge amounts of young small fish for the production of fish meal for animal feed, and the second is to treat the huge quantities of shrimp waste around the world to produce value-added protein sources for animal feed.

3.5. In Vitro Protein Digestion

The in vitro digestion using a multienzyme system to estimate the protein digestibility is shown in Fig. 5. It consisted of trypsin (type IX-S, porcine pancreases), chymotrypsin (type II, bovine pancreases) and a bacterial protease (type XIV, Streptomyces griseus). All were from Sigma-Aldrich, USA. The pH change was recorded through 17 minutes of digestion. The pH of protein suspensions from hydrolysate protein products of all fermented GSW treatments, especially of the Sterilized GSW, declined more immediately than the albumin and CFM within 1 h after mixing with the multienzymes. These effects were caused by freeing amino acid carboxyl groups from protein chains by proteolytic enzyme digestion activities. The control treatment of GSW fermented without the addition of L. plantarum 541 inoculant showed a smaller decline in pH than the other groups that used L. plantarum 541 cultures as inoculants. This indicates that fermentation using L. plantarum 541 lactic bacteria was very effective in polypeptide hydrolytic digestion. Unlike the pH of CFM, it dropped slightly and maintained its value at ~ 7.4 until the end of the 17-min process. Albumin used as a standard protein showed that its pH value dropped to a value of ~ 7.1, lower than that of CFM, but higher than ~ 6.2–6.9 of the fermented GSW hydrolysate products. The results clearly show that the hydrolysate protein products obtained from this study have higher digestibilities than those of CFM and albumin. As sterilization under high temperature at 121°C and pressure 15 lb/in² for 15 minutes helped free amino acids, the sterilized GSW treatment product showed the highest digestibility. Additionally, sterilization of raw GSW at 121°C for 15 minutes showed the highest digestibility [32]. This improved the digestibility of proteins by opening their structures through denaturation and by degrading protease inhibitors. Protein hydrolysis in the control treatment of GSW fermented without the addition of L. plantarum 541 occurred from endogenous enzymes and natural inborn microorganisms of the raw shrimp head. The hydrolytic activities of the remaining three treatments were from endogenous enzymes and lactic acid bacteria L. plantarum 541.

The results of in vitro digestibility show that the more acidity there is, the more free amino acids or shortened polypeptides there are. Therefore, the protein hydrolysate products of all the fermented GSW treatments were more digestible than CFM and albumin. As the CFM released lower amounts of free amino acids or shortened polypeptides through digestion, this implies that the CFM has lower quantities of low molecular weight proteins (see SDS-PAGE result in Fig. 8) than other hydrolysate protein products. The hydrolysate protein products from fermented GSW in this study are expected to contain highly digestible proteins for the replacement of animal feed CFM. L. plantarum 541, by its proteolytic system, is expected to hydrolyze those proteins in GSW. The proteolytic system of lactic acid bacteria was composed of a cell envelope-associated proteinase, peptide transport systems, and intracellular peptidases [51]. It hydrolyzes proteins to small peptides and amino acids, which can be imported for rapid growth.

3.6. SDS-PAGE of Protein Molecular Weights

Figure 6 shows the SDS-PAGE protein patterns of the markers (Lane 1) with different molecular weights and the molecular weights of the hydrolysate protein products from fermented GSW (Lane 2–6). The patterns of Lane 2 (control, GSW fermented with inborn microorganisms) were similar to those of Lanes 3 and 4 (GSW fermented with inoculants from MRS and PA cultures), which showed five major protein bands with molecular weights (MWs) of 77,000, 60,000, 50,000, 35,000, and 26,000 Da. The molecular weight bands of the hydrolysate protein product of the sterilized GSW fermented with inoculant from MRS disappeared (Lane 5) because sterilization degrades those large MW proteins to too small undetectable molecules. This result agrees well with the results of the in vitro digestion shown in Fig. 7. The molecular weight bands of CFM (Lane 6) were hardly seen or almost disappeared and showed only the band MW of 60,000 Da, which was larger than those of the hydrolysate protein products.

Also reported, the autolysis of waste from shrimp heads was related to proteolytic reactions and involved the soluble endogenous enzymes and the insoluble substrate of shrimp head proteins. The autolysis of shrimp hydrolysate by gradually increasing the temperature up to 70°C within 3 h showed a protein MW of < 5,000 Da [41]. Again, that is why the protein bands of the sterilized GSW above disappeared. The hydrolysate was derived from oat bran protein concentrate (OBPC) hydrolyzed using trypsin [52]. The major protein components in OBPC were large peptides (MW = 29,000–33,000 Da) and small peptides (MW < 20,000 Da). In addition, some peptides of MW < 10,000 might exist. However, they are too small to be detectable under their electrophoresis conditions for the same sterilized GSW reason above. Shrimp waste hydrolysate peptides with MWs ranging between 1,000 and 10,000 Da, 10.2% smaller than 1,000 Da, and 3.44% larger than 10,000 Da have also been reported [13].

The shrimp hydrolysate protein products were obtained from the GSW digested by proteolytic enzymes from L. plantarum 541 in the fermentation processes operated well by the addition of L. plantarum 541 from MRS and PA inoculants. The proximate compositions of protein, lipid, and ash in the hydrolysate protein products from L. plantarum 541 fermentation were found to be lower than those of the CFM and nonfermented GSW, as they were partially used for their cell growth. Although they were lower, they were comparable concentrations. However, the quality of shrimp waste hydrolysate proteins from fermentations with the MRS and PA inoculants were excellent in rich amino acid contents and in terms of their profiles superior to those of the CFM and the nonfermented GSW because of the shorter polypeptide chains, the easier digestibility to free amino acids and to assimilate into the animal cells during digestions. Therefore, the hydrolysate protein products from GSW fermentations by lactic bacteria and from the nonfermented GSW can be essentially used as alternative protein sources and to replace the fishmeal proteins in animal feed. This can save the environment by utilizing waste and saving small fish fishing from fish meal industries. In addition, pineapple juice application as the medium for the production of L. plantarum inoculant is efficient and economical to reduce the cost of using MRS medium. This protein production can be commercially operated on an industrial scale.

Author Contributions: Conceptualization, J.K.; methodology, investigation, analysis, K.R. and writing—original draft preparation, resources, validation, visualization, and data curation, S.C.; writing—review and editing, validation, visualization, supervision, project administration and funding acquisition.

Funding: A research grant was from The Coastal Aquatic Feed Research Institute (CAFRI), Thailand. This work was also supported by the Biochemical Engineering Pilot Plant, Biological Science Graduate Program, Faculty of Science, Burapha University, Chonburi, Thailand.

Acknowledgments: The authors would like to thank Dr. Kwanruthai Malairuang and Mr. Jatuporn Sukna for their help in completing this research paper by adding and editing the contents, tables, and figures.

Conflicts of interest: The authors declare no conflicts of interest. The funder had no role in the design of this study, analyses, or interpretation of data; in writing of the manuscript, or the decision to publish the experimental results.

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Nutritive Values of Protein Hydrolysate Products from Shrimp Waste Utilization by Fermentation with Lactobacillus plantarum

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Methods

2.2.1. Raw Material Preparation

2.2.2. First Inoculum Preparation

2.2.3. MRS Broth Inoculant Preparation

2.2.4. Pineapple Juice Inoculant Preparation

2.2.5. Viable Cell Number Density Concentration during Fermentation (CFU/g)

2.2.6. Protein Hydrolysis by Fermentation

2.2.7. Proximate Analysis of the Compositions

2.2.8 Amino Acid Composition Analysis

2.2.9. In vitro Protein Digestion Analysis

2.2.10. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

3. Results And Discussion

3.1 Growth of Lactobacillus plantarum 541 and Total Bacteria

3.2. The pH Change during Fermentation

3.3. Proximate Compositions of Moisture, Protein, Carbohydrate, Lipid, Fiber, and Ash

3.3.1. Protein Contents

3.3.2. Carbohydrate Contents

3.3.3. Lipid Contents

3.3.4. Fiber Contents

3.3.5. Ash Contents

3.4. Quantitative Analysis of Amino Acid Components

3.4.1. Comparative Study of Each 16 Amino Acid among the Four Treatments and Comparators

3.4.3. Studies of Total Amounts and their Total Fractions

3.5. In Vitro Protein Digestion

3.6. SDS-PAGE of Protein Molecular Weights

4. Conclusions

Declarations

References

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